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Marine Biological Laboratory Library E

Woods Hole, Mass.

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Presented by

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Protozoology

R. P. HALL

New York University

New York

PRENTICE-HALL, INC.

1953

PRENTICE HALL ANIMAL SCIENCE SERIES
H. Burr Steinbach, Editor

PRENTICE HALL, INC.

70 Fifth A\'enue, New York

All rights reserved. No part of this book mav be repro-
duced in any form, by mimeograph or any other means,
without permission in writing from the publishers.

L.C. Cat. Card No.: 52-14030

PRINTED IN THE UNITED STATES OF AMERICA

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ACKNOWLEDGMENTS

The writer is much indebted to several colleagues
for their patience in reading portions ot the manu-
script and tor their helpful suggestions, and also to
the many investigators whose contributions of re-
prints have greatly eased the task of reviewing the
literature.

R. P. Hall

Contents

CHAPTER PAGE

I. General Morphology of the Protozoa 1

II. Reproduction and Life-Cycles 54

III. The Classification of Protozoa 103

IV. The Mastigophora 116

V. The Sarcodina 201

VI. Sporozoa 269

VII. Ciliophora 332

VIII. Physiology 428

IX. Heredity in Protozoa 506

X. Host-Parasite Relationships 527

XI. Protozoa of the Digestive and Urogenital Tracts . 544

XII. The Blood Flagellates 574

XIII. Malaria 597

XIV. Immunity and Resistance 627

Index 654

67853

General Morphology
of the Protozoa

Variations in form of the body

Colonial organization

Non-colonial groupings

Cortex, secreted coverings and skeletons

Pseiidopodia

Flagella and associated structures
Flagella
Axostyles

Costa, cresta, pelta and aciculum
The parabasal apparatus
Multiple karyomastigonts and mastigonts

Cilia and their derivatives

Fibrillar systems

Neuromotor apparatus
Silver-line system
Neuroneme system
Infraciliary network
The infraciliature
Sensory bristles

Significance of fibrillar systems
Silver-line systems of flagellates

Myonemes and contractile stalks

Trichocysts and nematocysts

The cytostome and associated structmes

\'acuoles of Protozoa
Contractile vacuoles
Sensory vacuoles
Vacuoles in flotation

Chromatophores, pigments, pyrenoids,
photoreceptors
Chromatophores
Pyrenoids
Pigments
Photoreceptors

Cytoplasmic inclusioirs
Cytoplasmic food reserves
Chromidia
Mitochondria
\'acuome
Osmiophilic inclusions and organelles

Nuclei of Protozoa
Vesicular nuclei
Nuclear dimorphism
Dispersed nuclei

Literature cited

HE Protozoa include a variety of microorganisms which, by
general agreement of protozoologists, are currently assigned to the phy-
lum. More specific characterization of the Protozoa is difficult and even
the name of the phylum, as applied to the groups it conventionally in-
cludes, is not entirely appropriate. Many flagellates — those usually listed
as Phytomastigoda, Phytomastigina, or Phytomastigophora — are com-
monly considered algae by botanists. Also, the Mycetozoida (Mycetozoa)

2 General Morphology of the Protozoa

of j^rotozoologists are nothing else than the slime-molds of botanists, and
the Sarcosporidia, usually considered Sporozoa, are believed by some
workers to be molds.

This situation, which suggests that protozoologists are unable to dis-
tinguish animals from plants, is somewhat disconcerting to those who
favor consistency in taxonomy. Consequently, various taxonomic reforms
have been suggested. The old term. Protista, recalls such an effort by
Haeckel, but the Protista were only a heterogeneous collection of micro-
organisms with the plant and animal labels obscured. A more positive
reform was proposed by Calkins (17) in his decision to eliminate the
chlorophyll-bearing flagellates from the Phylum Protozoa. On the face
of it, the proposal seemed to be an admission that zoologists had been
in error in laying claim to the "Phytomastigophora." However, some of
the more interesting colorless phytoflagellates were saved from a botanical
fate by arbitrary transfer to the "Zoomastigophora." The resulting mix-
tures could not be justified on the basis of sound taxonomic criteria;
hence, this innovation has not been generally accepted. The basic classifi-
cation of Copeland (33) recognizes a separate Kingdom Protoctista which
includes the Protozoa and various groups of algae and fungi. While this
suggestion sidesteps the problem of deciding which Protozoa are animals
and which are plants, it seems to imply that such Protozoa as the ciliates
are more closely related to the red algae and related organisms than they
are to the Kingdom Animalia.

At present, many protozoologists continue to list the phytoflagellates
and slime-molds as Protozoa, although they realize that botanists have no
objections to placing these groups in the plant kingdom. While the cur-
rent practice is a bit confusing taxonomically, there is the advantage that
botanists and protozoologists can legitimately maintain equal interest in
these groups which apparently represent mergers of the plant and animal
kingdoms.

From the morphological standpoint. Protozoa are often referred to as
unicellular animals, in contrast to the multicellular Metazoa. The small
size and simple structure of many Protozoa tend to justify this designa-
tion. On the other hand, some Protozoa are not so small and are measur-
able in millimeters, or even centimeters, instead of microns. Furthermore,
the uninucleate condition is far from universal. Many species possess more
than one nucleus, and the numbers range from two to many hundreds.
Examples are found in each of the major taxonomic groups. Structural
complexity often extends beyond a mere increase in number of nuclei.
Mycetozoan protoplasm, as noted in Physarum (167), is traversed by chan-
nels through which a liquid, containing many granules, flows back and
forth in a sort of primitive circulatory system. Multiplicity of flagellar
units is associated with multinuclearity in Mastigophora. The result may
be many nucleo-flagellar units (karyomastigonts), as in certain Calonym-

General Morphology of the Protozoa 3

phidae (Fig. 1. 10, D). In addition to normally multinucleate Protozoa,
many species are uninucleate in one phase of the life-cycle and multi-
nucleate in another.

Such structural diversity has led protozoologists into difficulties with
the Cell Theory. Dobell (45), who suggested that Protozoa are non-cel-
lular organisms, was one of the first to revolt against strict application of
the Cell Theory to this group. Such an interpretation has appealed to
some zoologists. A different concept, favored by Kofoid (138) for example,
is that some Protozoa are unicellular while others are multicellular.
Protozoan "multicellularity" is considered analogous to metazoan multi-
cellularity as seen in syncytial tissues. According to this view, the Protozoa
are the phylum in which multicellularity originated in animals.

The evolutionary transition from Protozoa to Metazoa involved dif-
ferentiation beyond the separation of reproductive and somatic cells.
Hyman (98) has stressed the characteristic establishment of an axis along
which morphological and physiological differentiation has occurred. Such
colonial types as Volvox, in spite of their specialized somatic and repro-
ductive "cells," are usually considered Protozoa. The distinction is mainly
one of degree, since Volvox has several attributes of an organism in the
metazoan sense. The colony moves as a unit, with apparently coordinated
flagellar activity, and exhibits some degree of polarity with functional
differentiation. The colony may produce daughter colonies asexually or it
may develop gametes. The zygote develops into a young colony in a man-
ner not unlike that in which a fertilized egg produces a young metazoan
individual. The Myxosporida, another exceptional group, show somatic
differentiation in that some cells produce spore-membranes while others
give rise to the polar capsules of the myxosporidian spore. In other words,
the separation of Protozoa from Metazoa in borderline cases may involve
somewhat arbitrary decisions influenced to some extent by factors of
taxonomic convenience.

VARIATIONS IN FORM OF
THE BODY

Protozoa range from approximately spherical forms to bizarre
shapes not readily explained on a functional basis. Symmetry is often
poorly defined. Most active swimmers show spiral torsion in some degree
and this tendency toward asymmetry is presumably correlated with the
usual spiral course in locomotion (62, 136). However, universal sym-
metry and radial symmetry may be noted in various floating and sessile
species, respectively, and bilateral symmetry is apparent in such genera
as Giardia and Octomitus. In Protozoa which are not spherical, form of
the body may be rather characteristic of a given species under particular
conditions. However, form is often relatively constant rather than abso-
lutely so, and within specific limits, may be modified by environmental

4 General Morphology o£ the Protozoa

conditions and activities of the organism. Even the nature and quantity
of the available food may influence form as well as size of the body. Such
a relationship is striking in Tetrahymena vorax (Fig. 1. 1) when strains
are fed on different diets (118). In addition to the usual variations, attrib-

Fig. 1. 1. Influence of diet on form and size in Tetrahymena vorax.
A. Organism from young broth culture (saprozoic nutrition). B. Speci-
men from older broth culture. C. A ciliate fed on Aerobacter cloacae.
D. A ciliate fed on killed Tetrahymena geleii. E. A large carnivore from
a culture fed living T. geleii. F. A carnivore after transfer to a culture
of living yeast. Ingested food, peristomial area, and contractile vacuole
are indicated diagrammatically but cilia are not shown. x450 (after
Kidder, Lilly, and Claff).

utable to environmental or inherent factors, dimorphic and polymorphic
life-cycles include two or more different morphological stages. Naegleria
gruberi (Chapter V), for example, exhibits both flagellate and amoeboid
stages. Although adaptive trends may be assumed, specific correlation of
form with habitat is impossible in many instances. Yet certain generaliza-

General Morphology of the Protozoa 5

tions are permissible for sessile, floating, swimming, and creeping types.
Floating types, free from the usual stresses of locomotor activity, often
approach a spherical form. Active swimmers are usually elongated, with
a major axis more or less parallel to the path of locomotion. Creeping

Fig. 1. 2. AC. Gonium sociale: side view (A); surface view (B); colony
with superficial continuous matrix (C); x900 (after Pascher). D, E. Gonium
sp., portions of colonies showing supposed protoplasmic connections impreg-
nated with silver; x760 (after Klein). F. Syncrypta volvox; x580 (after Stein).
G. Protoplasmic connections of somatic flagellates in Volvox; xl800 (after
Janet).

forms are frequently flattened and may show differentiated dorsal and
ventral surfaces. Sessile ciliates and flagellates are often more or less
conical, attached to the substratum directly or by a secreted stalk.

In individual Protozoa, form of the body may be maintained by a
thickened cortex (the differentiated outer zone of cytoplasm), by various

6 General Morphology of the Protozoa

secreted layers (pellicle, theca, lorica, test, and shell of particular groups),
and by internal structures such as radiolarian skeletons. The gross mor-
phology of protozoan aggregates and colonies depends upon the means
by which the individual organisms are bound together.

COLONIAL ORGANIZATION

The usual colony consists of similar organisms joined together in
some particular jDattern so that the form of the mature colony is char-
acteristic of the genus or species. As a rule, any member of the colony
may undergo fission or budding. In the Phytomonadida, this is true in
Gonium, Pandorina, and Platydorina but apparently not in Eudorina,
Pleodorina, and Volvox. However, flagellates isolated from colonies of

Fig. 1. 3. Arboroid colonies. A. Phalansterium digitatum, branching ma-
trix; x290 (after Lemmermann). B. Zoothamnium adamsi, portion of colony
showing stalk with continuous branching fibril; diagrammatic (after Stokes).
C. Hyalobryon ramosum, loricate type; x720 (after Awerinzew). D. Poterio-
dendron petiolatum; each lorica with stalk; x290 (after Lemmermann).
E. Cladomonas fruticulosa with continuous branching "lorica"; x290 (after
Lemmermann).

General Morphology of the Protozoa 7

Eudorina, Gonium, and Pandorina may undergo fission and produce
daughter colonies (11). The component flagellates of the Volvox colony
are differentiated into somatic and reproductive individuals and the
former are believed to lose their reproductive ability when the colony
reaches maturity.

Protozoan colonies are usually classified on the basis of their organiza-
tion. Spheroid and discoid colonies, containing a matrix secreted by the
associated organisms during development of the colony, are represented
by such ciliates as Ophrydium and various flagellates — Syncrypta, Go-
nium, Pandorina, Volvox, and others. In Gonium sociale, for example, the
matrix shows two components (Fig. 1. 2, C), a "cell wall" enclosing each
flagellate and a continuous outer gelatinous layer. In some specimens (Fig.
1. 2, A, B) the outer layer is lacking. Each flagellate in the Volvox colony
is enclosed in a thin cell wall and a thick outer sheath. Except in V.
aureus, the boundaries of the individual sheaths are readily distinguished.
The flagellates appear to be joined by protoplasmic strands in certain
species of Volvox (Fig. 1. 2, G) and apparently also in Eudorina, Gonium,
and Pandorina (11). Dried colonies of Gonium (Fig. 1. 2, D, E), after
silver impregnation, show "silver-line" connections between adjacent
flagellates (131).

In arboroid colonies (Fig. 1. 3), the individual organisms are arranged
in a branching pattern. Stalks are characteristic of many arboroid colonies.
In different species, each organism may have its own stem which is at-
tached to a common stalk, or each stalk of the framework may bear more
than one organism. Such stalks may be gelatinous or sometimes solid and
relatively firm, and in certain cases they are elastic tubes containing
contractile fibrils. In other arboroid types, colonial organization is main-
tained by attachment of one lorica to another (Fig. 1. 3, C, D), or by a
continuous tubular "lorica" in which the organisms are located at the
tips of the branches (Fig. 1. 3, E).

NON-COLONIAL GROUPINGS

Certain other aggregates are not colonies in the strict sense. So-
called catenoid colonies have been described in dinoflagellates (Fig. 1.
4, D) and certain astomatous ciliates (Fig. 1. 4, C). These chains arise in
repeated fission without prompt separation of daughter organisms and
are temporary groupings rather than true colonies. Palmella stages (Fig. 1.
4, A) of certain flagellates develop in much the same manner as spheroid
and discoid colonies. However, the palmella does not show a well defined
range in size, the number of organisms varies with size of the mass, and
the flagellates lack flagella. The term, gleocystis stage, is sometimes applied
to similar aggregates in which an individual gelatinous layer surrounds
each organism (Fig. 1. 4, B).

8 General Morphology of the Protozoa

Fig. 1. 4. A. Palmella stage, as seen in Haematococcus and related
Phytomonadida; diagrammatic (after Wollenweber). B. Gleocystis stage, as
found in various Chlamydomonadidae; diagrammatic (after Goroschan-
kin). C. Chain ("catenoid colony") of Haptophrya niichiganensis; x90
(after Bush). D. Chain formed in fission of Gonyaulax catenella; x580
(after Whedon and Kofoid).

CORTEX, SECRETED COVERINGS,
AND SKELETONS

No well developed cortex is apparent in simple flagellates or
typical amoebae. The superficial cytoplasmic layer of Amoeba proteus
is formed from, and gives rise to endoplasm continuously during amoe-
boid activity and thus lacks the relative permanence of the cortex in more
specialized Protozoa. However, some amoeboid organisms have a thin
pellicle similar to that of Amoeba verrucosa. In this species, the pellicle
maintains itself under mechanical stress in microdissection (96).

At the other extreme, the relatively thick cortex of a ciliate may con-
tain basal granules, fibrils, myonemes, mitochondria, and other inclu-
sions, and sometimes trichocysts. Although often flexible, the layer is at
least firm enough to maintain a typical body form in the swimming
ciliate. The pellicle covering the surface of ciliates seems to be a distinct

General Morphology of the Protozoa 9

layer, and Blepharisma undulans is said to shed its pellicle after treat-
ment with strychnine. The cilia are withdrawn and the body retracted,
leaving a space beneath the pellicle, and the ciliate later emerges through
the old cytostomal area or the region of the posterior contractile vacuole
(169).

Surface layers of flagellates range from a delicate periplast or pellicle,
similar to that of certain amoebae, to thick tests or shells. The flexible

Fig. 1. 5. A, B. Ventral and dorsal thecal plates in Gonyaulax acatenella;
x560 (after Whedon and Kofoid). C. Vaginicola longicoUis, optical section of
lorica; xl40 (after Penard). D. Stokesiella lepteca, stalked lorica; xl060 (after
Stokes). E. Test of Euglypha alveolata; x350 (after Leidy). F. Difflugia corona;
xl35 (after Leidy). G. Tintinnopsis nucula, optical section of lorica; diagram-
matic; x570 (after Campbell).

periplast of many Euglenida permits a characteristic euglenoid movement
("metaboly"), but tends to maintain a characteristic form in the swim-
ming flagellate. This periplast presumably is a secreted layer, since it
becomes separated from the underlying cytoplasm in plasmolysis (22).
Thickened pellicular layers, as seen in LepocincUs and Phacus, may be so
firm that the body shows little change in shape. Such membranes are
often decorated with ridges, papillae or other markings.

The theca of many Phytomonadida and Dinoflagellida is a secreted

10 General Morphology of the Protozoa

covering applied directly to the surface of the body and is comparable to
the thick cell wall found in higher plants. The flagella emerge through
pores in the theca. A theca may be somewhat flexible, allowing slight
changes in form, or it may be rigid. The firmness imparted by cellulose
or pectins is sometimes increased by impregnation with inorganic salts
to produce a hard covering, as in Phacotus, Trachelomonas, and some of
the dinoflagellates. The theca of many dinoflagellates is differentiated into
a number of plates (Fig. 1. 5, A, B), the pattern varying with the species.
Lorica, test, and shell are terms applied to coverings which often fit
less closely than the theca and hence are less comparable to the typical
cell wall of plants. A lorica (Fig. 1. 5, C, D) is usually a tubular or vase-
like structure with an opening through which the anterior part of the

^r^'

Fig. 1. 6. Groups of myxopodia (A) and axopodia (B); diagrammatic.

body or its appendages can be extended. The base of the lorica, in sessile
species, may be attached directly to the substratum or may end in a
stalk. In colonial types (Fig. 1. 3, C, D), one lorica may be attached to
another directly or by means of a stalk. A lorica may be composed en-
tirely of secreted material or may be reinforced with diatom shells, sand
grains, or other foreign particles.

The tests (or shells) of many Sarcodina vary widely in form and com-
position. Some appear to be homogeneous. Others consist mainly of sep-
arate elements cemented together, as in Euglypha and Difflugia (Fig. 1.
5, E, F). The test of Euglypha is composed of plates, formed within the
body prior to fission; that of Difflugia is made of sand grains embedded
in a secreted cement. The comparable arenaceous tests of certain Foram-
iniferida (Chapter V) are built of sand grains, discarded tests, sponge

General Morphology o£ the Protozoa H

spicules, or other materials cemented together over a thin chitinous test.
The composition of other foramiferan tests varies from group to group.
That of the AUogromiidae is typically chitinous, while the majority of
the multichambered tests are calcareous. Siliceous tests also have been re-
ported in a few Foraminiferida. In many species at least, the foraminif-
eran test is not really external; instead, it is normally enclosed within a
thin layer of cytoplasm.

The simplest skeletons of Radiolarida are represented by scattered
siliceous spicules, while the more complicated types are structures unique
among the Protozoa (Chapter V). In the Acantharina long spines radiate
in definite patterns from the center of the body. To these elements is
often added a lattice-work shell, joining and supported by the spines.
Siliceous skeletons of other Radiolarida are quite varied in structure.
Spherical types may be composed of several concentric lattice-work shells,
and sometimes of spicules in addition. Bilateral types, conical forms, and
other departures from radial symmetry are fairly common.

PSEUDOPODIA

Pseudopodia are temporary organelles which can be retracted and
formed anew, depending upon activities of the organism. Four major
types may be distinguished — lobopodia, filopodia, myxopodia, and
axopodia.

Lobopodia, which have relatively dense outer layers and more fluid
inner zones, are relatively broad pseudopodia with rounded tips. Short
or slender lobopodia may be hyaline, but larger ones usually show a clear
ectoplasm enclosing a granular endoplasm. Lobopodia are characteristic
of amoebae, certain flagellates, and certain testate rhizopods (Fig. 1. 5, F).

Filopodia are slender hyaline pseudopodia which taper from base to
pointed tip and also tend to branch and anastomose. In addition, filo-
podia may fuse locally to produce thin webs of cytoplasm. The absence
of circulating granules helps to distinguish filopodial from myxopodial
nets.

Myxopodia (rhizopodia, or reticulopodia), characteristic of the Foram-
iniferida, are filamentous structures (Fig. 1. 6, A) which branch and
anastomose into complex networks often covering a wide area. Such nets
are efficient food-traps and are fairly effective locomotor organelles. In
addition, the digestive activities of myxopodia are usually marked in
Foraminiferida (Chapter V). The comparatively dense inner zone of the
myxopodium has been considered fibrillar in structure (198). The fluidity
of the outer layer is indicated by the active circulation of cytoplasmic
granules, as illustrated by Elphidium (Polystomella) crispum (103).

Axopodia (Fig. 1. 6, B) tend to radiate singly from the surface of more
or less spherical organisms (Heliozoida, Radiolarida). The axial filament
of a typical axopodium has been described as a fibrillar tube enclosing a

12 General Morphology of the Protozoa

homogeneous core (193, 195). In contrast to the axial filament, the outer
cytoplasm is a sol, as indicated by the movement of inclusions. Axial
filaments may converge in a central granule (Acanthocystis and related
genera) or they may end separately in the cytoplasm (Actinosphaerium).

FLAGELLA AND ASSOCIATED
STRUCTURES

Flagella

These organelles are found in Mastigophora and in flagellate stages
of Sarcodina and Sporozoa. A typical fiagellum is composed of a sheath,
which may be circular, elliptical, or flattened in cross-section, and an
inner axoneme. The latter, according to some workers, is the active por-
tion of the fiagellum while the sheath is merely protective. Others think
that the axoneme is only an elastic support for a contractile sheath. The
axoneme arises from a granule, the blepharoplast . and may or may not
extend beyond the sheath as a distal end-piece (Fig. 1. 7, F). A terminal
knob (Fig. 1. 7, H), instead of a filament, is evident in silver preparations
of Trypanosoma rhodesiense (127). The anterior flagella of Hexamitus
pulcher (130) also are unusual in that they arise from external rod-like
structures (Fig. 1. 7, E) of uncertain significance.

The finer structure of the fiagellum^ is incompletely known, although
investigations with the electron microscope (13, 56, 180, 199) have sup-
plemented earlier observations. The axoneme may be composed of one,
two (Astasia, Euglena), three {Peraneyna), or perhaps more fibrils, while
the sheath apparently contains a spirally coiled filament in certain species.

The sheath in some flagella shows lateral filaments (Fig. 1. 7, A, C),
the mastigonemes (43) or "Flimmer," the nature of which is uncertain.
Although observed in living Mallomonas acaroides in dark-field (217),
they may be artifacts (173) or may represent fibrils of the sheath which
are frayed out laterally under certain conditions (180). At any rate, such
filaments appear consistently in some species and not in others. In the
stichoneynatic fiagellum (43), a single row of filaments extends along one
side of the sheath (Fig. 1. 7, A), as in Astasia and Euglena (13). In the
pantojiematic type there are two or more rows of mastigonemes. Only a
terminal filament is present in the acronematic, or "lash" fiagellum (174),
while the pantacronematic type shows both a terminal filament and one
or more rows of mastigonemes. A simple type, found in Cryptomonadida
and Dinoflagellida (174), shows neither terminal filament nor masti-
gonemes. These characteristics of the fiagellum seem to be constant within
various groups and may furnish significant information in studies on
taxonomy and phylogeny (174, 217).

In the majority of flagellates, the flagellum extends forward from its

^ This subject has been reviewed in several papers (13, 174, 180, 217).

General Morphology o£ the Protozoa 13

origin, whereas a trailing flagellum (Fig. 1. 9, F) arises anteriorly but is
trailed posteriorly in swimming. A trailing flagellum may be of the con-
ventional type, or it may be ribbon-like as in Macrotrichomonas pulchra
(126). The undulating rnembrajie of Trichomonas and related genera
(Fig. 1. 7, G, I) contains a marginal flagellum which originates in an
anterior blepharoplast and extends posteriorly, sometimes beyond the end

niiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiir^ "^

Fig. 1. 7. A-D. Deflandre's types of flagella: stichonematic (A), acrone-
matic (B). }3aiUonematic (C), pantacioneniatic (D). E. Hexarnitus pulcher,
flagella with rod-like basal portions; protargol; x3460 (after Kirby and
Honigljerg). F. Acronematic flagella of Moiiocercomonoides piUeata; pro-
targol; \3960 (after Kirby and Honigberg). G. Ribbon-like flagellum of
undulating menil,rane in Trittichomoims imiris; protargol; x2810 (after
Kirby and Honigberg). H. Terminal knob on flagellum of Tiypaiwsoma
brucei; protargol; xI790 (after Kirbv). I. Undulating membrane in Penta-
trichomonas honiinis; x2660 (after Wenrich). J. Axial flagellum and ribbon-
like transverse flagellum of Gyrodinium dorsum x470 (after Kofoid and
Swezy). Key: a, axostyle; c, costa; /, flagellum in undulating membrane.

of the membrane. This marginal flagellum is sometimes ribbon-like, as in
Tritrichomonas muris (130). The undulating membrane of Trypanosoma
originates near the posterior end of the body and extends to the anterior
end (Fig. 1. 7, H).

The majority of species have only one or two flagella. More than four
are rare in free-living flagellates, although not in parasites. When several
flagella are present, they may differ in size, structure, and activity.
Pentatrichomonas hominis (Fig. 1. 7, I), for instance, has four relatively

14 General Morphology of the Protozoa

short anterior flagella, a longer fifth flagellum, and an undulating mem-
brane. A typical dinoflagellate (Fig. 1. 7, J) has a transverse flagellum,
lying in a spiral groove (the girdle), and an axial (or longitudinal) flagel-
lum extending posteriorly from a lateral or postero-lateral origin.

Axostyles

The axostyle (Fig. 1. 8) varies from a filament to a thick hyaline
rod, usually joined to a blepharoplast and extending posteriorly along
the major axis of the body. The axostyle may end in the body or may
project externally, sometimes tapering to a filament which may serve for
attachment to the host (124). The anterior end is often expanded into a

Fig. 1, 8. Axostyles. A. Capitulum and anterior portion of axostyle in
Hyperdevescovina insignita; xl800 (after Kirby). B. Slender axostyle in
Monocercomonoides pilleata; x3600 (after Kirby and Honigberg). C. Mul-
tiple axostyles of Snyderella tabogae; diagrammatic; x350 (after Kirby).
D. Tritrichomonas augusta, axostyle with inclusions; xl950 (after Kofoid
and Swezy). E. Axostyle with capitulum in BuUanympha silvestri; x750
(after Kirby). Key: a, axostyle; b, blepharoplast; c, capitulum; ct, cortex;
m, mastigont; n, nucleus; t, trailing flagellum; u, undulating membrane.

General Morphology of the Protozoa 15

capitulum (Fig. 1. 8, A, E). Many multinucleate species contain a num-
ber of axostyles, one for each mastigont, and the distal portions of the
axostyles form a bundle extending posteriorly as in Snyderella (Fig. 1.
8, C).

Staining reactions of the axostyle vary in different species. Iron-hema-
toxylin stains the axostyle of Monocercomonoides pilleata (Fig. 1. 8, B)
but not that of certain other flagellates. The organelle appears homo-
geneous in some species, shows a sheath and a core in others (Fig. 1. 8,
A), and sometimes contains stainable granules (Fig. 1. 8, D). The axo-
style of Trichomonas termopsidis (124) is stained brown in iodine solu-
tion.

Costa, cresta, pelta, and aciculum

The costa (Fig. 1. 7, G; 1. 8, D) arises from a blepharoplast and
extends along the base of the undulating membrane in various tricho-

Fig. 1. 9. AC. Pelta, different views, Hexamastix citelli; x6500 (after
Kirby and Honigberg). D. Aciculum of Cryptobia helicis; kinetoplast indi-
cated diagramraatically; x5330 (after Kozloff). E. Cresta, small type, Cadu-
ceia kofoidi; x3060 (after Kirby). F. Large cresta, Macrotrichornonas emer-
soni; shelf-like unguis attached; trailing flagelluni ribbon-like; xl425 (after
Kirby). Key: a, axostyle; ac, aciculum; cr, cresta; ct, cortex; k, kinetoplast;
71, nucleus; p, pelta; t, trailing flagellum; u, unguis.

16 General Morphology of the Protozoa

monad flagellates. The function of the costa is uncertain, although it may
add firmness to the cytoplasm underlying the undulating membrane.

The cresta (Fig. 1, 9, E, F), possibly a homologue of the costa, is
present in Macrotrichomonas and related genera. This organelle is a
somewhat triangular membrane, often visible in the living organism and
apparently capable of independent movement (125). The broad anterior
end is usually joined to a blepharoplast, while the rest of the cresta ex-
tends posteriorly with its outer margin near the periplast. The length, in
different species, ranges from about 1.5[x to almost that of the body. A
trailing flagellum, sometimes loosely adherent to the periplast anteriorly,
may parallel the cresta (Fig. 1. 9, F) and thus simulate the relationship
between the undulating membrane and the costa.

The pelta (Fig. 1. 9, A, C), demonstrable by the Bodian silver tech-
nique, is a crescentic membrane lying anterior to and separate irom the
blepharoplasts in certain flagellates. The pelta may be homologous with
a membranous extension of the axostylar capitulum in certain devesco-
vinid flagellates (128).

The acicuhim (Fig. 1. 9, D) of Cryptobia helicis, a needle-like structure
lying opposite the kinetoplast and extending approximately to the origin
of the anterior flagellum, is detectable in living material but is best
demonstrated by the Bodian silver technique (141).

The parabasal apparatus

In many parasitic and a few free-living flagellates a parabasal
apparatus, an organelle of unknown function, forms part of the mastigont
(Fig. 1. 10). The simplest type is a small compact body, often attached
by a rhizoplast to a blepharoplast. At the other extreme, the apparatus
may be a large branched structure or may be composed of separate
elements.

The index of refraction of the parabasal body is approximately that
of the cytoplasm and vital staining is rather slow (71); consequently,
the organelle is not readily seen in the living flagellate. The apparent
internal structure may vary with the species as well as with methods of
fixation and staining (47, 123).

The parabasal apparatus of free-living flagellates shows little variety
(Fig. 1. 10, H, L, N-P). One or two small parabasal bodies have been
described in several species; one or more long slender bodies, in certain
others. In the tetranucleate Polykrikos schwartzi (27), each band-like
parabasal body is attached to a ring encircling the intracytoplasmic por-
tion of an axial flagellum. The parabasal body of Codosiga elegans (196)
is of special interest because it closely resembles a structure (Fig. 1. 10,
M) described in choanocytes of calcareous sponges (218).

Among parasitic flagellates, the complexity of the parabasal apparatus
varies widely. The small kinetoplast of Trypanosoma hnicei (Fig. 1. 10,

Fig. 1. 10. Parabasal apparatus in different flagellates. A. Tetramitus bu-
fonis; x2200 (after Duboscq and Grasse). B. Pseudodevescovitia imiflagellata;
xllOO (after Kirby). C. Single mastigont of Snyderelln lahogae (see Fig.
1. 8, C): diagrammatic (after Kirby). D. Stephaiwnyinfyha nehimbitim. dia-
grammatic optical section showing karyomastigonts; x750 (after Kirby). E.
Karyomastigont of 5. nelumbium; xI870 approx. (after Kirby). F. Hyper-
devescovina torquata; xl050 (after Kirby). G. Macrotriclionionas ramosa;
xl360 (after Kirby). H. Bodo caudatus; x3600 (after HoUande). I. Leptomonas
ctenocephali; diagrammatic (after A. and M. Lwoff). }. Trypanosoma brucei,
kinetoplast seen on edge; protargol; diagrammatic (after Kirby). K. T. brucei,
surface view of kinetoplast (after Kirby). L. Codosiga elegans, a choanoflagel-
late; diagiammatic (after de Saedeleer). M. Choanocyte of a sponge, Clathrina
coriacea; diagrammatic (after Volkonsky). N. Chilomonas Paramecium; x2850
(after Hollande). O. Polytoma uvella; diagrammatic (after Volkonsky). P.
Cercobodo heimi; x3450 approx. (after Hollande). Key: a, axostyle; ab, apical
body; b, blepharoplast; co, collar; cr, cresta; f, parabasal filament; k, kineto-
plast; Aw, karyomastigont; n, nucleus; p, parabasal apparatus; pn, para-
nuclear body; r, periflagellar ring; rh, rhizoplast.

18 General Morphology o£ the Protozoa

J, K) is fairly typical of the Trypanosomidae, although the mastigont
of Leptomonas ctenocephali (151) is less simple. In addition to the kine-
toplast, a periflagellar ring in L. ctenocephali gives rise to a long para-
basal filament (Fig. I. 10, I). A simple elongated parabasal body is found
in certain uninucleate Trichomonadidae (Fig. 1. 10, A) and in each
complete mastigont of such multinucleate genera as Stephanonympha,
Calonympha, and Snyderella (Fig. I. 10, C-E). In certain flagellates a long
parabasal body is coiled around the axostyle (Fig. 1. 10, F), while the
apparatus of Macrotrichomoyias ramosa (126) is branched (Fig. 1. 10, G)
and that of Pseudodevescovina uniflagellata is compound (Fig. 1.10, B).
A complex apparatus, often including many separate elements, occurs
also in various Hypermastigida (47).

The special term, hinetoplast (127), has been applied to the parabasal
body of trypanosomes and related flagellates. This usage seems justified.
Kinetoplasts are Feulgen-positive (104, 152, 188, 192) and are demon-
strable by methods of fixation and staining which are unsatisfactory for
the trichomonad parabasal body. Finthermore, the kinetoplast divides
in fission whereas this is rarely the case in other types of parabasal
apparatus.

Multiple karyomastigonts and mastigonts

The kinetic elements of many multinucleate flagellates have in-
creased in number along with their nuclei. Each flagellar unit (mastigont)
is associated with a nucleus in Coronympha and Stephanonympha (Fig.
1. 10, D). Such flagellates thus contain a number of karyomastigonts, each
composed of a nucleus and associated blepharoplasts, flagella, parabasal
body, and axostyle. This appears to be the primitive condition in such
flagellates. Two sets of flagella are associated with each of the four
nuclei in Polykrikos sclnvartzi (25); the flagellar apparatus has doubled
independently of the nucleus without otherwise disrupting the basic
karyomastigont (Fig. 4. 20, G). Caloyiympha represents an intermediate
condition showing both karyomastigonts and mastigonts, the latter being
far more numerous. A degree of specialization rare in flagellates — inde-
pendence of nucleus and mastigont — is represented by Snyderella tabogae
(Fig. 1. 8, C), in which the several dozen nuclei are all dissociated from
the hundreds of mastigonts.

CILIA AND THEIR DERIVATIVES

Cilia are structurally similar to flagella but are shorter and more
restricted in movement and are generally present in greater numbers.
Prorodon teres, for example, is equipped with about 11,600 cilia (231). A
cilium, like the flagellum, apparently consists of a sheath and an axoneme
ending in a basal granule. A "sensory" component has been described as

General Morphology of the Protozoa 19

a thin argentophilic layer covering the axial filament and tapering distally
to a granular "end-organ" (132), Electron micrographs indicate that the
unfixed, dehydrated axoneme is composed of fibrils in Paramecium,
whereas a sheath is suggested merely by possible remnants of an envelop-
ing layer (102, 199).

In certain ciliates an accessory "ciliary corpuscle" (30) is attached to
the basal granvde; in some instances, the accessory body may be mito-
chondrial in nature (26). Two accessory granules have been reported in
certain ciliates (132). A slender fibril, the ciliary rootlet, extends inward
from the basal gianule in some ciliates, but is said to be absent in certain
primitive species (29). From many of the basal granules in Opalijia
obtrigonoidea (Fig. 1. 11, G), fibrils extend dorso-ventrally through the
cytoplasm to end in basal granules on the opposite side of the body (34).
Whether these fibrils are homologous with the ciliary rootlets of other
ciliates is uncertain.

Cilia lie in meridional or spiral rows in the less specialized ciliates.
Although such a pattern is usually rather constant within a species,
changes from spiral to meridional to spiral, and even a reversal of the
spirals, occur in certain species with complex life-cycles (150). Individual
cilia, in ciliates with sculptured pellicles, may emerge from grooves, from
the margins of such grooves, or from individual pits in different cases.

The simple cilium is the primitive locomotor structure in ciliates.
Many species possess compound organelles which have arisen by fusion of
cilia in longitudinal or transverse rows, or in tufts. Such organelles are
known as undulating membraries, memhranelles and cirri. An undulating
membrane, formed by the fusion of one or more longitudinal rows of
cilia, lies in the peristome (oral "groove") of various species. Rippling
movements of the membrane drive particles to the cytostome. This organ-
elle may not be permanent in quite the same sense that cilia are so
considered, since the membrane of Blepharisma undulans may break up,
spontaneously or after injury, into individual cilia. The cilia eventually
fcuse again into an undulating membrane (24). Memhranelles, which are
more or less triangular flaps formed by fusion of two or more transverse
rows of cilia, are found especially in the peiistomial area (Fig. 1. 15, H).
Each membranelle of Spirostomum ambiguum contains a double row of
cilia whose basal granules end in a plate parallel to the surface of the
body (Fig, 1, 11, E), A basal lamella, extending inward from the plate,
tapers to an end-thread which joins a basal fibril in the endoplasm (8),
Cirri, characteristic of the Hypotrichina, consist of tufts of cilia probably
embedded in a matrix (Fig. 1, 11, C, D). The number of cilia varies with
the size of the cirrus — in Oxytricha fallax, for example, three or more in
the small marginal cirri and 8-18 in the ventral, frontal, and anal cirri
(146).

General Morphology of the Protozoa

T^^^T^ %t vo;

es-

©/ ^®y<s

..?:(?Ji.?/</..l!ly.Ai

Fig. 1. 11. A. Longitudinal fibril joining basal gianules in Entorhi-
pidiiim echini; longitudinal section of cortex; xl890 (after Lynch). B. Trans-
verse connecting fibrils in E. echini; cross-section of cortex; xl800 (after
Lynch). C. Frayed cirrus of Oxytricha fallax showing component cilia; x2025
(after Lund). D. cirrus of O. fallax; xl725 (after Lund). E. A membra-
nelle of Spiiostomum anihiguimj; diagrammatic (after Bishop). F. Basal
granules and connecting fibrils in Tillina canalifera; diagrammatic (after
Turner). G. Dorso-ventral fibrils joining basal granules in Opalina obtri-
gonoidea; longitudinal section; xllOO (after Cosgrove). Kev: b, basal fibril;
c, cilium; e, end-thread; es, endoplasmic spherule; j, longitudinal fibril; g,
basal granule; gi, primary basal gianule; gs, secondary basal granule; I,
basal lamella; m, membranelle; p, basal plate; t, transverse fibril; tr, tricho-
cyst.

FIBRILLAR SYSTEMS

The basal granules in each longitudinal row of cilia are joined by
a fibril. Transverse fibrils may also link the basal granules in some species
(Fig. 1. 11, A, B). Tillina canalifera (123) is unusual in that longitudinal
and transverse fibrils join secondary basal granules, which in turn are
connected by rhizoplasts to superficial primary basal granules from which
the cilia arise (Fig. 1. 11, F). In Opali?ia obtrigorioidea, oblique fibrils
join basal granules in different longitudinal rows but longitudinal fibrils
cannot be detected (34). This situation suggests possible modification of
the primitive symmetr)' during the evolution of opalinid ciliates.

The longitudinal fibrils in certain ciliates seem to be morphologically
independent (29). In other species, the fibrils are joined in complex

General Morphology o£ the Protozoa 21

fibrillar systems (64, 132, 211) referred to as neuromotor apparatus,
silver-line system, neuroneme system, and infraciliature by different
workers. These "systems" have been demonstrated by various techniques,
so that it is difficult to correlate each one with all the others. In general,
however, the neuromotor system seems to be both endoplasmic and
ectoplasmic while the other fibrillar systems occupy a superficial position.

Fig. 1. 12. Silver-line systems (after Klein). A. Prorodon teres; narrow-
mesh type with some orientation of filjrils. B. Primitive narrow-mesh type.
C. Striation-system in Cyclidiutn glaucoma. D. Double striation-system in
Cinetochilum marmritaccum.

Neuromotor apparatus

The neuromotor system of Euplotes (79, 209, 212, 238) includes a
relatively small number of fibrils (Fig. 1, 13, G). Those from the anal
cirri converge anteriorly in a "motorium," from which a membranelle
fibril passes anteriorly and to the left, and then posteriorly beneath the
peristomial membranelles. In addition, groups of fibrils extend from the
basal plates of the frontal and ventral cirri into the endoplasm. Although
the first and second frontal cirri are joined by such fibrils, no intercon-
nections have been demonstrated for the other cirri (79). A comparable
neuromotor apparatus has been described in other ciliates (211).

Silver-line system

The observations of Klein (132, 134) and others have revealed a
silver-line system, in many ciliates. The name of the system is derived from
Klein's technique, in which reduced silver is deposited on superficial
structures. The argentophilic "silver-lines" are assumed to be plastic
structures having the capacity to grow, split, undergo resorption, and
then reappear (132).

The primitive system is a narrow-mesh (0.75-1. 0|j.) network containing
the basal granules (Fig. 1. 12, B). The fibrils themselves extend through
the interstices of the ectoplasmic alveoli. Since a narrow-mesh network
has been reported in Dileptus, Oxytricha, Epalxis, Spirostomum, Stentor,

22 General Morphology o£ the Protozoa

Fig. 1. 13. A. Myonemes in anterior half of Stentor coeruleus; dia-
grammatic (after Dierks). B. Portion of a myoneme (S. coeruleus) showing
cross-striations; diagrammatic (after Dierks). C. A neuroneme in Para-
mecium; diagrammatic (after Gelei). D-E. Myonemes in Monocystis agilis,
longitudinal and transverse sections; diagrammatic (after Roskin and
Levinson). F. Sensory bristle of Euplotes patella; diagrammatic (after
Hammond). G. Neuromotor system of Euplotes (patella) eurystomum,
showing major fibrils and incisions made in microdissection; x680 (after
Taylor). Key: c, cilium; cf, anal-cirrus fibril; cm, circular myoneme; g,
basal granule; Ic, longitudinal canal containing myoneme; Im, longitudinal
myoneme; m, motorium; /«/, fibril to membranelles; n. neuroneme; p,
pellicle; r, rodlet of "rosette"; s, "sensory" fibril; t, trichocyst; tg, tricho-
cyst-granule; 1-5, approximate planes of incisions in Taylor's operations.

General Morphology of the Protozoa 23

and also in Podophrya (Suctorea), this primitive system obviously is not
limited to unspecialized ciliates.

Specialization of the silver-line system involves first an increase in
diameter of the mesh, so that a single mesh comes to enclose a group of
ectoplasmic alveoli. As the mesh widens, the silver-lines decrease in num-
ber and begin to parallel the rows of basal granules (Fig. 1. 12, A). In
meridional ciliation, the system may be reduced to ineridians bearing the
basal granules and sometimes joined by transverse commissures (Fig. 1.
12, C) — so-called striation-systems. Each meridian is sometimes double
(Fig. 1. 12, D). Or the silver-line may be a bundle of fibrils. In Colpidiitm
colpoda (132), each meridian is said to split just behind the cytostome
into primary and secondary meridians. The basal granules lie very close
together in the fused anterior meridians. Posteriorly, the basal granules
in the primary meridians are spaced at fairly short and rather regular
intervals; those in the secondary meridians, at longer and more irregular
intervals.

In addition to the basal granules of cilia, the silver-line system includes
trichocyst-granules and protrichocyst-granules, all three types being con-
sidered '"relator-granules" which relate the corresponding structures to
the silver-lines. The protrichocyst-granules also have been considered
tec tin-granules (12), which presumably are extruded through secretory
pores in the pellicle (65). The relator-granules, especially the basal gran-
ules, may persist after their organelles have disappeared in phylogeny,
and thus represent persistent traces of much more primitive conditions
(132).

Both the fibrils and the relator-granules are said to lie at the same
level in the ectoplasm (132). However, the network ("indirect system"
of Klein) apparently does not occupy the same plane as the basal gran-
ules and longitudinal fibrils in Paramecium caudatum, since the latter are
not in focus in photomicrographs which show the network clearly (67).
Lund (145) concluded that the peripheral "network" in P. multimicro-
nucleatum represents pellicular ridges upon which silver is deposited in
dried specimens. The correlation between pellicular markings and the
silver-line pattern also has been stressed by Jacobson (100). Therefore,
the exact nature of Klein's superficial network remains uncertain. The
silver-line meridians, which join the basal granules, appear to be sub-
pellicular.

Neuroneme system

This system (64, 67), demonstrable by the techniques of Gelei
and Horvath (68), joins the basal granules and possibly corresponds to
the meridians of Klein. In sectioned material, the neuronemes of Para-
mecium caudatum (Fig. 1. 13, C) appear as zigzag lines joining the basal
granules to the more superficial trichocyst-granules (64). The neuronemes
are not continuous with the superficial silver-line network of Klein.

24 General Morphology of the Protozoa

Infraciliary network

This system lies at the level of the basal granules or somewhat
deeper, between the alveolar and the inner ectoplasmic layers (62).
Neither the basal granules nor the trichocysts are directly connected with
this system, and no connection with the outer network has been observed.
The longitudinal fibrils of the infraciliary system generally follow the
pattern of the ciliary rows.

The infraciliature

The basal fibril, or kinetodesma, of Chatton's infraciliature is
considered to be separate from the ciliary meridian of Klein. The silver-
line fibril is said to lie on the left of the basal granules while the
kinetodesma lies on the right (26, 29, 214). However, such regularity in
position of the silver-line is not apparent in some of Klein's figures, and
in ciliates showing "circular fibrils" in the silver-lines, basal granules may
lie on the circular fibril or may be enclosed by it (133).

Sensory bristles

So-called sensory bristles, apparently associated with the fibrillar
systems of certain ciliates, are well developed on the dorsal surface of
Euplotes (79, 212). Each bristle arises from a granule at the base of a pit
which is surrounded by a "rosette" of rodlets (Fig. 1. 13, F). On the
ventral surface, two or three similar rosettes without bristles lie near
the base of each cirrus. Analogous structures, supposedly sensory in func-
tion, have been described in Didinium and certain other genera (63).

Significance of fibrillar systems

According to various workers, the fibrillar systems of ciliates are
contractile fibrils, supporting or skeletal structures, organizers in the onto-
genetic development of related organelles (134), coordinating systems,
and delicate circulatory systems for transporting such materials as nucleo-
proteins to the basal granules and trichocysts (86),

Except for whatever support these hypotheses may derive from mor-
phological relationships, the data bearing on functions of fibrillar systems
are meager. The results of microdissection suggest a coordinating func-
tion in Euplotes (209). The adoral membranelles in this hypotrich are
important in swimming, while the anal cirri play a major part in creep-
ing. Cutting the membranelle-fibril (Fig. 1. 13, G) destroyed coordination
of the membranelles so that swimming movements were abnormal. Sever-
ing the fibrils to the anal cirri affected both creeping and swimming,
while destruction of the motorium disturbed the coordination of the anal
cirri and membranelles. Incisions not severing the neuromotor fibrils
failed to modify swimming or creeping movements. Similar experiments

General Morphology of the Protozoa 25

(236) indicate that coordination of the ciliary beat in Paramecium is
dependent upon impulses transmitted longitudinally through the ecto-
plasm. These findings seem to eliminate Klein's superficial network as a
coordinating system in Paramecium and suggest, instead, such a function
for the longitudinal fibrils (superficial fibrils of the neuromotor appa-
ratus, neuronemes of Gelei, kinetodesmas of Chatton and possibly the
silver-meridians of Klein). More recently, it has been concluded that the
cortical localization of acetycholinesterase in Tetrahymena pyriformis
supports the hypothesis that conduction by the fibrillar system is similar
to conduction along nerve fibres (201a).

Silver-line system of flagellates

Among the dinoflagellates, Polykrikos schwartzi (28) and such
gymnodinioid types as Gyrodinium pavillardi and Gymnodinium splen-
dens show an argentophilic surface network, while impregnation merely
blackens the sutures of the thecal plates in peridinioid species (6). Im-
pregnation of Gonium, Eudorina, and Volvox (Fig. 1. 2, D, E) demon-
strates silver-lines in the individual flagellates (131), while the silver-lines
of various Euglenida (106, 131) apparently correspond to the pellicular
striations visible in living material. In addition, the flagella of Pyrso-
nymphidae (105), as well as the pellicular ridges and the margin of the
undulating membrane in Trypanosoma rotatoriuyn (106), are impreg-
nated with silver.

MYONEMES AND CONTRACTILE
STALKS

Myonemes are well developed in various large ciliates which are
capable of changing form rapidly. The band-like and cross-striated
myonemes of Stentor coeruleus (44) extend from the posterior end of the
body to the adoral zone, sometimes branching to follow the rows of cilia
(Fig. 1. 13, A, B). Posteriorly, the myonemes turn inward and anteriorly
as a bundle which finally branches into fibrils that disappear in the
endoplasm. Among the flagellates, swimming of the medusa-like Lepto-
discus and Craspedotella is attributed to myonemes which bring about
rhythmic contractions of the body (177). Pellicular ridges in such large
trypanosomes as Trypanosoma rotatorium have been considered myo-
nemes, but their contractile nature is uncertain. Some of the larger
gregarines apparently possess both circular and longitudinal myonemes
(Fig. 1. 13, D, E) enclosed in individual ectoplasmic canals (194). Many
Protozoa have no myonemes but the absence of such structures does not
eliminate contractility. This fundamental property is exhibited by many
species which seem to show no appropriate differentiations at the micro-
scopic level.

Well developed myonemes (stalk-muscles, or spasmonemes) are found

26 General Morphology of the Protozoa

also in the stalks of certain ciliates. The stalk-muscle of Zoothamnium
extends spirally within a sheath continuous with the protoplasm of the
body. Between the sheath and the surface of the stalk, there is a matrix
filled with elastic fibrils which arise from a differentiated area, the
scopula, in the aboral body wall (182). The stalk-muscle extends almost
to the base of the stalk, where it is attached by a fibrillar bundle to the
basal disc. The individual stalk-muscles of the Carchesium colony are
attached to the bases of their own stalks, so that each stalk-muscle
contracts independently.

TRICHOCYSTS AND NEMATOCYSTS

Trichocysts are cortical structures reported in certain ciliates and
flagellates. Trichocyst-bearing Holotrichida are represented by several
dozen genera. In addition, trichocysts have been reported in some Het-
erotrichina (e.g., Blepharisma) and in Strombidiiun among the Oli-

# ^

Fig. 1. 14. A. Nematocyst of Polykrikos; xl450 (after Kofoid and Swezy).
B. Fusiform and spherical trichocysts of Gonyostomum semen; diagram-
matic (after Chadefaud). C, D. Trichocysts of Dileptus anser before and
after discharge; x3080 (after Hayes). E. Developing and mature trichocysts
in Parayneciiim caudatum; diagrammatic; xl280 (after Jacobson). Key: c,
cortex; ch, chromatophore; /, fibril (A), fusiform trichocyst (B); 5, spherical
trichocyst; st, stylet; t, trichocyst; td, developing trichocyst.

gotrichina (200). Trichocysts may be widely scattered over the body
(Parameciiwi), limited to the peristomial area (Dileptus), or borne on
tentacles or papillae [Actinobolina, Legendrea). Development of tricho-
cysts from macronuclear granules has been reported. However, this
phenomenon has not been confirmed (100), although developing tricho-
cysts (Fig. 1. 14, E) appear in the cytoplasm of Frontonia leucas and
Paramecium caudatum. The outgrowth of trichocysts from "trichocysto-
somes," granules produced by division of basal granules, also has been
reported (150). So-called protrichocysts of various ciliates have been in-
terpreted also as deposits of tectin, used in formation of the cyst mem-
brane or the lorica (12, 200).

General Morphology of the Protozoa 27

The trichocysts of Dileptus gigns (215) show no internal organization
and form no detectable structures upon discharge. These trichocysts
(toxicysts), which change shape during contortions of the ciliate and be-
come almost spherical under pressure of a coverslip, are believed to
contain a fluid. Comparable trichocysts are found in the tips of retrac-
tile tentacles in Actinobolina (225), and the flask-shaped trichocysts of
Conchophthirhis mytili (116) also may be similar to those of D. gigos.

Trichocysts of certain other ciliates are discharged as recognizable
structures. Such is the case in Legendrea, Frontonia, Paramecium, Pror-
odon, and Dileptus anser. In electron micrographs (101, 102), discharged
trichocysts of Paramecium show a pointed tip resembling a golf tee, and
a transversely striated shaft. The trichocyst of Dileptus anser consists of
a thread-like extension into the cytoplasm and a bulbous portion which
tapers to a subpellicular granule (Fig. 1.14, C). Upon discharge (Fig. 1.
14, D), the positions of these components are reversed, the trichocyst-
granule adhering to the pellicle (81). Discharge apparently involves turn-
ing the trichocyst inside out.

Trichocysts have been interpreted as offensive and defensive weapons
and as organelles of attachment. Under artificial stimulation, trichocysts
of Entorhipidium pilatum are often expelled from the body but they
sometimes backfire into the endoplasm (148). Therefore, the behavior of
trichocysts under artificial conditions should be interpreted cautiously. A
protective function is often suggested but has not been adequately dem-
onstrated in Paraynecium. Another suggestion for Paramecium (197) is
that the trichocyst, which hardens after extrusion so that only the tip
remains sticky, serves in anchoring the ciliates. The stimulus to natural
discharge, in which only a portion of the trichocyst is discharged, is said
to be contact with solid objects. In contrast to the trichocysts of Para-
?7iecium, those of Dileptus gigas apparently are offensive and defensive
weapons. They paralyze some organisms, induce cytolysis of others, and
cause vigorous reactions in additional Protozoa (215). Paralysis of flagel-
lates and small ciliates is produced by trichocysts of Dileptus anser, and
even large rotifers react vigorously (81). Contact of rotifers with the
tentacles of Actinobolina vorax also may be followed by paralysis (225).

Trichocysts have been reported in various flagellates (77). Two types
have been described in Gonyostominn (Fig. 1. 14, B) — spindle-shaped
trichocysts and small spherical ones, both distributed in the cortex. The
former become long filaments when discharged; the latter give rise to
short delicate filaments (21). The "trichocysts" of Chilomonas, repre-
sented by refractile bodies lining the pharyngeal groove, are discharged
as long slender threads. Another type, possibly represented by small
cortical inclusions, gives rise to short thin filaments. Filamentous struc-
tures also have been interpreted as discharged trichocysts in species of
Polykrikos, Peridinium, Diplopsalis, and Ceratium (142).

28 General Morphology of the Protozoa

Certain subcuticular inclusions of Euglenida — the cortical globules of
Euglena archaeoplastidiata, which are expelled and stained brown in
iodine solution (22), and the rod-like bodies beneath the pellicular
striations of Peranema trichophorum (23) — also have been homologized
with trichocysts of ciliates. These inclusions are demonstrable by mito-
chondrial techniques in Peranema trichophorum (74). It has been sug-
gested (88) that such subcuticular bodies of Euglenida are merely
substances accumulated for the secretion of cyst membranes and similar
layers.

Among the dinoflagellates, Polykrikos and Neynatodiniu^n contain
nematocysts resembling those of coelenterates (27, 136). The similarity
is so close that some workers have considered such nematocysts (Fig. 1.
14, A) to be foreign bodies ingested by the flagellates. However, this in-
terpretation is not supported by the regtdar occurrence of nematocysts in
certain species and their absence in others feeding on the same plankton.
The nematocysts lie free in the cytoplasm and nothing is known about
their possible discharge under natural conditions.

THE CYTOSTOME AND ASSOCIATED
STRUCTURES

The ingestion of solid food by organisms with a well developed
cortex usually occurs through a cytostome, which often opens into a
cytopharynx. The area leading to or surrounding the cytostome often
forms a specialized peristome in ciliates. Although a typical cytostome
and cytopharynx ("gullet") are to be expected only in holozoic organisms
with a well difl:erentiated body wall, interestingly similar structures occur
in certain amoebae (Fig. 1. 15, C, E, F) — Amoeba vespertilio and Hart-
manella sp. (99), and also Dientajnoeba fragilis (230), and Entamoeba
muris (227). Aside from their greater permanence, the cytostome and
cytopharynx of various flagellates represent little advance beyond the
condition seen in these amoebae. In ciliates, however, the peristomial
area may be equipped with an undulating membrane, a row of mem-
branelles, or differentiated zones of cilia.

The peristome of Paramecium multimicromicleatum (Fig. 1. 15, G)
is lined with cilia, while the pharynx is equipped with a dorsal zone of
long cilia and the penniculus, a band of closely set cilia extending spirally
from an antero-dorsal origin to the ventral pharyngeal wall. Activity of
these specialized cilia drives particles into a zone of paraoesophageal
fibrils which are continued from the wall of the cytopharynx into the
endoplasm as the postoesophageal fibrils. When enough particles are
trapped, a food vacuole develops as a bulge in the dorsal wall of the
gullet. After separation from the gullet, the vacuole is guided into the
endoplasm by the surrounding postoesophageal fibrils which exert a sort
of "peristaltic" effect (147). The peristomial area, or "oral groove," serves

General Morphology of the Protozoa 29

/o )

■^

u---

L.'^ A

//.■

; : /

'"*- D

: • 1

'■•.■■. w

Fig. 1. 15. A. Section across the peristome of Oxytriclia jallax; xl225
(after Lund). B. Section through the middle of the peristome in Euplotes
aediculatus; x445 (after Pierson). C. Gullet-like structure in Amoeba iuve-
nalis after ingestion of a flagellate; diagrammatic (after Ivanic). D. Pharyn-
geal-basket in Chilodonella labiata; diagrammatic (after MacDougall). E, F.
Gullet-like structures in Dientamoeba fragilis; xl930 (after Wenrich). G.
Gullet (peristomial area) in Paramecium midtimicronucleatiim; diagram-
matic; x670 (after Linid). H. Peristome of Stylonychia; diagrammatic; x720
(after Lund). Key: b, pharvngeal-ljasket; c, cirrus; d, dorsal cilia; f, postoe-
sophageal fibrils; g, basal granule of cilium; m, row of membranelles; p,
basal granules of penniculus; u, undulating membrane; v, food vacuole; vd,
developing food vacuole.

as a scoop which directs water toward the cytostome. The cytostome and
associated structures in Paramecium thus form an efficient mechanism for
concentrating small particles and delivering them to the food vacuole.
In Oxytricha (146) and Stylonychia (147) the left margin of the peri-
stome bears a row of adoral membranelles, while an undulating mem-
brane extends along much of the right peristomial wall (Fig. 1. 15, A,

General Morphology of the Protozoa

H). In addition, dorsal and lateral fibrils extend forward in the peri-
stomial cortex and, by their contractions, produce undulations of the
cortex near the cytostome. Both sets extend, as the postoesophageal fibrils,
past the base of the pharynx and deep into the cytoplasm, their move-
ments guiding each newly formed food vacuole, much as in Paramecium.
The small undulating membrane and the lack of cytostomal fibrils

ct-l

Fig. 1. 16. A. Contractile tube of Haptophiya michiganensis; xlOO (after
MacLennan). B. Contractile vacuole and pore in Eudiplodinium maggii;
x830 (after MacLennan). C. "Sensory" vacuole of Blepharoprosthium; dia-
grammatic (after Dogiel). D. Row of "statocysts" in Loxodes rostrum; body
cilia omitted; xl60 (after Penard). E. Single "statocyst" of L. striatus; dia-
grammatic (after Penard). F. Contractile vacuole and canals in Paramecium
miiltimicronucleatum; x720 (after King). G. Contractile vacuole and canals
in Tillina canalijera, diagrammatic (after Turner). Key: cp, pellicular cap;
ct, cortex; fe, endoplasmic fibril; ^v, fibrils at surface of vacuole; g, granules
in vacuole; s, "statocvst."

General Morphology o£ the Protozoa 31

suggest that Euplotes harpa, and perhaps other Euplotidae, cannot con-
centrate small food particles very efficiently (147), although the peri-
stomial area is partially enclosed ventrally by an extension of the right
peristomial wall (Fig. 1,15, B). In cultures, E. harpa thrives best on food
particles larger than bacteria (147).

In such ciliates as Chilodonella and Prorodon the pharynx is sur-
rounded by a conical or cylindrical "pharyngeal basket" (Fig. 1. 15, D)
which undergoes dilation during ingestion (179). The basket is com-
posed of rods which are probably protein in nature (153) and, in certain
species, may represent fused bundles of slender trichites (179). Conver-
gence and apparent fusion of the rods posteriorly may be noted, as in
Chilodofiella (153). There are circumpharyngeal trichites in Spathidium
(235) and Didiniwn (179) also, although a compact basket is lacking.
The paralysis of small ciliates after contact with the cytostomal region of
S. spathula (235) suggests that the trichites function much like tricho-
cysts of certain other ciliates.

The pharyngeal-rod apparatus ("Staborgan") of Perayieyna and similar
holozoic Euglenida (Chapter IV) includes two longitudinal rods extend-
ing posteriorly from the cytostome and a smaller curved element at the
rim of the cytostome (23). The conical "siphon" of Ryitosiphon (23) is
possibly a derivative of the rod-apparatus but its homologies are un-
certain.

VACUOLES OF PROTOZOA

Contractile vacuoles

Contractile vacuoles, characteristic of fresh-water species, are ab-
sent in most parasitic and marine Protozoa. The position, number, and
accessory structures of the contractile vacuoles vary in different Protozoa.
In such genera as Amoeba, the position of the vacuole changes with
movements of the organism. Differentiation of the cortical layer is gen-
erally accompanied by a relatively fixed position of the contractile
vacuole.

The origin of a new vacuole after discharge (systole) usually involves
the appearance of a few minute vacuoles in the area where the new con-
tractile vacuole will develop. These small vacuoles fuse into a single
larger one. Later increase in volume (diastole) of the contractile vacuole
involves various processes. In certain ciliates, the young vacuole is fed by
one or more canals (Fig. 1, 16, F, G). The newly formed vacuole in
Spirostomum ambiguutn (42), for example, fuses with a long canal which
in turn receives fluid by fusion with small vacuoles. The new vacuole in
Paramecium multimicronucleatiim (119) is fed in similar fashion by sev-
eral canals. Growth of the new vacuole in Amoeba (80), Euplotes (210),
and many other Protozoa depends, to some extent at least, upon fusion

32 General Morphology of the Protozoa

with smaller vacuoles. In fact, growth of the vacuole in spurts, instead
of a steady increase in volume, suggests that fluid enters the contractile
vacuole of Amoeba proteus almost entirely through the fusion with ac-
cessory vacuoles (161). In Eudiplodinhim, however, growth of the vacuole
in late diastole apparently is not dependent upon accessory vacuoles,
and the vacuolar membrane presumably is responsible for the segrega-
tion of fluid (161).

Systole usually involves discharge of the contents directly to the out-
side of the body, but there are exceptions. The vacuoles of Euglenida
empty into the reservoir ("gullet"); those of Epistylis and related ciliates
(55) empty into the pharynx, often termed the "vestibule" in view of its
several functions. The point of discharge is often a differentiated pore as
in Parnmeciutn (119) and the Ophryoscolecidae (154). Except during
systole, this pore is sealed by a membrane (Fig. 1. 16, B, F) apparently
derived from the wall of the preceding vacuole.

A long contractile tube (Fig. 1. 16, A), instead of a contractile vacuole,
extends throughout most of the body between the endoplasm and dorsal
ectoplasm in Haptophrya (160). This tube is an apparently permanent
structure which is divided in binary fission. Excretory canals extend to
dorsal pores, which vary in number with length of the ciliate. Systole
involves one or more waves of contraction, but the wall of the tube does
not collapse completely, and does not disappear. After systole, small
vacuoles appear in the wall of the tube and then fuse to form a continuous
lumen.

Sensory vacuoles

A supposedly sensory vacuole, located anteriorly under a pellicular
cap (Fig. 1. 16, C), occurs in parasitic ciliates belonging to the families
Biitschliidae and Paraisotrichidae (46). Fibrils on the wall of the vacuole
converge toward the pellicular cap, while the vacuolar cavity contains a
number of granules ("statoliths"). Superficially similar vacuoles (Fig. 1.
16, D, E), forming a row near the aboral surface of the body, have been
described in Loxodes. These vacuoles ("Miiller's vesicles") have been in-
terpreted both as statocysts (179) and as "excretion-vacuoles" (187).

Vacuoles in flotation

Cytoplasmic vacuoles may play an important part in flotation. In
Radiolarida (Chapter V), the foamy outer cytoplasm (calymma) is filled
with vacuoles which maintain the organisms at a particular depth in the
ocean. Under appropriate stimulation, collapse of the vacuoles and re-
traction of pseudopodia increase the specific gravity and the organisms
sink. When new vacuoles develop in the calymma, the organisms rise
again. An analogous phenomenon has been described in Arcella (10).
The appearance of gas bubbles in the peripheral cytoplasm, supposedly

General Morphology of the Protozoa 33

induced by reduction in oxygen tension of the medium, causes the organ-
ism to rise toward the surface. Perhaps the zone of vacuoles beneath the
theca of Ceratium (53) also functions in flotation.

CHROMATOPHORES, PIGMENTS, PYRENOIDS,
PHOTORECEPTORS

Chromatophores

Chromatophores, found in many phytoflagellates, vary in number,
size, color, and form in different groups. Some flagellates contain one
large cup-shaped chromatophore, or an H-type in which two large lobes
are joined by a connective (Fig. 1. 17, A, E, F, I). The chromatophores
in Peridininm umbunattim form an anastomosing network with lobes
extending into the endoplasm (Fig. 1. 17, J). Some of the Euglenidae
contain many small flattened chromatophores arranged in a peripheral
layer (Fig. 1. 17, G).

Pigment-free leucoplasts, homologous with chromatophores, have been
reported in Polytoma (219) and Polytomella (185). Since Pringsheim
(183) has pointed out that peripheral mitochondrial networks have
sometimes been interpreted as leucoplasts, the status of leucoplast-bearing
species requires further investigation.

Pyrenoids

These structures (Fig. 1. 17, B, C, J, L), which are usually asso-
ciated with chromatophores, vary from solid bodies to aggregates of
granules, around which starch or paramylum may accumulate. Each
pyrenoid in Euglena americana (Fig. 1. 17, L), for example, consists of
two plano-convex masses adherent to the chromatophore and covered by
paramylum (88). The extraplastidial "pyrenoids" of certain Crypto-
monadida are termed amphosomes by Hollande (88), who believes that
homologous structures are unknown in other flagellates. The amphosome
of Cryptomonas dangeardii (Fig. 1. 17, N) consists of two chromophilic
plates separated by chromophobic material, and is sometimes surrounded
by starch grains. The functional significance of the amphosome is un-
known.

Pyrenoids have been interpreted as reserves of protein, as structures
involved in the synthesis of polysaccharides, and even as intracellular
symbiotes. The second assumption is consistent with the frequent occur-
rence of starch or similar materials immediately surrounding the pyre-
noids. According to various reports, pyrenoids may be resorbed occasion-
ally, they may be reduced in number during formation of cysts and
zygotes, and they may arise by division or be formed de novo. Such appar-
ent variations in behavior are of little assistance in functional inter-
pretations.

34 General Morphology of the Protozoa

Fig. 1. 17. A, E, F, I. Chiomatophores in Chlamydomonas agloeformis,
C. umbouata, C. inversa and C. bicocca; diagrammatic (after Pascher).
B, C. Compound pyrenoids of Pyramidomonas montana, with and without
starch deposits; xl200 (after Geitler). D. Granular stigma of Eiiglena;
diagrammatic. G. Chromatophores in Euglena geniculata (?); xl500 (after
Hollande). H. Ocellus with granular pigment in Protopsis neapolitana;
x550 (after Kofoid and Swezy). J. Chromatophore "network" in Peri-
dinium umbonatuni; diagrammatic optical section (after Geitler). K. Pe-
ripheral chromatophores in Cnlacium vesiculosum; xl380 (after Johnson).
L. A chromatophore with pyrenoid and paramylum shell in Euglena
americana; diagrammatic (after Hollande). M. Ocellus with pigment-cup
in Erythropsis cornuta; x320 (after Kofoid and Swezy). N. Amphosorae
in Cryptomonas dangeardii; chromatophores omitted; x2700 (after Hol-
lande). Key: a, amphosome; cli, chromatophore; cp, pigment-cup; g, gran-
ular pigment; /, lens; n, nucleus; p, paramylum; py, pyrenoid; s, starch;
st, stigma.

General Morphology of the Protozoa 35

Pigments

The pigments of chromatophores vary in different groups of the
phytoflagellates, and may have some phylogenetic as well as taxonomic
significance. Chlorophyll of one variety or another presumably is always
present although the green color may be masked by other pigments to
produce shades of greenish-yellow, yellow, brown, and rarely blue. Blue
chromatophores have been reported (143) in Cyanomonas coeruleus,
Chroomonas setoniensis, Cyanomastix morgani, and Gymnodinium lim-
netician.

In addition to the pigments of chromatophores, various red, yellow,
violet, brown, blue, and green pigments are found in the cytoplasm of
certain Protozoa, many of them species without chromatophores. The
blue-green granules ("stentorin") of Stentor coeruleus, which contain
lipoproteins and resemble mitochondria, lie mainly in longitudinal ecto-
plasmic bands. These granules usually disappear after 24-48 hours of
starvation (223). The pink pigment of Blepharisma undulans, which is
also peripheral (169), is bleached after exposure to light, and is regen-
erated in darkness (69). This pigment is quite toxic to species of Para-
mecium, various other ciliates, and also to rotifers (70).

Chromatographic techniques (18, 204, 205, 206) have been used in the
identification of certain pigments. In Dinoflagellida, chlorophylls a and
c, ^-carotene ("yellow haematochrome"), and several xanthophylls (di-
noxanthin, diadinoxanthin, neodinoxanthin, peridinin) have been dis-
tinguished. Peridinin, which may be limited to dinoflagellates among the
Protozoa, is possible identical with sulcatoxanthin isolated from certain
sea anemones and probably derived from their symbiotic algae (82).
Chrysomonadida contain chlorophyll a (but apparently no chlorophyll
b), ^-carotene and the xanthophyll, lutein. Euglenida contain chloro-
phylls a and b, ^-carotene and also red haematochrome (euglenarhodon),
a xanthophyll closely related to the astacene of Crustacea. Phytomonadida
contain chlorophylls a and b and, in such species as Haematococcus plu-
vialis, a red haematochrome similar to that of Euglenida.

The functional significance of most protozoan pigments is unknown.
The absorption of energy in photosynthesis is, of course, dependent
mainly upon the chlorophylls, of which chlorophyll a is probably most
important. However, carotenoid pigments also may serve in absorbing
the predominantly blue-green light of low intensity received by marine
species in fairly deep water (50, 205). The red pigment of Euglejia rubra
may be protective in reflecting light from the red end of the spectrum.
This species thrives in shallow water reaching a temperature of 35-45°
in bright sunlight. Under such conditions the pigment forms a layer
just outside the chromatophores (107). In the laboratory, peripheral
migration of the pigment occurs at temperatures of 30-40° in either dark-

General Morphology of the Protozoa

/.'•%•■

(2--' B

/ /"'N

p---f •(■•;'

\ 0. .; /

1
I

k// 1

-gv

ill

t
if

H '^-^.J^

Fig. 1. 18. A. Bacilliform paramylum bodies in Euglena acus; x750
(after Deflandre). B. Small paramylum bodies of Distigma proteiis; x3640
(after Hollande). C. Paramylum bodies (p) of Euglena spirogyra; x850
(after Dangeard). D. Polysaccharide reserves in Cliilomonas parameciinn:
x4500 (after Hollande). E. Chromatoid bodies and glycogen "vacuole" in
Entamoeba invadens; x2350 (after Geiman and Ratcliffe). F. Skeletal plates
and glycogen granules in cross-section of Polyplastron multivesiculatum;
diagrammatic; endoplasm omitted (after MacLennan). G. Cross-section of
right dorsal skeletal plate, P. multivesiculatum ; x4720 (after MacLennan).
H. Starch grains at smface of chromatophore in Cryptomonas ovata; x2250
(after Hollande). Key: c, chromatophore; ch, chromatoid body; g, glyco-
gen blocks; gg, glycogen granule; gv, glycogen "vacuole"; m, macro-
nucleus; n, nucleus; p, pharynx; 5, starch; sk, skeletal plate.

General Morphology of the Protozoa 37

ness or light and also after irradiation with infrared or visible light. With
the effects of temperature controlled, light from the blue end of the
spectrum is more effective than that of longer wave lengths (108). The
accumulation of a similar red pigment in Haematococcus pluvialis has
been attributed to the exhaustion of nitrogenous or other foods. Massive
production of this pigment occurs also in young cultures exposed to light
in a medium containing acetate; salts of butyric and certain other acids
show no such effect (152). In acetate-free medium, intensely red "haemato-
cyst" stages are commonly developed in bright sunlight but not in dim
light (52).

Photoreceptors

A stigma is characteristic of many chlorophyll-bearing flagellates,
and occurs also in certain colorless phytoflagellates. The stigma of Eu-
glenida is typically a flattened mass of reddish granules embedded in a
matrix (Fig. 1. 17, D), whereas a granular organization is not apparent
in typical phytomonad and chrysomonad flagellates. The stigma of Volvox
and related colonial types is said to contain a concave mass of pigment
and a hyaline lens (164). The stigma is usually located near the anterior
end of the flagellate, but lies near the middle of the body in some species.
The typical position in Euglenida (Fig. 1. 17, D) is near the wall of the
reservoir.

Certain dinoflagellates (Pouchetiidae) possess an ocellus composed of
a hyaline lens and a dark mass of pigment (melanosome) partially cover-
ing the lens. In certain species the melanosome can be extended over
the surface of the lens or contracted toward the base (Fig. 1.17, M). The
melanosome may be homogeneous except for a core of red pigment at
the base of the lens, or may be merely a loose aggregate of granules as in
Protopsis neapolitana (Fig. 1. 17, H).

CYTOPLASMIC INCLUSIONS

Cytoplasmic food reserves

Foods of various kinds are frequently stored in the cytoplasm of
Protozoa and, in any one species, the amount and type of reserves may
vary with environmental conditions. Starch is the major reserve in young
cultures of Polytoma iivella; lipids predominate after 15-30 days (220).
In P. iiveUa, and probably in Protozoa generally, less food is stored in
rapidly growing than in slowly growing cultures.

Polysaccharide reserves of phytoflagellates occur as granules or as larger
bodies sometimes of characteristic shape and size. Synthesis of these car-
bohydrates is not dependent upon chlorophyll and may be expected in
both colorless and pigmented species. The supposed presence of colorless
"chromatophores" (leucoplasts) in non-pigmented phytoflagellates has
been questioned (183). Paramylum (or "paramylon") of Euglenida occurs

General Morphology of the Protozoa

mainly as refractile endoplasmic bodies (Fig. 1. 18, A-C), but may be
found also at the surface of the pyrenoid (Fig. 1. 17, L) in chlorophyll-
bearing species. In addition, glycogen-like inclusions occur in Euglena
(22) and Peranema (23). The leucosin of Chrysomonadida (Chapter IV)
is stored as bodies (Fig. 4. 1, D-F) which are often relatively large. Starch
may be deposited as fine granules or larger bodies in Cryptomonadida,
Phytomonadida, and Dinoflagellida. Starch grains are deposited on the
inner surface of the chromatophore in Cryptomonas ovata (Fig. 1.18, H),
while the reserves of Chilomonns, which are composed of ^-amylose and
amylopectin (97), are scattered refractile bodies (Fig. 1.18, D). In Phyto-
monadida and Dinoflagellida, starch occurs both as scattered granules and
as deposits around pyrenoids.

Glycogen, or a similar material, is common in groups other than the
phytoflagellates. Little is known about the chemical nature of these in-
clusions. Such reserves are distributed as fine granules in Paramecium
(186), but may be concentrated posteriorly in Stentor (222, 240). In the
Ophryoscolecidae, the skeletal plates (Fig. 1. 18, F, G) contain much of
the glycogen (155), although cytoplasmic granules may occur also (154).
Glycogen or paraglycogen is generally deposited during heavy feeding
and consumed in starvation, as traced in Stentor (222, 223). Such reserves
also may be deposited before encystment — as in Dileptus (207), Ich-
thyophthirius (156), and certain Endamoebidae — and consumed before
excystment. The paraglycogen of Ichthyophthirius is formed as small
granules within a mitochondrial sphere which disappears after the para-
glycogen mass reaches a diameter of 5-6[jl (156). A similar development
has been observed in gregarines (109), whereas glycogen is deposited in
association with the parabasal body of Cryptobia helicis (48).

Lipids are probably stored by most, if not all. Protozoa, under certain
conditions. The accumulation of fat in Stentor has been attributed to
low oxygen tensions (240), and stored lipids are characteristic of old
rather than young cultures of Polytoma uvella (220). Lipids may be
distributed through the endoplasm or else concentrated in one region
as in Anoplophrya (51). In Stentor coernleus (223), these inclusions vary
from small granules to bodies as large as the macronuclear nodes. So-called
bodies of Maupas, believed to contain at least some lipids, occur in vari-
ous Cryptomonadida as two refractile ellipsoidal inclusions (88). In Ich-
thyophthirius, the fatty acids and glycerol which reach the cytoplasm are
first segregated into globules within which neutral fat is formed (156).
A similar process has been described in Opalina (114).

Protein reserves have been described as basophilic granules, metachro-
matic granules, chromatoid bodies, albuminoid reserves, and chromidia.
Such reserves occur as scattered granules through the endoplasm, they
may be stored in peripheral globules in Opalina (115), or they may be
deposited as fairly large masses. Chromatoid bodies (Fig. 1,18, E), repre-

General Morphology of the Protozoa 39

senting the third condition, are refractile inclusions present in young
cysts in certain Endamoebidae. Little is known about the origin of protein
reserves. However, protein granules are extruded from the macronucleus
of Ichthyophthirius multifiJiis after the ciliates invade a host and begin
to feed (156). Similar achromatic bodies in the macronucleus of Blepha-
risma midulans also have been interpreted as protein reserves (239). The
chromatoid bodies of Entmnoeba histolytica on the other hand, are
formed by the coalescence of clear vacuoles which appear in the cytoplasm
(90). Dietary factors may influence the synthesis of protein reserves. Stor-
age of protein granules in Polytoma uvella, for example, is extensive in
a medium containing butyrate, but is much less noticeable with acetate
(220).

Volutin granules, known in many Protozoa, presumably should be con-
sidered nitrogenous reserves (158). Although the term has been used
rather loosely, volutin may be considered metachromatic material which
is Feulgen-negative but is stainable by a modified technique which omits
preliminary hydrolysis (158, 188). The disappearance of volutin in try-
panosomes and haemogregarines after digestion with ribonuclease in-
dicates that the granules contain ribonucleic acid (5). Such granules are
resorbed in old cultures and during induced starvation of Polytoma uvella
(220); they accumulate during active feeding of Oxymonas dimorpha and
decrease in the motile phase (32), and are apparently a reserve food in
Pelomyxa caroUnensis (233).

Crystalline inclusions are found in various species. The crystals of
Amoeba proteus apparently contain amino acids, presumably derived
from digested food (165, 166), but the composition of such inclusions in
many other Protozoa remains to be determined. As traced in Paramecium
bursaria (232), crystals accumulate during holozoic feeding and then
disappear gradually as the supply of bacteria in the culture is exhausted
and the ciliates become increasingly dependent upon their symbiotic
algae.

Chromidia

Originally, chromidia were defined as granules derived from the
nucleus. Certain "generative" chromidia were considered extranuclear
chromatin granules with a potential ability to form aggregates and de-
velop into nuclei. In other cases, extrusion of chromidia from the nucleus
was believed to be a means of restoring a normal nucleo-cytoplasmic ratio.
In more recent literature, a variety of inclusions — probably including
mitochondria, volutin granules and protein granules ("albuminoid re-
serves")— have been referred to as chromidia. Since the older chromidial
theories are no longer accepted, and the identities of the more modern
"chromidia" are so varied, it seems advisable to drop the term as a
designation for cytoplasmic inclusions of Protozoa.

40 General Morphology of the Protozoa

Mitochondria

Mitochondria (or chondriosomes), which seem to be generally
present in Protozoa, were observed as early as 1910 in Chilomonas, Cryp-
tomonas, and Noctihica (54). Subsequently, mitochondria have been
described in many species (76, 77, 158). Mitochondria are to be expected
in Protozoa during the active phase of the life-cycle, but may be absent in
the cyst, as reported for Ichthyophthirhis multifiUis, in which the mito-
chondria disappear rapidly after encystment (156, 159). Reportedly mito-
chondria-free sporozoites of Monocystis (94) may be an analogous case.
In addition, mitochrondria have not been found in active stages of a
marine amoeba, FlabeUula mira (89).

Mitochondria may occur as granules, short rods, filaments, or fila-
mentous networks. The form is more or less characteristic of a species,
although some variation may be expected. Filamentous mitochondria
(Fig. 1. 19, A), apparently less common than other types, have been
described in Chlorogonium, Chlamydomonas, and Polytoma. Such fila-
ments are often anastomosed in a superficial network. Mitochondrial nets
also have been observed in green and colorless strains of Euglena gracilis
(95). Similar networks, which occasionally break up into short rods, occur
in Glenodiyiium sociaJe and other dinoflagellates (7). These inclusions
are usually stainable vitally with Janus green B, although the reaction
may be less intense than that of metazoan mitochondria. Good results
have also been reported with Janus red (91). Special fixatives, such as
the chromate-osmic mixtures, are advisable for good permanent prepara-
tions.

The cytoplasmic distribution of mitochondria varies with the species.
Random cytoplasmic distribution (Fig. 1. 19, B) is common, but aggrega-
tion around food vacuoles is sometimes noted (166). The mitochondria
of Chilomonas (Fig. 1. 19, D) and Cryptomonas, as well as those of Phy-
tomonadida and Euglenida, are believed by Hollande (88) to be entirely
peripheral. Certain endoplasmic inclusions of these flagellates, previously
interpreted as mitochondria after demonstration with mitochondrial tech-
niques (76, 77), are said to be cytoplasmic vacuoles. Peripheral mitochon-
dria of ciliates may be oriented in rows, as in Nyctotherus cordiformis
(93). Association of peripheral mitochondria with the basal granules has
been described in Colpidiiim colpoda and other ciliates (26), but this is
not the case in Tillina canalifera (213). Mitochondria of Bursaria truri-
catella are mostly peripheral during conjugation but are scattered through
the cytoplasm in other phases of the life-cycle (181).

Various functions have been assigned to the mitochondria. Morpho-
genetic interpretations include a mitochondrial origin of pyrenoids (20)
and the derivation of Golgi material, parabasal bodies, blepharoplasts,
and the stigma from mitochondria (1). Supposed physiological activities

General Morphology of the Protozoa 41

.r--n

/.O.a

(va:55w^

> ' '.-..•.%

/^\.

/:.\

E F G ~-^^

"-■. \-y/

t..^ y

Fig, 1. 19. A. Mitochondrial network in Polytuiiia uvellu; \2700 (after
HoUande). B. Mitochondria and one nucleus in Protoopalina hylarnm; dia-
grammatic (after Richardson and Horning). C. Granules stained with neu-
tral red in Chlamydomouas variabilis: \1170 (after Dangeard). D. Nfitochon-
dria in Chilomonas Paramecium; x270O (after HoUande). E. Neiitral-red
granules in Paramecium caudatum; x3I0 (after Dunihue). F. Neutral-
red granules in EugJena polymorpha; x635 (after Dangeard). G. Osmio-
philic inclusions in associated "gametocytes" of Gregarina cuneata; x635
(after Jo\et-Lavergne). H. Osniiophilic inclusions in P. caudatum; x245
(after Dunihue). I. Osniiophilic inclusions in Protoopalina hylarurn; dia-
grammatic (after Richardson and Horning). Key: c, chromatophore; ^v, food
vacuole; n, nucleus; v, developing food vacuole.

are even more varied: association with the deposition of lipids in grega-
rines (112); a causative role in amoeboid movement (19); mitochondrial
origin of digestive enzymes (92); transportation of waste products to con-
tractile vacuoles (165); transportation of enzymes to food vacuoles and
of digested materials away from the vacuoles (166); and association with
the deposition of paraglycogen (156). A belief that protozoan mitochon-
dria are involved in oxidations is in accord with the demonstration that
mitochondria contain most of the succinic dehydrogenase in liver cells
(87). Joyet-Lavergne (113) reported the capacity of mitochondria in gre-

42 General Morphology of the Protozoa

garines to oxidize leuco-derivatives of various dyes, and suggested that
such oxidations are effected partly with the aid of glutathione and vita-
min A, previously detected in mitochondria (35, 111). The localization
of cytochrome oxidase in mitochondria also has been determined for
Stentor coeruleus (224).

Vacuome

The term, vacuome, was introduced as a collective designation for
the vacuoles in plant cells (36). According to later views (72), the vacuome
is distinct from the mitochondria and shows several characteristic proper-
ties. It may be stained vitally with dilute solutions of neutral red and
certain other dyes which do not stain mitochondria. Furthermore, the
vacuome is not reliably demonstrated by mitochondrial techniques, al-
though often impregnated by Golgi methods.

Cytoplasmic inclusions of Protozoa were probably first referred to as
a vacuome by Dangeard (37), although neutral-red-stainable granules
had been described much earlier. The vacuome, in microorganisms gen-
erally, consists of small globules or granules rather than obvious vacuoles
(39). The reverse is true in higher plants. The vacuome of Protozoa in-
cludes small inclusions (Fig. 1. 19, C, E, F) which are distinguishable from
mitochondria in vital staining with mixtures of neutral red and Janus
green B. In certain species, it is evident that the elements of the vacuome
are normal inclusions of the living organism. The available data (76, 77,
158) suggest that a vacuome is generally present in Protozoa, although
apparently lacking in Conchophthirius mytili (116) and disappearing
during encystment of Ichthyophthirius multifiliis (156). Elements of the
vacuome are scattered through the endoplasm in many species. In certain
gregarines (110), however, the distribution varies in different stages of the
life-cycle. Adhesion of neutral-red granules to newly formed food vacuoles
(Fig. 1. 19, E) also occurs in certain ciliates.

The ability to segregate neutral red apparently is not limited to one
type of inclusions. Dangeard (38) stained not only the usual vacuome but
also the cortical "mucous granules" (sometimes called trichocysts) in cer-
tain Euglenida. Bush (16) also found two types of neutral-red granules in
Haptophrya michiganensis. Food vacuoles of Protozoa also are stainable
with neutral red but they are usually not considered a part of the vac-
uome, in view of their different origin and behavior.

Guilliermond (72) has pointed out that the vacuome of plants func-
tions in segregation and storage of metabolic products, and should be
considered a part of the deutoplasm, or paraplasm, rather than living
protoplasm. The vacuome may have comparable functions in Protozoa.
As shown by micro-incineration, the vacuome of Paramecium caudatum
segregates appreciable quantities of minerals (162), and the number of
neutral-red granules decreases in this species during starvation (49). The

General Morphology of the Protozoa 43

vacuome of Opalina is said to serve for the storage of proteins (114). In
addition, the vacuome contains volutin in Chilomonas Paramecium (59),
Peranema trichophorum (23), Polytoma uvella (219), and species of
Euglena (22, 178).

The neutral-red granules which collect on the food-vacuole in certain
ciliates (Fig. 1. 19, E) have been called digestive granules. Prowazek (184)
suggested that they enter the food-vacuoles of Paramecium aurelia and
participate in digestion. Similar conclusions have been reported for P.
caudatum (192), Vorticella (218), and Tetrahymena pyriformis (221).
Both Koehring (135) and Dunihue (49), while confirming aggregation
at the surface, have denied that the neutral-red granules penetrate the
food-vacuole in Paramecium. Such granules apparently get into food-
vacuoles of Ichthyophthirins without penetrating a membrane. The gran-
ules collect on a freshly formed food-vacuole, a new membrane is de-
veloped around the mass, and the original vacuolar membrane then
disappears (156). Although no such relationship has been detected in
certain other ciliates (75, 78), the behavior of these inclusions in Parame-
cium and Ichthyophthirins may justify their designation as digestive
granules.

Osmiophilic inclusions and organelles

A number of osmiophilic structures and inclusions have been in-
terpreted as protozoan Golgi material. The nature of such material is
undoubtedly varied, and complete agreement has not been reached in
regard to the identity of protozoan Golgi apparatus.- Even a single species
has sometimes been credited with two or more kinds of Golgi material.
This situation is not surprising because the Golgi techniques are not
absolutely specific. Furthermore, selection of the appropriate inclusions
is handicapped by the lack of a precise concept of protozoan "Golgi
material" and specific criteria for identifying such material.^

Protozoan Golgi material apparently was first described as osmiophilic
rings and crescents in Monocystis ascidiae (84). Comparable inclusions
(Fig. 1. 19, G-I) have been reported subsequently in many species of
Mastigophora, Sarcodina, Sporozoa, and Ciliatea. The distribution and
relative number of such "Golgi bodies" apparently vary within a species.
Golgi material may even disappear in the cyst and arise de novo after
excystment, as in Ichthyophthirins (157) and Protoopalina (189). Young

-This subject has been discussed in several reviews (76, 77, 83, 121, 158, 202, 208).

^This situation in protozoan cytology merely reflects the unstable position of "Golgi
material" in metazoan cytology. Some workers maintain that ". . . the Golgi apparatus
is a gross artifact" (176). According to another view, "a tissue lacking the full com-
plement of Golgi substance would be unable to function normally" (237). Likewise,
the statement that "efforts to demonstrate a Golgi apparatus in living, or fresh, somatic
cells have been unsuccessful" (175), may be contrasted with the conclusion that "the
Golgi apparatus can be seen in most, and perhaps all, living animal cells" (237).

44 General Morphology of the Protozoa

stages of Monocystis agilis contain comparatively few Golgi bodies; the
older stages show more numerous inclusions (85). Changes in cytoplasmic
distribution also occur at different stages in the life-cycles of gregarines
(109).

7 he parabasal apparatus of flagellates has been homologized with meta-
zoan Golgi material (48). The stigma of Euglena also has been considered
Golgi apparatus (71) on the basis of its supposed homology with the
parabasal body of certain other flagellates. Mangenot (163) has objected
that the stigma is more probably a modified plastid and that impregnation
has no significance beyond the fact that carotenoid pigments will reduce
osmium tetroxide. The endoplasmic spherules of Opalma also have been
considered ecjuivalent to the parabasal apparatus of flagellates and hence
to represent Golgi bodies (122). However, the endoplasmic spherules are
distinct from the Golgi material described in Protoopalina (189). Even
the recognition of authentic parabasal bodies as Golgi material has been
opposed on several grounds (123, 208).

Inclusions superficially resembling Golgi networks have been described.
A simple "net" was produced in Plasmodium praecox by the fusion of
osmiophilic globules (35). A more complicated net, supposedly arising
from the food vacuole, has been reported in Entamoeba gingivalis (19),
while the Golgi apparatus of Peranema trie hop horiitn (14) has been pic-
tured as fibrils resembling the silver-line systems of certain euglenoid
flagellates.

The membrane of the contractile vacuole, which is osmiophilic in
Chilomonas Paramecium and certain ciliates (51, 170, 171), also has been
considered Golgi apparatus. Gatenby and Singh (58) have extended this
concept to the wall of the reservoir (gullet) in Copromonns suhtilis
(Euglenida). If the wall of the contractile vacuole is to be homologized
with the Golgi apparatus, it probably should be osmiophilic in Protozoa
generally. Such is not the case, since the contractile vacuole of various
ciliates and flagellates is not osmiophilic (15, 75, 84, 168).

Another suggestion (35, 75, 110) is that neutral-red granules may be
recognized as Golgi material because elements of the vacuome are im-
pregnated by Golgi techniques in a number of species. Sound objections
to this generalization have arisen. Attempts to impregnate the neutral-
red granules of several species by the usual Golgi methods have failed
(16, 40, 124, 139, 154, 157, 213). Furthermore, the osmiophilic bodies of
certain gregarines move toward the centripetal pole in the ultracentrifuge,
whereas the neutral-red granules are not noticeably displaced (40). Also,
the neutral-red granules oi Paramecium (120) and Ichthyophthirius (159)
remain stratified with the food vacuoles, although other cytoplasmic in-
clusions are displaced. The significance of the results obtained with the
ultracentrifuge is uncertain. In the case of metazoan Golgi bodies, the

General Morphology of the Protozoa 45

centrifuge frequently separates chromophilic and chromophobic sub-
stances (190), sends the chromophilic elements to either the centrifugal
pole (191) or to the centripetal pole (3), sometimes stratifies the Golgi
bodies in different zones (237), and sometimes separates the "vacuome"
and the Golgi bodies (237).

NUCLEI OF PROTOZOA

Under the general term, nucleus, are included the micronucleus
and the macronucleus of ciliates and the vesicular nuclei of other Pro-

5"><

.4.2: • Vi

Fig. 1. 20. Nuclei. A. Heteronema acus; x4290 (after Loefer). B. Mul-
tiple endosomes in H. acus; chromatin omitted; x3740 (after Loefer). C.
Haematococcus pluvialis; x3280 (after Elliott). D. lodamoeba biUschlii;
x4000 (after Wenrich). E. Chilomastix magna; x9360 (after Kirby and
Honigberg). F. Entamoeba histolytica; x3900 (after Wenrich). G. Zelleriella
elliptica; x2340 (after Chen). H. Pelomyxa carolinensis; x2070 (after
Kudo). Key: c, chromatin; e, endosome; g, peripheral "chromatin" gran-
ules; n, nucleolus.

tozoa. On the basis of nuclear equipment, two types of Protozoa may thus
be recognized. In one group, the nuclei in binucleate and multinucleate
species are of the same kind, so far as structure can be determined and

46 General Morphology of the Protozoa

functions inferred. In Ciliophora, with the apparent exception of the
Protociliatia,^ nuclei are differentiated into micronuclei and macronuclei
which differ in size, in structure, and in behavior during fission and
conjugation.

Vesicular nuclei

The vesicular nuclei of Mastigophora, Sarcodina, and Sporozoa
vary so much in structure that morphological classifications are neces-
sarily arbitrary. However, it is possible to recognize two general types —
those with an endosome and those without. In the endosome-type (Fig. 1.
20, A-D, F) the chromatin lies between the nuclear membrane and a more
or less central body, the endosome. The endosome apparently does not
contribute directly, at least in a morphological sense, to the formation of
chromosomes. A negative Feulgen reaction, indicating the absence of
desoxyribonucleic acid, has been reported for the endosome in Euglenida,
Phytomonadida and trypanosomes (104) and in Entamoeba coli, E. his-
tolytica, Endolimax nana, and lodamoeba hiltschlii (228). In encysted
Giardia Ia?nblia, however, the endosome is intensely Feulgen-positive
(144). The endosome of Entamoeba miiris also gives a positive reaction
(226). The nucleus in Entamoeba (Fig. 1. 20, F) contains a small endo-
some and relatively little chromatin; that of Endolimax and lodamoeba
(Fig. 1. 20, D), a large endosome and a small amount of chromatin. The
well defined peripheral granules, adherent to the nuclear membrane and
commonly considered chromatin granules, are Feulgen-negative in Enta-
moeba muris (226), E. coli, and E. histolytica (228). The discovery that the
chromosomes develop from a zone of minute Feulgen-positive "granvdes"
around the endosome of these amoebae emphasizes the need for critical
study of the smaller protozoan nuclei. The nucleus of Euglenida (Fig.
1. 20, A, B) contains abundant Feulgen-positive chromatin and a rather
large endosome which is sometimes fragmented. The endosome disap-
pears early in mitosis in the Endamoebidae and phytomonad flagellates,
but it persists and undergoes division in Euglenida and such dinoflagel-
lates as Oxyrrhis marina (73).

Nuclei without endosomes (Fig. 1. 20, E, G, H) may contain several
nucleoli which often disappear in mitosis, although they persist in Zelleri-
ella (31). The chromatin is usually distributed throughout the nucleus
and its appearance may suggest some sort of a nuclear framework or
"network." Such nuclei are characteristic of many Heliozoida, Radiola-
rida, Hypermastigida, Dinoflagellida, opalinid ciliates, and Sporozoa.

^Although Konsuloff (140) has maintained that the Feulgen-negative endoplasmic
spherules of Opalinidae are macronuclei, this interpretation has not been accepted.
Furthermore. Metcalf's "macrochromosomes," supposedly homologous with the macro-
nuclear chromatin of other ciliates, are merely Feulgen-negative nucleoli (31).

General Morphology of the Protozoa 47

Nuclear dimorphism

The Ciliophora are unique in that all species, except the sup-
posedly primitive opalinids, have both micronuclei and macronuclei —
unless Stephanopogon mesnili (149) is another valid exception. In S.
mesnili, all of the nuclei are similar in size and structure, and their
division closely resembles that of the micronucleus in typical ciliates.
Perhaps this case is analogous to that of Dileptiis (81, 216), discussed
below. In the typical ciliates, more than one micronucleus or macronu-
cleus may be characteristic of a species and the number of each type some-
times varies independently. Stentor coeriileus, for example, may show
10-42 micronuclei distributed irregularly along the 7-23 links of the macro-
nuclear chain (201). Both types of nuclei have a common origin from
the synkaryon formed in conjugation. The division-products of the syn-
karyon, presumably identical cytologically and genetically, undergo diver-
gent metamorphosis in conjugation. A developing micronucleus under-
goes reduction in size and often a decrease in staining capacity. The de-
veloping macronucleus increases in size, undergoes changes in internal
structure and may show extensive changes in form before reorganization
is completed. The nature of the changes involved in the development of
macronuclei is still unknown. On the basis of genetic data (203), it has
been suggested that the macronucleus is a compound nucleus composed
of many units, each with its own diploid set of genes. At each fission, the
macronucleus divides amitotically, contributing approximately half of
its units to each daughter ciliate. Subsequently, the normal number of
units is restored by mitotic processes within each reorganizing macronu-
cleus. This theory is interesting, but adequate morphological grounds for
such an interpretation are lacking. The ciliate micronucleus, in contrast
to the macronucleus, undergoes mitosis during reproduction of the or-
ganism.

Macronuclei vary considerably in form, size and number. The simplest
are spherical to ovoid bodies (Fig. 1. 21, C) containing many densely
staining granules perhaps embedded in an achromatic framework (159).
The macronucleus of Paramecium is stretched in the ultracentrifuge and
the contents are stratified in two zones, the chromatin granules appar-
ently being denser than the achromatic substance (120). The Feulgen
technicjue indicates that different types of granules are stainable with
hematoxylin. Uniformly small granules, scattered through the macro-
nucleus of certain ciliates, are Feulgen-positive; certain larger granules
give a negative reaction (57, 104, 156). The staining capacity of these
Feulgen-positive granules in Stentor coeruleiis is not affected by ribonu-
clease (223).

Macronuclei are not always compact. The macronucleus of Euplotes

48 General Morphology o£ the Protozoa

(Fig. 1, 21, B) and that of Vorticella wind through much of the endoplasm,
while that of Conchophthirius caryoclada (117) is irregularly lobate (Fig.
1. 21, A). The two slender macronuclei of Spathidiiim spathula (235) ex-
tend nearly the length of the ciliate and may sometimes be joined pos-
teriorly. In various species of Spirostomiim and Stentor (Fig. 1. 21, F)

Fig. 1. 21. Macronuclei. A. Conchophthir'nis caryoclada; diagrammatic;
x440 (after Kidder). B. Euplotes; diagrammatic; x460 (after Turner). C.
Ichthyophthirius multifiliis; x630 (after MacLennan). D, E. Nyctotherus
gyoeryanus, longitudinal and transverse sections; karyophore attached to
macronucleus; diagrammatic (after Grasse). F. Stentor type; diagrammatic.
Key: k, karyophore; m, macronucleus.

there is a chain of macronuclear nodes joined by filaments. In certain
ciliates, the macronucleus is suspended from the cortex by a fibrillar
"karyophore" (Fig. 1. 21, D, E).

The significance of nuclear dimorphism remains uncertain. It is usually
assumed that the macronucleus is involved in metabolic activities. In this
connection, the extensive resorption of the macronucleus during starva-
tion of Stentor coeruleiis (223) is of interest. The micronucleus is sup-

General Morphology of the Protozoa 49

posedly concerned mainly with reproduction and sexual phenomena and
therefore is primarily of genetic interest. The occurrence of apparently
amicronucleate strains in several species — Oxytricha hymenostoma (41),
Oxytricha fallax, Urostyla grandis (234), and Tillifia magna (4), among
others — suggests that the micronucleus is not actually essential to giowth
and fission. Observations on the regeneration of fragments (2) indicate
that the macronucleus is essential for complete regeneration of ciliates.
The importance of the micronucleus apparently varies with the species.
Some species fail to grow, or even to regenerate, without a micronucleus,
while macronucleate fragments containing no micronuclei have given rise
to amicronucleate races in other species — Stentor coeruleiis (201) and
Oxytricha fallax (9), for example.

Dispersed nuclei

So-called dispersed nuclei have been described in certain Protozoa,
although the older accounts have not been confirmed in more recent
investigations and such interpretations were undoubtedly based upon in-
adequate cytological techniques. However, the ciliate Dileptiis has been
cited for many years as an example in which chromatin granules, scat-
tered through the endoplasm, are the substitute for a nucleus. The con-
dition in Dileptus gigas has been analyzed by Visscher (216). During post-
conjugant reorganization the synkaryon divides into two nuclei, one of
which produces 32-64 micronuclei, and the other a comparable number
of macronuclei. The latter eventually divide further into the many scat-
tered inacronuclear derivatives characteristic of the normal ciliate. The
nuclear apparatus of Dileptus anser (81) may include as many as 200 small
macronuclei measuring 2-3[x and containing fine Feulgen-positive gran-
viles. A few of the macronuclei can usually be found in division at almost
any time, but they all seem to divide almost simultaneously just before
binary fission.

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General Morphology of the Protozoa 51

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52 General Morphology of the Protozoa

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Reproduction and Life-Cycles

Methods of reproduction
Binary fission
Budding and schizogony

Nuclear division

Eumitosis and paramitosis
The micronucleus of ciliates
The achromatic figure
The macronucleus

Life-cycles

General features
Cysts

Encystment
Excystment

Sexual phenomena

Varieties of sexual phenomena
Meiosis in relation to the life-cycle
Syngamy
Pedogamy
Autogamy
Conjugation

Factors inducing conjugation
Mating types in ciliates
Nuclear phenomena of uncertain sig-
nificance

The physiological life-cycle

Literature cited

,N iMANY Protozoa, reproduction occurs at frequent intervals,
with relatively short periods of growth intervening under favorable con-
ditions. In other cases, growth may extend over a period of several to
many days, so that reproduction occurs at comparatively long intervals.
Depending upon the species, reproduction may or may not be preceded
regularly by sexual phenomena. Of the species which do show sexual
activity, some normally undergo syngamy as a prelude to a reproductive
phase while others show sporadic sexual activity.

METHODS OF REPRODUCTION

The less complex Protozoa reproduce either by binary fission or
by simple budding. In either case, the nucleus undergoes mitosis; or
mitosis of the micronucleus and "amitosis" of the macronucleus occur in
Ciliophora. Cytoplasmic division is approximately equal in fission, un-
equal in budding.

Although reproduction in uninucleate species is comparable in some
respects to cell division in higher organisms, the structiual specialization
of many Protozoa introduces complications. The new organisms must be
equipped with various organelles, the nature of which varies with the

Reproduction and Life-Cycles 55

species. Parental organelles such as flagella are often inherited equally or
unequally by the daughter organisms which later produce enough new
structures to complete their equipment. Even the paraglycogen reserves of
Stentor coeruleus, normally stored posteriorly, are shifted to the middle

c e?" .:•■ '.•'"•I

W^.'

\:ji ■<<^^'\

•%#

Fig. 2. 1. A. Multinucleate stage (schizont) of Ovivora thalassemae;
superficial section; xlOOO (after Mackinnon and Ray). B. Schizogony in
O. thalassemae; xlOOO (after Mackinnon and Ray). C, D. Plasmotomy in
Pelomyxa carolinensis; division into two and three individuals; x40 (after
Kudo). E, F. Coronympha octonaria; xl650 (after Kirby); vegetative stage
showing nuclei and flagellar groups (E); nuclear groups at end of telo-
phase, just before plasmotomy (F),

Reproduction and Life-Cycles

of the body and then shared between the daughter organisms in transverse
fission (238). Blepharoplasts, basal granules, kinetoplasts, and sometimes
chromatophores and pyrenoids, are self-reproducing. Their duplication
during fission fills the needs of the daughter organisms. Other structures,
including the cirri of certain ciliates and the parabasal apparatus of cer-
tain flagellates, undergo resorption so that each daughter organism must
develop a set of its own — in the case of cirri, apparently by outgrowth
from inherited basal granules. The resorption of parental structures is
sometimes extensive. Reproduction may thus involve dedifferentiation of
the old body as well as the differentiation of new structures in the de-
veloping daughter organisms. The beginning of differentiation, in two
new centers of organization within the parental body, possibly supplies
the stimulus for subsequent dedifferentiation.

Reproduction of multinucleate Protozoa, or of multinucleate stages in
the life-cycle, may involve either budding or fission. In many Sporozoa a
young uninucleate stage grows, with repeated mitoses, into a multinu-
cleate Plasmodium (Fig. 2. 1, A) which then reproduces by schizogony.
Essentially, schizogony is multiple budding in which separation of uni-
nucleate buds from a residual mass of protoplasm is completed within a
short time (Fig. 2. 1, B). Certain multinucleate Protozoa normally divide
into several organisms, each of which receives some of the parental nuclei.
This process, not necessarily synchronized with nuclear division, is known
as plasmotomy. Both schizogony and plasmotomy have been described
in Coelosporidmrn (119), while plasmotomy is characteristic of Pelomyxa
(158). In the latter (Fig. 2. 1, C, D), plasmotomy produces 2-6 smaller
organisms, among which the parental nuclei are distributed at random.
Less variation is characteristic of Coronyinpha octonnria (151), in which
all eight nuclei usually undergo mitosis and the daughter nuclei separate
in groups of eight before plasmotomy occurs (Fig. 2. 1, E, F).

Binary fission

In Mastigophora, fission may occur in the active stage, within
a cyst, or in non-flagellated palmella stages (Fig. 2. 2, K). The plane
of fission is most frequently longitudinal and the division-furrow usually
appears first at the anterior end (Fig. 2. 2, I). Among the dinoflagellates,
however, fission is often oblique and may be almost transverse in late
stages (Fig. 2. 2, A-H). Spirotrichonympha bispira divides transversely,
although related species undergo longitudinal fission (61). Mitosis in
Trichomonadida is commonly followed by migration of the karyomasti-
gonts to opposite sides of the body and fission is then completed by cyto-
plasmic constriction (Fig. 2. 2, J).

Cytoplasmic structures^ may undergo division, resorption followed by
origin de novo, or partial resorption followed by growth and differentia-

^ The literature on several groups of flagellates has been reviewed by Kirby (152).

Reproduction and Life-Cycles 57

tion. Duplication of blepharoplasts, apparently by division, is character-
istic of fission in the flagellate stage. The behavior of blepharoplasts in
non-flagellated stages of Phytomastigophorea is mostly unknown, al-
though they persist as division-centers in Eudorina illinoisiensis (117).
The fate of other cytoplasmic structures in fission seems to be variable.

Fig. 2. 2. A-G. Fission and regeneration of missing portions of tfie body
and theca in Ceratium hiruncUnella; diagrammatic (after Entz). H. Late fis-
sion in Oxyrrhis mariym; nuclei and fiagella indicated diagrammatical!) :
xHOO (after Hall). I. Heteronema acus; division of body starting at anterior
end; endosomes shown, chromosomes omitted; xl395 (after Loefer). J. Late
fission in Tritrichomonas augusta; xl305 (after Kofoid and Swezy). K. Fission
in palmella stage of Haematococcus pluvialis; xl815 (after Elliott). L. Early
fission in a lophomonad flagellate; nucleus divided and new sets of organelles
developing; old organelles degenerating; diagrammatic; xl25 (after Kirby).

58 Reproduction and Life-Cycles

The stigma divides in Chlamydomonas nasuta (140), whereas the old
stigma passes to one daughter flagellate in Platydorma caudata (228).
Division of the chroma tophores has been reported in certain Euglenida
(115). Division of pyrenoids has been described in Eudorina illinoisiensis
(117); resorption of the old pyrenoid and differentiation of new ones occur
in Chlamydomonas nasuta (140).

Flagella probably do not split in fission and the few reports of such
a process are based upon inadequate evidence. Retention of the old
flagella has been described most frequently. In biflagellate and multi-
flagellate species, each daughter organism may receive one or more of the
original flagella and develop the necessary new ones, as in Heteronema
(163) and Trichonympha (152). However, flagellar resorption occurs in
Monas (210) and in Phytomonadida which divide within a parental theca.
The old flagella and associated structures also degenerate in Lophomonas
and related genera (Fig. 2. 2, L). The axostyle of trichomonad flagellates,
the pharyngeal-rod apparatus of Heteronema (163), the siphon of Ento-
siphon (115), and the cresta of devescovinid flagellates (152) undergo
resorption, whereas the costa of trichomonads apparently is retained by
one of the daughter flagellates. The kinetoplast of Trypanosomidae di-
vides but parabasal bodies of other flagellates usually do not. One of
the exceptions is Cliilomonas Paramecium in which each daughter re-
ceives part of the old parabasal apparatus (115). Parabasal bodies are
sometimes retained intact, as in Barbulanympha laurabuda (66); or partial
or complete resorption may occur. Although complete resorption of the
parabasal body sometimes occurs in Trichoynonas termopsldis and various
devescovinid flagellates, a portion often remains attached to its blepharo-
plast. In these cases, the parabasal of one daughter is regenerated from the
persisting fragment while that of the other is differentiated de novo (152).

The rigid theca of Ceratiiim (Fig. 2. 2, A-G) and related dinoflagellates
is divided in fission and the missing portions are regenerated. On the
other hand, such testate flagellates as Trachelomonas volvocina usually
undergo fission within the test, one daughter emerging to produce a new
test (99).

The simpler Sarcodina often show little of cytological interest aside
from division of the nucleus. However, the cytoplasmic changes in
Amoeba proteus (39, 162) indicate that the physical aspects of fission are
not particularly simple (Fig. 2. 3, A-C). The presence of a shell compli-
cates reproduction of many Sarcodina. In primitive genera {Cochlio-
podium, Pseudodiffliigia), the simple test is divided in fission. Euglypha
alveolata secretes reserve shell-plates and stores them (Fig. 2. 3, D) until
the next fission, when they are passed to one of the daughter organisms.
The other retains the old test. In typical Foraminiferida, schizogony has
replaced binary fission.

Fission in ciliates is typically transverse (Fig. 2. 4, H) and, in at least

Reproduction and Life-Cycles 59

yry:^^%

Fig. 2. 3. A-C. Surface changes during fission in Amoeba protcus; earlv
division (A); stage with nucleus in anaphase (B); shortly before constriction
of the body (C): diagrammatic fafter Chalkley and Daniel). D. Reserve shell-
plates stored in Euglypha; x8I0 (after Hall and Loefer). Key: /, ingested
food; 71, nucleus; s, reserve shell-plates.

certain species, there seems to be a definite division-plane which is not
displaced by amputations just before fission (256). However, the plane
of fission in Peritrichida passes from the oral to the aboral end and is
morphologically longitudinal (Fig. 2. 4, A-C). The plane of fission in
Opalinidae also is oblique or almost longitudinal. CyatJwdiniian piri-
jorme (Fig. 2. 4, D-F) is unusual, in that the plane of fission passes
through the originally longitudinal axis of the body but separates the
posterior ends of the daughter ciliates in late fission (164).

Reorganization in ciliates is often striking, and may involve macronu-
clei as well as cytoplasmic structures. The old cirri are resorbed in Urojiy-
chia (229), dedifferentiation of the peristomial area occurs in Bursaria
(212), and resorption of the peristomial membranelles in Fabrea (79).
In Chilodonella imcinatus (165), the old pharyngeal basket, cytostome,
and many body cilia are resorbed. On the other hand, Euplotes (107),

Reproduction and Life-Cycles

Colpidium, Glaucoma (49), and Stentor (238) retain the peristomial or-
ganelles. Division of the parental cytostome and peristome occurs in
CyclocJiaeta astropectinis (51) and possibly in other peritrichs.

The infraciliature shows genetic continuity through multiplication of
basal granules, as traced in Chilodonella (49), Foettingeriidae (48), Opa-
lina (42), and Ichthyophthirius (71), among others. In Tetrahymena and
similar ciliates (Fig. 2. 4, G, H), the development of a new mouth for the
posterior daughter involves the multiplication of basal granules at a
particular level in the stomatogenous row. These basal granules later give
rise to membranelles of the new peristomial area. The continuity of basal

Fig. 2. 4. A. Late fission in Opisthouecta heriueguyi, x410 (after Kofoid
and Rosenberg). B, C. Fission in Scyphidia ameiuri; ciliation not shown;
diagrammatic (after Thompson, Kirkegaard and Jahn). D-F. Fission in Cya-
thodiniuyn piriforme (after Lucas); two new sets of cilia move into the trans-
verse axis (D, E), and posterior ends of daughter organisms are separated in
fission (F); D, E, xl220; F, xll60. G, H. Fission in a hypothetical ciliate
similar to Tetrahymena; basal granules (indicated diagrammatically) multiply
in a particular region of the stomatogenous row (G) and liecome organized
into new adoral membranes (H).

Reproduction and Life-Cycles

granules is especially striking in Podophrya fixa, which shows the usual
ciliated larva and non-ciliated adult of the Suctorea. Basal granules per-
sist in the adult, and during reproduction, those in the cortex of the
bud multiply and form rows from which the cilia of the larva arise (50a).
All the cilia, and apparently their basal granules also, are resorbed in
Cyathodinium (164). New infraciliatures appear as endoplasmic units
which migrate to opposite surfaces of the body, where cilia then arise from
the new basal granules (Fig. 2. 4, D, E). This process resembles the
formation of new mastigonts in Lophomonas.

Budding and schizogony

In simple budding nuclear division is accompanied by unequal
division of the cytoplasm. Budding in ciliates is typically external, while
both internal and external budding occur in Suctorea. In internal bud-
ding of Tokophrya lemnarum (Fig. 2. 5), a slit-like cavity appears in the
endoplasm during division of the micronucleus, and is gradually extended
to cut out a spheroidal mass of cytoplasm following division of the macro-

Fig. 2. 5. Internal budding in Tokophrya lemnarum; tentacles not shown
(after Noble). A. Cytoplasmic cleft developing; macronucleus dividing and
micronuclear division completed; xl050. B. Completely separated bud en-
closed in pouch; ciliary bands developing; x660. C. Expulsion of bud from
brood pouch; x660. D. Ciliated larval stage; x715.

62 Reproduction and Life-Cycles

nucleus. After differentiation of cilia, the larva begins to rotate within
the brood pouch. Increasingly vigorous contractions of the parent finally
expel the larva (196). The development of sporoblasts in various Cnido-
sporidea (Chapter VI) also may be considered a form of internal budding.

In certain other Protozoa, budding may follow a series of nuclear di-
visions. Tritrichomonas aiigiista, although usually reproducing by fission,
sometimes develops into a somatella which undergoes budding (Fig. 2.
14, F, G). A similar process in Colacium vesiciilosum (Fig. 2. 14, A, B)
involves a multinucleate stage without flagella or reservoirs. These struc-
tures appear in each bud before it is separated from the parental somatella
(134).

Schizogony, involving the production of several to many buds more
or less simultaneously, is characteristic of certain Protozoa. This process
is especially efficient in many Sporozoa in which the plasmodium (Fig. 2.
1, A) often contains many nuclei before schizogony (Fig. 2. 1, B). A
schizont of Eimeria bovis, for example, may produce as many as 170,000
merozoites (108).

NUCLEAR DIVISION

Although mitosis has been reported in most species which have
been studied carefully, the small size of many nuclei has made it difficult
to interpret the structure of chromosomes in early mitosis and in the
interphase. The interphase chromatin of Cryptomonadida (115) and
ZellerieUa elUptica (Fig. 1. 19, G) has been described as fine granules dis-
persed on a network; that of Pelomyxa carolinensis (Fig. 2. 7, A), as
Feulgen-positive granules and short filaments. The Feulgen-positive inter-
phase chromatin of Euglenida, according to different reports (115), ranges
from periendosomal granules to a continuous spireme which in optical
section simulates separate gianules. Actually, it has been impossible to
find stages suggesting an achromatic network containing chromatin gran-
ules in some of the Euglenida and Dinoflagellida. Instead, beaded chromo-
somes seem to persist through vegetative stages. In general, however,
chromosomes of the later prophases seem to develop from some sort of a
"reticulum" and, in such favorable material as Pamphagus hyalinus (17),
the process has been traced in living material. In certain species of Enta-
moeba (Fig. 1. 20, F) and in Naegleria gruberi (207), the chromosomes
develop from a finely granular or reticular zone of Feulgen-positive mate-
rial around the endosome. The persisting "peripheral chromatin" gran-
ules, apparently adherent to the nuclear membrane in Entamoeba, may
give rise to chromosome-like bodies perhaps analogous to the nucleoli of
ZellerieUa (58). Interpretations are even more difficult in Endamoeba
blattae because the interphase nucleus is Feulgen-negative, although
Feulgen-positive chromosomes appear in mitosis (177).

The origin of chromosomes from an endosome or a karyosome, in

Reproduction and Life-Cycles

nuclei supposedly containing no interphase "chromatin granules," has
been reported in certain Protozoa. In some of these, such as Endolimax
nana, periendosomal material has been demonstrated in more recent in-
vestigations (240). Furthermore, Noble (200) reports a functional separa-
tion of endosomal granules and periendosomal chromatin in Entamoeba
gifigwalis, in spite of their intermingling during early prophases. How-
ever, the interphase precursors of the chromosomes have not yet been
identified with certainty in many Protozoa and much remains to be
learned about the earliest stages of mitosis in most species.

The observations of Cleveland (63) on Holoniastigotoides have shown
that the chromosomes persist as such throughout the mitotic cycle. The
diagrammatically clear behavior of the chromosomes in this genus sup-
j3lies a logical pattern for interpreting mitosis in smaller nuclei. Each
chromosome consists of a coiled chromoyiema embedded in a matrix. Only
the matrix is distinguished in heavily stained preparations, but both com-
ponents can be detected with phase-contrast microscopy and also by
ordinary microscopy in suitably stained preparations. The disappearance
of major coiling and the apparent lengthening of each chromonema, as
the chromosomal matrix disappears late in mitosis, result in long twisted
filaments. This stage, in small nuclei containing a nvnnber of chromo-
somes, would suggest the interphase "reticulum" described in various
species. If the uncoiled chromosomes are very slender, optical sections of
a small nucleus might suggest a granular organization of the chromatin.
Origin of chromosomes from such a "granular" or "reticular" interphase,
in the light of chromosomal behavior in Holomastigotoides, may involve
only a condensation of preexisting chromosomes. Each chromonema be-
comes more and more tightly coiled (in the "major coils" of Cleveland)
as a new matrix is developed. The result is the more or less compact
chromosome of the later prophases. According to Cleveland, duplication
of each chromonema occins before the development of the new matrix.

Eumitosis and paramitosis

Differences in chromosomal behavior and the structure of the
achromatic division-figure have formed the bases for various classifications
of protozoan mitoses. Among these systems, that of Belai- (20) has the
advantage of simplicity in recognizing two general types, eumitosis and
paramitosis.

Characteristic features of eumitosis are longitudinal splitting of the
chromosomes, the development of compact prophase chromosomes, and
the appearance of an equatorial belt of chromosomes within the spindle.
Many protozoan mitoses can be fitted into such a scheme.

Nuclear division in Dimorpha mutans (Fig. 2. 6) is representative.
Mitosis is initiated by division of the centrosome, and the subsequent
development of an amphiaster is accompanied by the formation of short

64 Reproduction and Life-Cycles

chromosomes from the interphase chromatin. The nucleus moves into
the spindle, the nuclear membrane is said to disappear, and the chromo-
somes form an equatorial plate. Additional examples are found in Actino-
phrys (18), Pelomyxa (Fig. 2. 7), and Zelleriella (58).

In paramitosis, condensation of the prophase chromosomes is less
marked and a typical equatorial plate is not developed. In Aggregata
eberthi (Fig. 2. 8, A-D), only one end of each chromosome extends into

Fig. 2. 6. Mitosis in Diniorpha mutans; euniitotic type; basal portions of
flagella and a few axopodia are indicated at the poles of the division-figure;
x3135 (after Belaf).

the equatorial zone of the spindle. Since the daughter chromosomes sep-
arate before they are shortened, later stages of mitosis suggest transverse
division of long chromosomes. Long chromosomes persist also in certain
Radiolarida (20), Dinoflagellida (29, 106), Euglenida (3, 115, 157), and in
Teratonympha (Fig. 2. 8, E, F).

The picture presented during separation of the daughter chromosomes
depends upon the position of the centromeres and the length of the
chromosomes. Terminal centromeres (Fig. 2. 10, H), which have been
demonstrated in Holojnastigotoides (63), probably occur in Aggregata

Reproduction and Life-Cycles

^'\ /'

/ « > *U'Vf.>i,i...f*--

•-^#»*# \

~ E

hi/in n^/H /

Fig. 2. 7. Mitosis in Pcloinyxa caroUnensis. A. Interphase; x2300. B.
Early prophase; x2300. C. Late prophase; x2645. D, E. Separation of daugh-
ter chromosomes; x2990. F. Chnnping of chromosomes in late anaphase;
x2990 (after Kudo).

and in Euglenida and Dinoflagellida. The appearance of Y-shaped daugh-
ter chromosomes during anaphases, as described in PleurotricJia lanceo-
lata (173), would suggest median instead of terminal centromeres.

Persistence of the nuclear membrane throughout mitosis is character-
istic of many Protozoa. However, a large portion of the old membrane is
discarded after division of the nucleus in Holomastigotoides (63) and
disappearance of the membrane in the prophase has been described in
Dimorpha mutatis (20).

The micronucleus of ciliates

The small size of the micronucleus increases the difficulty of inter-
preting chromosomal behavior. Longitudinal splitting of the chromo-
somes has been reported in some species and transverse division in others,
but decisions are difficult for the almost spherical chromosomes found in
certain ciliates. Longitudinal splitting has been described in Pleurotricha
(173), Stylonychia (119a), Concliophtliirius (144), and in pregamic di-
visions in Euplotes (234). In the last three cases, members of each pair
of daughter chromosomes slip past each other toward the poles of the
spindle (Fig. 2. 9).

66 Reproduction and Life-Cycles

if ^^^\

^. :«^'* i/^ A

Fig. 2. 8. AD. Mitosis in Aggregata eberthi; paramitotic type; x3790
(after Belaf). E, F. Long chromosomes in the dividing nucleus of Terato-
nynipha; x2400 (after Cleveland).

The achromatic figure

Both extranuclear and intranuclear achromatic figures have been
described in Protozoa. The extranuclear figure is sometimes represented
merely by the centrosomes and a paradesmose (156) which ranges from a
delicate fibril to a bundle of fibrils in different species (Fig. 2. 10, A-G).
The fibrillar paradesmose, as seen in Gigantomonas, differs mainly in

Reproduction and Life-Cycles 67

degree from the extranuclear spindle of Pseudotrichouymplia and similar
types (Fig. 2. 10, I, J). Comparable extranuclear spindles occur in certain
dinoflagellates (29, 195) and in Aggregata (20). Since the nuclear mem-
brane persists in such forms as Pseudotrichonympha, some of the astral
rays, during development of the spindle, make connections with the cen-
tromeres at the nuclear membrane. Each chromosome in HoJomastigo-
t aides (Fig. 2. 10, H), for example, ends in a terminal centromere which
remains anchored to the nuclear membrane. Duplication of the centro-
mere parallels that of the chromonema. In some of the Hypermastigida,
development of the spindle and its connections with the chromosomes has
been followed in living flagellates. Pulls exerted on the achromatic figure

Fig. 2. 9. Mitosis in the ciliatc, CoDchophthirius auodontae; x3995
(after Kidder). A. Longitudinal splitting of the chromosomes. B, C. Sepa-
ration of daughter chromosomes. D. Nuclear division nearly completed.

cause corresponding movements of the chromosomes; when the tension
is released, the fibrils and chromosomes snap back into place (59).

Intranuclear figures have been described in the micronuclei of ciliates,
in Actinophrys (IS), Monocystis (184), ^nd Euglyph a (120), among others
(Fig. 2. 11, E-L). In some of these cases, the spindle ends in centrosomes
which seem to be embedded in the nuclear membrane or else adherent to
it. An intranuclear spindle is typical of the dividing micronucleus (Fig.
2. 11, E, F), although little is known about division-centers in ciliates.
The spindle sometimes extends into achromatic masses ("polar caps")
which may or may not contain "centrioles." Only a granule has been
described at each pole in certain ciliates, and even the granules seem to
be missing in others.

68 Reproduction and Life-Cycles

Fig. 2. 10. A, B. Paradesmose in Tritrichomonas augusta; x2390 (after
Kofoid and Swezy). C-E. Paradesmose in Metadevescovina cuspidata; early
division (C), x2160; nuclens divided and other organelles duplicated (D),
xl800; later stage with very long paradesmose (E), xl440 (after Kirby). F, G.
Fibrillar paradesmose in Giga)itonw>ias herciilea; late anaphase (F), xl710:
nuclear division completed (G), x725 (after Kirby). H. Centromeres in Holo-
mastigotoides; portions of chromosomes indicated diagrammatically; xl260
(after Cleveland). I, J. Extranuclear spindle in Pseudotrichony?npha; chromo-
somes moving toward poles (I), x935; later stage (J), chromosomes not shown,
x735 (after Cleveland). Key: a, axostyle; c, cresta; ce, centromere; p, parades-
mose.

The significance of the persisting endosome in Euglenida and certain
dinoflagellates (Fig. 2. 11, A-D) is uncertain. Although this structure oc-
cupies an axial position and is divided in mitosis, there is no good evi-
dence that the endosome is analogous to an intranuclear spindle.

The macronucleus

The simpler macronuclei often divide by mere elongation and
constriction into approximately equal parts, although unequal division
occurs occasionally (71). Division of the compact macronucleus is not

Reproduction and Life-Cycles

always simple, however. A regular elimination of material (Fig. 2. 12)
from the macronucleus during division, or from the daughter nuclei
afterward, has been described in such genera as Ancistruma (142), Col-
poda (145), Tillina (14), Chilodonella (167), Colpidiinn, Glaucoma, and
Urocentnun (146). The significance of this process is unknown.

Ciliates with more than one macronucleus and those with long beaded
or band-like macronuclei may show more complicated nuclear changes.

Fig. 2. 11. A-D. Behavior of the endosome during mitosis in Heteronema
acus; A, B, D, x2890; C, x2270 (after Loefer). E. Intranuclear spindle, micro-
nucleus of Steutor coeruleus; x2365 (after Mulsow). F. Intranuclear spindle,
micronucleus of Stylonychia pustulata; xI730 (after IvaniC). G-I. Intra-
nuclear spindle in Oxymonas grandis; xl330 (after Cleveland). J-L. Intranu-
clear spindle in Pyrsonympha; early stage in development (J), x2920; later
stages (K, L), xl98r) (after Cleveland).

Reproduction and Life-Cycles

The C-shaped macronucleus of Euplotes (Fig. 2. 13, A-D) is shortened
and thickened, and undergoes changes in staining reactions which sug-
gest progressive internal changes. The two macronuclei of Stylonychia
pustiilata (227) fuse into a single body which then divides. The macro-
nuclear chains of Spirostornum, Stentor, and Blepharisma also undergo

Fig. 2. 12. Elimination of chromatin during macronuclear division in
Colpidium colpoda. A. Central chromatin mass evident just before divi-
sion. B-E. Stages in division. F. Separation of discarded mass from a daugh-
ter macronucleus. A-E, x510; F, x700 (after Kidder and Diller).

extensive condensation. In Spirostomiim ambiguum (22) and Stentor
coeriileiis (238) the macronuclear nodes gradually fuse into a compact
central body, which then undergoes moderate elongation and a final
constriction (Fig. 2. 13, E-I). In Blepharisma imdiilans, the anterior and
posterior macronuclear nodes fuse into two masses, while the middle
nodes gradually disappear. The anterior and posterior masses then fuse
into one body which elongates and undergoes division (238, 255).

Reproduction and Life-Cycles 71

LIFE-CYCLES

General features

The simple life-cycles of many species include only an active phase
and a cyst. With the cyst apparently eliminated, the "cycle" reaches the
limit of simplicity in such types as Entamoeba gingivalis and Pentatricho-
monas hom'mis. Other modifications of this basic pattern include: (a)
the development of two or more stages in the active phase; (b) the intro-
duction of sexual phenomena, which may appear in a sexual phase alter-
nating with an asexual phase in the cycle.

Two or more active stages occur in the life-cycles of many Protozoa. In
addition, immature and adult forms of a single organism may be quite

Fig. 2. 13. AD. Changes preceding division of ilie macronucleus in
Eiiplotes; stages in condensation (AC); elongation jnst before division (D);
x485 (after Tnrner). E-I. Division of the macroiniclens in Slentor coeruleus;
diagrammatic (after \Veisz).

72 Reproduction and Life-Cycles

Fig. 2. 14. A, B. Soniatella and reproduclion by budding in Colacium
vesiculosum (after Johnson); A, xl840; B, xl380. C-E. Metamorphosis of flag-
ellate into amoeboid stage in Tetramitus rostratus; x2080 (after Bunting).

F, G. Somatella and formation of bud in Tritrichomonas aiignsta; F, x2100;

G, xl200 (after Kofoid and Swezy). H-J. Gigmitomonas Jierculea, flagellate
stage (H), xlOOO; uninucleate amoeboid stage (I), x385; multinucleate amoe-
boid stage (J), xl35; diagrammatic (after Kirby). Key: c, chromatophore; cr,
crcsta; r, rhizostyle; t, trailing flagellum.

Reproduction and Life-Cycles 73

different in appearance and behavior. Examples include the ciliated larva
and non-ciliated adult of Suctorea and the stalkless telotroch and the
stalked adult of vorticellid ciliates. Dimorphism sometimes involves the
alternation of amoeboid and flagellate stages (Fig. 2. 14, C-E; H-J).
The flagellate stage may be temporary, as in Naegleria; or it may be the
dominant stage, as in Tetramitus and certain Chrysomonadida. The flag-
ellate, Gigantomonas herculea, shows amoeboid-flagellate dimorphism in
which reproduction is limited to the amoeboid phase. Reproductive stages
in Haematococciis and related genera also are typically non-flagellated.
The dominant phase in Colaciiim (134) is a non-flagellated form which
occasionally produces flagellate buds (Fig. 2. 14, A, B). Dimorphism also
may involve the alternation of a gamete-producing stage and one which
undergoes asexual reproduction, as in Foraminiferida. Life-cycles char-
acterized by more than two active stages are found in certain Trypano-
somidae, in many Sporozoa, and in some of the Ciliophora.

Protozoan life-cycles may be considered adaptive in that they represent
responses to changes in the environment, and perhaps favor, or insure
survival when such changes occur. Occurrence of a cycle as such probably
is dependent directly upon the environment. This seems evident in
parasitic species which must reach a susceptible host in order to complete
the cycle, or in many instances even to survive for more than a short time.
Within a suitable host, there is often reasonable security during comple-
tion of a life-cycle, but establishment in a host does not necessarily insure
independence of external conditions. For example, the development of
Plasmodium vivax in the mosquito may be retarded or prevented by un-
favorable temperatures. A modification of environmental conditions may
induce a marked change in the cycle, in parasitic as well as free-living
species. Maintenance of Plasmodium- gallinaceum in chick-tissvie cultures
has caused a normal stock to lose its ability to produce pigmented ery-
throcytic stages. Chicks inoculated from such cultures always died from
exoerythrocytic infections, always without showing normally pigmented
erythrocytic stages, and often without any erythrocytic parasites at all
(160). In some cases it has been possible to eliminate cyclic changes by
strict control of environmental conditions, as in the prevention of con-
jugation and encystment in ciliates by Woodruff, Beers, and others. Such
elimination of cyclic changes does not necessarily mean that the particular
life-cycles have no significance. Since a given cycle presumably adapts a
species to changing environments it may normally encounter, a perfectly
uniform environment may fail to evoke the cycle.

Cysts

Encysted stages, in which the organism is enclosed Avithin a cyst
membrane, are a common feature of protozoan life-cycles. On rhe basis

74 Reproduction and Life-Cycles

of apparent functions, protective and reproductive cysts have been dis-
tinguished.

Protective cysts may be developed directly from active stages, from
zygotes in Volvox and Gregarinida, or from sporoblasts (division-products
of the zygote) in Coccidia. Such cysts usually possess rather firm walls
(Fig. 2. 15, AD), the composition of which varies from group to group.

Fig. 2. 15. A. Cyst of Ceratium liiruncUnella; x385 (after Hall). B. Pro-
tective cyst of Didinium nasuhim; outer (ectocyst) and inner (mesocyst)
membranes evident; x310 (after Beers). C. Protective cyst of Bursaria
truncatella; xl35 (after Beers). D. Encysted zygote of Volvox globator;
diagrammatic (after Janet). E. Reproductive cyst in Gyrodinium sp.; x240
(after Kofoid and Swezy). F. Reproductive cyst in Colpoda citcuUus; x735
(after Kidder and Claff). Key: c, chromatophore; e, developing ectocyst;
71, nucleus in syncytial layer enclosing zygote; zn, nucleus of zygote.

The cyst membranes of many ciiiates are probably composed largely of
proteins (172), although the inner meinbrane (endocyst) may be carbo-
hydrate in nature (100). In Endamoebidae and Giardia, the properties
of the cyst wall resemble those of keratins (155). Siliceous cyst walls are
characteristic of Chrysomonadida, and walls composed largely of sand
grains are produced in Difflugia (203). Many of the thick-walled cysts
show spines, ridges, or other surface markings. A compound cyst wall
(Fig. 2. 16, C), composed of two or more membranes, is not uncominon.

Reproduction and Life-Cycles 75

In such cases one of the membranes — the ectocyst of Bursoria (16), the
mesocyst of Didiniimi (12), the outer membrane of Volvox (122) — is often
thicker and more rigid than the others. This heavy membrane may be
continuous like the others, or it may, as in Bursaria (Fig. 2. 17, A), con-
tain an "emergence-pore" closed by a thin membrane. The two-layered
cyst of Naegleria contains several analogous pores (207). Double or mul-
tiple resting cysts are sometimes produced in Colpodidae. The double cyst
of Tillina magna shows only one ectocyst, but each of the contained
ciliates has its own mesocyst and endocyst (11).

The protective qualities of cysts vary with the species. Dried cysts of
Colpoda ciiciilhis have remained viable for more than five years (69).
Cysts of Naegleria gruberi also withstand drying (207). Drying at room
temperature prolongs the life of protective cysts of Stylonethes sterkii but
kills those of Euplotes taylori (90). Cysts of Didinium nasutum do not
survive desiccation although they have remained viable for ten years in
sealed containers of hay infusion (10). Cysts of Endamoebidae also do
not survive drying. However, cysts of Entamoeba histolytica, kept moist
under refrigeration, have remained viable for 46 days (245). Woodruffia
metabolica produces two types of resting cysts, a stable one which resists
desiccation, and an unstable type which does not (136). Resistance of
protective cysts to unfavorable temperatures is sometimes striking. Thor-
oughly dried cysts of Colpoda have resisted exposure to dry heat at 100°
for three hours (23), and immersion in liquid air for 13.5 hours (230).

Reproductive cysts are those in which fission, budding, and sometimes
gametogenesis and syngamy occur m different species. However, repro-
ductive activities are not limited entirely to reproductive cysts, since
mitosis occurs in the protective cysts of Giardia and various Endamoe-
bidae. The wall of the reproductive cyst, although sometimes compound,
is usually thin and has relatively little protective value. Such cysts are
known in various dinoflagellates (Fig. 2. 15, E) and in certain free-living
and parasitic ciliates. Fission within a cyst is characteristic of Colpoda
ciicullus and related species (Fig. 2. 15, F). A similar cyst serves also for
attachment of Ichthyophthirius m,ultifiliis to the substratum (172). The
gametocyst of gregarines probably should be included in this type. Cysts
which are presumably of the reproductive type have been referred to as
"feeding cysts" in certain dinoflagellates, because they are formed after
the organisms ingest a large amount of food.

Encystment

Precystic changes in the organism usually precede secretion of a
cyst wall. Material for the membrane sometimes accumulates as globules
in the peripheral cytoplasm. Food vacuoles may be eliminated, as in
Endamoebidae, and cytoplasmic reserves such as starch or glycogen are
often stored in abundance. Since cysts usually approach a spherical form.

76 Reproduction and Life-Cycles

there is a corresponding change in shape o£ the body. In such genera as
Euplotes (90), softening of the pellicle must precede this change in form.
Partial or complete resorption of locomotor organelles is common. As
traced in Woodruffia metabolica, the cilia begin to shorten as the organ-
ism rounds up, and shortening is completed after 22-24 hours. The endo-
cyst is not secreted until after the cilia have disappeared (136). Encystment
apparently involves some loss of water, with a corresponding increase in
density of the protoplasm. The resistance to desiccation, noted in stable
cysts but not in unstable cysts of W. tnetabolica, is attributed to a lower
water content of the former (136).

The occurrence of encystment has been correlated with various envi-
ronmental changes. Encystment of Euplotes taylori seems to be related to
evaporation of the culture medium (90), while Bursaria truncatella en-
cysts when transferred singly or in groups to food-free spring water (16).
Encystment of Didinium nasutum is induced by crowding, either with or
without a food supply (15). Colpoda (duodennria) steinii encysts when
starved, and the percentage of cysts increases with the number of organ-
isms present. Encystment of this ciliate has been attributed to the inac-
tivation of essential enzyme systems by metabolic products (231). The
lack of materials for synthesis of such enzymes should produce the same
effect, and encystment of C. steinii in pure culture has followed elimina-
tion of thiamine, pyridoxine, nicotinamide, or pantothenic acid from the
standard medium, or the omission of foods known to contain several
B-vitamins (91). An abundance of food has been considered essential for
encystment of some species, but such a food supply would favor rapid
multiplication with subsequent crowding. Encystment of ciliates also has
been related to an unusually low or high pH of the medium (68), al-
though Didinium nasutum encysts most frequently within the range
favorable to growth (7).

The varied data on encystment obviously hinder selection of any one
factor as the key to this process. However, such a theory as that of Taylor
and Strickland (231) lends itself to possible correlation with several envi-
ronmental changes. The inactivation of a critical enzyme system might
result from accumulation of metabolic poisons — induction by waste prod-
ucts and by crowding. Inactivation might be accelerated by a deficiency
of materials for synthesizing such enzymes — induction by starvation and
crowding. Also, the inactivation of an enzyme system might occur more
rapidly at one pH than at another.

Excystment

Excystment often includes the regeneration of peripheral organ-
elles as well as a certain amount of internal reorganization. Rupture of
the cyst membranes may involve two different mechanisms. The more
important seems to be the absorption of water by the protoplasm early

Reproduction and Life-Cycles 77

in excystment. The resulting increase in volume forcibly ruptures rigid
membranes. In ciliates, the absorbed water may accumulate in a large
excystment-vacuole (Fig. 2. 16, A), apparently identical with the con-
tractile vacuole of Euplotes taylori (90) and Didinium nasutum (12), or
in a number of vacuoles as in Tillina magna (11). The second mechanism
involves the secretion of enzymes which digest the endocyst and perhaps
other flexible membranes. This enzymatic action, first described in Col-
poda ciicuUus (100), probably occurs also in Tillina and Didinium.
The first signs of excystment in Didinium nasutum (Fig. 2. 16) are the

Fig. 2. 16. Exc)stment in Didinmm nasutum; x275 (after Beers). A.
Appearance of excystment-vacuole. B. Ectocyst and mesocyst almost rup-
tured. C. Endocyst protruding from ruptured outer membranes. D. Organ-
ism after discharge of excystment-vacuole. E, F. Active ciliate in endocyst,
which increases in diameter. G. Escape of ciliate. Key: ec, ectocyst; en,
endocyst; in, mesocvst; v, excystment-vacuole.

beginning of cyclosis and the appearance of a small posterior vacuole.
When the vacuole grows to about half the volume of the body, a bulge
appears at the opposite pole of the cyst. A little later, the mesocyst and
ectocyst are ruptured and the organism slips out, still within the endo-
cyst. The ciliate soon becomes very active within the endocyst, which
gradually increases in diameter. The membrane becomes thinner and
thinner, and finally seems to dissolve in the medium. Excystment is com-
pleted within four hours. At emergence, the meridionally arranged cilia
extend from the anterior ciliary girdle about halfway to the posterior
end of the body. Later on, the posterior cilia of the longitudinal rows

78 Reproduction and Life-Cycles

develop into a posterior girdle, while the intermediate cilia disappear.
The primitive ciliary pattern of Holotrichida is thus recapitulated to
some extent during excystment of D. nasutiim (12).

Excystment of Biirsaria truncatella (Fig. 2. 17) is strikingly different.
Cyclosis begins early, and a hyaline area of cytoplasm just beneath the
emergence-pore becomes more apparent. After a time, the opercular mem-

Fig. 2. 17. Excystment in Bursaria truncatella; x200 (after Beers).
A. Appearance of a "hyaline cap" in the cytoplasm. B, C. Emergence of
the ciliate through the ruptured opercular membrane. D. Young excysted
ciliate. E. Older stage with developing peristomial membranelles. Key:
b, "bridge" joining endocyst and ectocyst; ec, ectocyst; en, endocyst; h,
hyaline cap; o, opercular membrane; p, emergence-pore.

brane bulges outward, and then breaks suddenly as a column of cytoplasm
erupts through the pore. The endoplasm streams into the protruded part
of the body and ciliary activity, which now increases, tends to move the
body through the pore in repeated thrusts. Emergence is completed,
posterior end first, and the immature organism swims away. During the
next hour the peristomial groove and membranelles are differentiated,
and the adult form is gradually assumed (16).

Reproduction and Life-Cycles 79

The physiological aspects of excystment are probably no less compli-
cated than the morphological changes. Excystment of Colpodo steinii
involves several stages. In an initial phase, the length of which is in-
fluenced by temperature but not by oxygen tension, essential organic
substances are absorbed from the medium. The activities of three later
periods, distinguishable by varying susceptibility of the cysts to X-rays,
are influenced by oxygen tension but not by organic components of the
medium (25). Weyer (241) suggested that excystment of Gastrostyla
steinii is induced solely by organic substances elaborated by bacteria in
the medium. Excystment of DicUniiim nasutum, in various media, de-
pends upon the presence of living bacteria. Previously bacterized culture
fluids are inactive after being heated or filtered to remove the bacteria
(13). Entamoeba histolytica will excyst in the absence of living bacteria,
but only at a low oxidation-reduction potential (216).

Barker and Taylor (5) apparently were the first to show that excyst-
ment can be induced specifically by adding certain animal or plant ex-
tracts to basal media. Some substance or group of substances was active
for Colpoda steinii in dilutions as high as 1: 100,000,000. In attempts to
isolate these factors, two concentrates from hay extract were found to be
active separately, and also to show complementary effects in combinations
(232). The activity of hay extracts was next related to salts of organic
acids (acetic, citric, fumaric, malic, and tartaric), the effectiveness of
which was quadrupled by a co-factor prepared from hay and replaceable
by certain sugars in dilute solutions (105). Tavo crystalline substances,
prepared from corn leaves, proved active at concentrations of 2.0-4.0 x
10-^ gm/ml in the presence of suitable co-factors. The co-factors, pre-
pared from corn extract and essentially inactive themselves, could be
replaced in part by a sugar solution and certain combinations of thiamine,
nicotinic acid, nicotinamide, adenylic acid, citrate, and malate (206). A
later report (226) indicates that potassium ions, not replaceable by so-
dium ions, are essential to excystment of C. steinii. Several vitamins pro-
duced no significant effect, although certain carbon sources (citrate,
glutamate, malate, and propionate) and adenosine triphosphate showed
some activity.

Requirements for excystment are less complex in certain other ciliates.
Distilled water induces excystment of TilUna magna (11) and Colpoda
cucullus (147), and dilution of the original medium is effective for
Euplotes taylori (89).

SEXUAL PHENOMENA

Varieties of sexual phenomena

Although sexual processes are not necessarily a prerequisite to
reproduction as they so commonly are in Metazoa, and although many

Reproduction and Life-Cycles

Protozoa undergo such activity at irregular intervals, the life-cycles of
certain species cannot be completed without syngamy. For example, the
mosquito phase of the life-cycle in Plasmodium must be initiated by
gametogenesis and syngamy. The same thing is true for the formation of
spores (protective cysts) in Eimeria and related genera.

Various kinds of sexual phenomena have been described in Protozoa.
Syngamy, in which two gametes fuse completely to form a zygote, may
involve gametes which are similar in appearance (isogamy), or are of two
types (anisogamy). Pedogamy appears to be an unusual type of syngamy
in which the two gametes are not more than one or two cell-generations
removed from a single gametocyte. Autogamy involves the formation of
two gametic nuclei, and their subsequent fusion to form a synkaryon
(zygotic nucleus) within a single organism. Parthenogejiesis, or the de-
velopment of a gamete without syngamy, has been reported but its status
in Protozoa is uncertain. Typical conjugation involves the exchange of
haploid pronuclei (gametic nuclei) between two paired organisms, the
formation of a synkaryon in each, and then nuclear reorganization.

Meiosis in relation to the life-cycle

A reduction of the chromosomes to the haploid number may occur
in gametogenesis {gametic meiosis), in an early division of the zygote

TABLE 2. 1. TYPES OF MEIOSIS IN PROTOZOA

Gametic

Zygotic

Mastigophora

Notila proteus (65a)
Paradinium poucheti (40)

Sarcodina

Actinophrys sol (18)

Foraminiferida (185, 186, 187, 188)

Gregarinida

Monocystis spp. (36, 184, 190)
Urospora lagidis (189)

Haemosporidia

Plasmodium falciparum, P. vivax,
probably gametic (168)

Cnidosporidia

Ceratomyxa blennius (198)
Guyenotia sphaerulosa (194)
Myxidium gasterostei (199)
M. incurvatum (193)
Myxobolus guyenoti (192)
Sphaeromyxa sabrazesi (193)
Triactinomyxon ignotum, T. legeri (169)
Tetractinomyxon intermedium (118)
^schokkella rovignensis (92)

Mastigophora

Euconympha imla (65b)
Glenodinium bibiniensijorme (74)
Oxymonas doroaxostylus (64)
Phytomonadida (202, 213, 213a, 257)
Saccinobaculus ambloaxostylus (65)
Trichonympha (62)

Gregarinida

Actinocephalus parvus (237)
Apolocystis elongata (204)
Diplocystis schneideri (121)
Gregarina blattarum (225)
Stylocephalus longicollis (103)
^gosoma globosum (197)

Coccidia

Adelea ovata (102)
Adelina cryptoceri (254)
A. deronis (112)
Aggregata eberihi ild, 11)
Karyolysus zuluetei (209)
Klossia helicina (191)
Ovivora thalassemae (170)

Reproduction and Life-Cycles 81

(zygotic meiosis), or in one of the pregamic divisions in conjugation
(conjugant meiosis). The type of meiosis varies in different Protozoa
(Table 2. 1). Available data indicate that the Heliozoida, Foraminiferida,
Cnidosporidia, and Ciliophora are diploid throughout most of the life-
cycle. Among the Mycetozoida, some of the Plasmodiophorina are said
to be predominantly haploid. Nuclear fusion, supposedly occurring at
the end of the vegetative phase, may be followed immediately by meiosis
(116, 236). In such cases, meiosis might be considered zygotic, although
the uninucleate haploid products promptly encyst, becoming "spores."
Some of the Eumycetozoina are believed to undergo syngamy just before
development of the plasmodium begins, and presumably are diploid
throughout the vegetative phase. In such cases, meiosis apparently pre-
cedes the formation of "spores," which give rise to the gametes after ex-
cystment. The Coccidia and a number of the Gregarinida are haploid
organisms, although a few of the gregarines seem to be diploid. Among the
flagellates, gametic meiosis has been reported in two species, and zygotic
meiosis in a number of others.

Syngamy

In addition to many established cases of syngamy (Table 2. 1) in
Protozoa, a nimiber of descriptions need confirmation. The lack of critical
evidence does not in itself justify dismissal of such reports. Syngamy in
Zoomastigophorea was described occasionally in the older literature but
most protozoologists remained unconvinced. The investigations of Cleve-
land (62, 64, 65, 65a, 65b) have supplied cytological evidence that was
previously lacking. Certain descriptions of syngamy in trypanosomes (86,
87) do not approach the cytological standards set by Cleveland. However,
the trypanosomes are not particularly favorable material for studying
chromosomal behavior and the accumulation of adequate evidence will
be correspondingly difficult.

The status of sexual phenomena in Phytomastigophorea other than
the Phytomonadida remains uncertain. A fairly recent description (21)
of syngamy in Euglena has not been confirmed, and the often cited case
of "Copromonas subtilis" (75) is questionable. In "C. subtilis" the so-
called reduction-divisions involved the extrusion of small granules ("polar
bodies") from the nucleus, whereas meiosis, as demonstrated in many
Protozoa, is a genuine nuclear division. Other reports of syngamy in
Euglenida also offer inadequate evidence. Among the Dinoflagellida,
syngamy has been reported in Ceratium hirundinella (85), Coccodinium
mesnili (41) and Noctiluca milaris (104). Syngamy and formation of
zygotes have been described in Glenodinium lubiniensijorme , a hetero-
thallic species which apparently undergoes zygotic meiosis (74). These
accounts receive additional support from a brief account of meiosis in
Paradinium poucheti (40).

82 Reproduction and Life-Cycles

Among the Sarcodina, descriptions of syngamy have been published for
several Testacida (20-)) and Amoebida. Careful studies of chromosomal
behavior have not been reported. Some supposed instances of syngamy in
Amoebida have appeared in peculiar life-cycles which seem to be elim-
inated by the use of pure-line cultures (135) and the occurrence of sexual
phenomena in this order is still unproven.

Although isogamy has been reported in some Sarcodina, gregarines and
Phytomonadida, certain of these examples involve gametes which are
similar in size and form but are distinguishable by vital staining or other
means. Miihl (183) noted that members of each pair, in syzygy of certain
gregarines, show different staining reactions with neutral red. These ob-
servations have since been confirmed and extended (138). Such differences
in staining reactions have been related to differences in oxidation-reduc-
tion potentials of the two gametocytes, which may differ also in the
quantity and distribution of cytoplasmic inclusions (138, 139).

Physiological differentiation of similar gametes also has been reported
in Chlamydomonadidae. Species of Chlamydomonas may be homothallic
(synoecious) or heterothallic (heteroecious). In homothallic species a
single culture will develop gametes of both "sexes." Every motile flag-
ellate in such a culture is a potential gamete capable of uniting with a
flagellate of the opposite sex in the same culture. As observed in the
laboratory, syngamy occurs in heterothallic species only when two cultures
containing gametes of opposite sexes are mixed imder favorable condi-
tions. Moewus (180, 181, 182) has attributed such differentiation to
specific substances produced by CJilamydomonas.- The original assump-
tions were based upon certain effects produced by fluids from cultures.
(1) A motility factor in culture fluid stimulates rapid formation of flag-
ella upon addition to a culture containing palmella stages. (2) Termones
determine the sex of the gametes derived from palmella stages in hetero-
thallic races. Gynotermones cause production of female gametes; andro-
termones, the production of male gametes. (3) Gamones, concerned mainly
with mutual attraction of the gametes, modify sexually inactive flagel-
lates so that they can undergo syngamy. Androgamones from male cul-
tures cause agglutination of female gametes under favorable conditions;
gynogamones from female cultures have a comparable effect on male
gametes.

Spectroscopic analysis of active substances, concentrated from large
volumes of culture filtrates, indicated that they were carotenoid deriva-
tives. In subsequent tests, the effects of culture filtrates were more or less
duplicated by certain derivatives of protocrocin. Accordingly, it was as-
sumed that protocrocin, synthesized by the flagellates, is broken down in
the presence of light in a series of reactions, each controlled by a par-
ticular gene. The products include picrocrocin, which in turn yields

^ The work of Moewus and his colleagues has been reviewed by Sonneborn (220, 221).

Reproduction and Life-Cycles

safranal and glucose, and crocin, which is decomposed into gentiobiose
and cis- and i)77?f5-dimethylcrocetin esters. Crocin (or a related glycoside
of crocetin) seems to be the motility factor, active for C. eugaynetos in
dilutions as high as 4 x 10-^^. The action of gynotermones was duplicated

i-?r:

Fig. 2. 18. A-E. Forniatioii of luicrogamctes in Ovivora tlialasscmae; A, B,
\1000: C-E, x2690 (after Mackinnon and Ray). F. Female gametocyte of O.
thalassemae; x900 (after Mackinnon and Ray). G. Microgamete of I'olvox
aureus; xl900 (after Janet). H. Macrogamete of Volvox globator shortly after
entrance of the microgamete (/??); diagrammatic (after Janet).

by picrocrocin; that of androtermones, by safranal. The gamones were
believed to be mixtures of the cis- and /rfl?75-crocetin esters and the in-
tensity of "maleness" or "femaleness" exhibited by gametes was attributed
to the cis-/trans- ratio in a given mixture.

The behavior of certain American stocks of Chlatnydojnonas differs to

84 Reproduction and Life-Cycles

some extent from that reported by Moewus. Strains of C. reinhardi, C.
minutissima, and C. intermedia, for example, become motile and develop
sexual activity in darkness as well as in light (214, 215). However, light
seems to be required for clumping and pairing of C. moewusi (161).

Prior to the work of Moewus, Schreiber (213) had described -|- and —
strains in Goniurn and Pandorina, mixtures of different clones producing
zygotes in some combinations but not in others. Tests with lines started
from division-products of zygotes indicated that differentiation occurred
in the first or second postzygotic fission.

Aside from such biochemical differentiation of similar gametes, the
development of minor structural differences apparently preceded the
evolution of marked gametic dimorphism. Among the gregarines, for
example, primitive anisogamy may involve differences in size of the nuclei,
differences in shape, and slight differences in size of the gametes. This
trend culminated in the development of small microgametes, resembling
spermatozoa in their low cytoplasmic content, and relatively large macro-
gametes containing appreciable amounts of stored food. Such extreme
differentiation is characteristic of certain Sporozoa (Coccidia, Haemo-
sporidia) and Volvox (Fig. 2. 18).

Pedogamy

In this process, attributed to Actinophrys and Actinosphaerium, a
single organism encysts and then divides into two or more "gametocytes."
After meiosis occurs, the resulting gametes undergo syngamy. In repeat-
ing earlier observations on Actinophrys sol, Belaf (18) described a reduc-
tional division in each gametocyte, followed by degeneration of one of
the two haploid nuclei. Fusion of the uninucleate gametes was then fol-
lowed by encystment of the zygote. The occurrence of syngamy in Helio-
zoida seems unquestionable but the validity of "pedogamy" may be less
certain. It has been suggested that, as in certain Foraminiferida (185),
two associated "gametocytes" secrete a common cyst membrane. However,
such an interpretation is not supported by Belaf's data.

Autogamy

The older literature (109) contains numerous descriptions of au-
togamy. In a typical account, the nucleus of an encysted amoeba divides
and each daughter nucleus undergoes meiosis. The haploid nuclei then
fuse in pairs. Or, fusion may be preceded by degeneration of all except
two haploid nuclei, so that only one synkaryon is produced. Believing
that such cases are open to more plausible explanations, protozoologists
generally had considered autogamy a highly dubious process.

The cjuestion was reopened by Diller's (70) report of autogamy in
Paramecium aurelia. Autogamy, followed by meiosis of the synkaryon,
was reported shortly afterward in Phacus pyrnm (157), although this

Reproduction and Life-Cycles 85

account has not been confirmed. Diller's observations on P. aurelia have
been followed by descriptions of autogamy in P. bursaria (5o) and P.
trichium (72). Cases of autogamy in which ciliates form a conjugant pair
but fail to exchange pronuclei have been referred to as cytogamy in P.
caudatum (243). In addition, certain genetic data (Chapter IX) agree with
the cytological evidence for autogamy in Paramecium. Up to a certain
point, nuclear behavior in autogamy parallels that in conjugation. Mat-
uration divisions are normal and pronuclei are formed. Instead of recip-
rocal transfer, however, fusion of two pronuclei occurs within the same
ciliate. It is uncertain whether autogamy is a normal process in its own
right or merely abortive conjugation. Chen (55) has found that, in con-
jugating trios of P. bursaria, a small area of cytoplasmic contact will
initiate autogamy in the odd member which is left out of the normal
pairing.

Conjugation

The onset of conjugation in mass cultures of certain ciliates is
indicated by a tendency for the organisms to adhere on contact, some-
times forming clumps containing many individuals. The nature of this
mating reaction is uncertain, although such a process suggests that the
ciliates develop sticky surfaces. This initial reaction in Paramecium
bursaria (126) seems to involve chance contact which leads to clumping.
In general, such a preliminary reaction seems to be independent of later
pairing and may be insignificant, or may not occur at all, in certain clones
of P. bursaria and in various other ciliates. The stalked conjugant of
Vorticella microstoma seems to exert some sort of attraction for motile
microconjugants passing within a distance of a millimeter (88).

Clumping in P. bursaria is followed, after a half hour or so, by gradual
breaking up of the aggregates. At the end of several hours, only pairs and
single ciliates remain as a rule. Groups of three or four persist occasion-
ally, but only two members of each group are properly paired for con-
jugation (55). Pairing seems to depend upon favorable conditions and
may be influenced by temperature and intensity of light.

The positions assumed by the paired conjugants (Fig. 2. 19) and the
extent of cytoplasmic fusion vary with the species. Contact commonly in-
volves the peristomial areas of the two conjugants. However, fusion at
the posterior ends occurs in Ancistrocoma myae (154), and fusion of oral
to aboral surface in Kidderia mytili (141). Among the Peritrichida, the
microconjugant becomes attached near the aboral end of the body in
Opisthonecta (211) and Vorticella, but near the oral end in Scyphidia
(233). In certain Apostomina, conjugants in lateral contact undergo re-
peated fission to produce chains and conjugation then proceeds between
corresponding members of the chains (47). The extent of fusion in con-
jugation apparently is influenced by the nature of the body wall. In

86 Reproduction and Life-Cycles

ciliates with a firm cuticle, fusion, or sometimes merely adhesion, may
involve a limited area of the body such as the left margin of the peristome
in Euplotes (234).

As a rule, the micronucleus undergoes three pregamic divisions (Fig.

Fig. 2. 19. Pairing in conjugation. A. Nyctotlienis cordiformis; x430
(after Wichterman). B. Pleurotricha lanceolata; x275 (after Manvvell). C.
Ancistroconm niyae, fusion of posterior ends; x2395 (after Kofoid and
Bush). D. Cycloposthium bipalmatuin, adoral organelles omitted; diagram-
matic (after Dogiel). E. Scyphidia ameirui; diagrammatic (after Thompson,
Kirkegaard and Jahn). F. Vorticella microstoma; x700 (after Finley). G.
Euplotes (patella) eurystomiis: x346 (after Turner).

41, A-D). More commonly the second, but sometimes the first (101) of
these, is reductional. However, exceptions to the usual pattern have been
noted. The third pregamic division is sometimes omitted in Paramechim
trichium (72), and micronuclei may even be exchanged just after the first
division (73). When several or many micronuclei are present, the num-

Reproduction and Life-Cycles

ber participating in the pregamic divisions varies with the species. Only
one of the many micronuclei undergoes the first division in Dileptus
gigas (235), but two or more may do so in other species. Comparable dif-
ferences in nuclear behavior have also been reported for the second and
third divisions. Furthermore, variation may occur within a single species.
For instance, 2-5 (and possibly 1-5) products of the second pregamic di-
vision may complete the third division in Paramecium aurelia (70). In

Fig. 2. 20. A. First pregamic division, early anaphase, Kidderia mytili;
xl875 (after Kidder). B. Late anaphase, second pregamic (reductional) divi-
sion, K. mytili; x2100 (after Kidder). C-G. Chilodonella uiiciimtiis: nuclei
just before the third pregamic division (C); late third division (D); fusion
of pronuclei (E); first division of the synkaryon (F, G); diagrammatic (after
MacDougall).

any case, the nuclei which do not undergo a particular division in the
series soon degenerate.

In typical conjugation the third pregamic division produces two or
more pronuclei. One of these, a migratory pronucleus, passes into the
opposite conjugant and fuses with a stationary pronucleus to form a
synkaryon (Fig. 2. 20, E). The actual exchange of pronuclei, which has
been questioned occasionally, is supported by recent cytological and
genetic data and has been observed in living specimens of Paramecium
bursaria (244).

Fusion of the pronuclei is followed by a reorganization in which the

Reproduction and Life-Cycles

synkaryon divides one or more times. Some or all of the resulting nuclei
may differentiate into macronuclei and micronuclei. Only one nuclear
division precedes differentiation in Nyctotherus cordifortnis (242), several
species of Chilodonella (167), and a few other ciliates (143). Differentia-
tion occurs after the second division in Paramecium aurelia (70), Eiiplotes
eurystomus (234), and about twenty other species (143). Differentiation
follows the third division in Bwsaria truncateUa (205), O pisthonecta
henneguyi (211), Parachaenia myae (154), Vorticella microstoma (88),
Paramecium bursaria (57), P. trichium (72), P. caiidatum, and a number
of other species (143). Differentiation after a fourth postzygotic division
has been reported in Kidderia mytili (143) and Parameciuvi multimicro-
nucleatum (159). Behavior of the nuclei in ciliates showing two or more
postzygotic divisions differs from species to species. All of the nuclei may

Fig. 2. 21. Development of a new macronucleus following conjugation in
Nyctotherus cordiformis; A-D, xllSO; E, x765 (after Wichterman).

remain functional, or some of them may degenerate. Variations may occur
also in individual species, as in P. caiidatum (71) and P. trichium (72).

Development of the micronucleus usually involves a decrease in size,
whereas a differentiating macronucleus grows and often undergoes ex-
tensive changes in form as well as internal organization. The young
macronucleus of Nyctotherus cordiformis (Fig. 2. 21) soon becomes finely
granular and stains more intensely. Later, the granules give rise to threads
during growth of the nucleus and then, as differentiation nears comple-
tion, the threads are replaced by the granules characteristic of the mature
macronucleus. The early stages of differentiation are similar in Euplotes
eurystomus. After the threads are replaced by granules the developing
macronucleus elongates, extends posteriorly, and makes contact with a
remnant of the old macronucleus. Fusion results in a complete macro-
nucleus (234).

Depending upon the species, postconjugant fissions may or may not be

Reproduction and Life-Cycles

necessary to restore the normal nuclear situation. Therefore, the final
result of typical conjugation is the formation of 2-8 reorganized ciliates
from a pair of exconjugants. In Metopus sigmoides (201), the pronucleus
of one conjugant (the "donor") is accompanied by a large amount of
cytoplasm during migration. After separation of the conjugants, the donor
eventually dies. Conjugation in Opisthonecta (211), Urceolaria (67), and
Vorticella (88) also produces only one functional exconjugant. One con-
jugant is a microconjugant, produced by budding, and the other is a
macroconjugant. In Vorticella ynicrostoma (Fig. 2. 22), a microconjugant

Fig. 2. 22. Conjugation in Vorticella microstoma. A. Formation of micro-
conjugant by budding. B. Fusion of microconjugant and macroconjugant;
micronucleus of former in the first pregamic division; second pregamic divi-
sions of the microconjugant have produced four nuclei. D. Two spindle-
shaped pronuclei are distinguishable. E. Synkar)'on and remnants of de-
generating macronuclei. F. One micronucleus in division; seven developing
macronuclei. xl050 (after Finley).

90 Reproduction and Life-Cycles

becomes attached near the aboral end of a macroconjiigant. Fusion then
occurs and the endoplasm of the microconjugant giadually flows into the
macroconjugant, leaving the pellicle behind. Pregamic divisions and for-
mation of a synkaryon then occur much as in other ciliates.

Conjugation is often considered an orderly process which, once started,
goes through a fixed series of nuclear activities. This is not always the
case and variations are striking in several species. Furthermore, conjuga-
tion between particular strains of a species may be abnormal. For instance,
in conjugation of certain Russian strains (variety IV) with several Amer-
ican strains of P. biirsaria, the first pregamic division is usually not com-
pleted and all conjugants die before or after separation. The lethal effect
is produced after cytoplasmic fusion, but before the exchange of pro-
nuclei (57, 132). Mixtures of certain abnormal strains of P. hursaria
with normal strains undergo typical pairing, but separation occurs after
a few hours. The micronucleus enlarges slightly but does not start the
first pregamic division (56). Polyploidy seems to have arisen frequently in
P. hursaria, probably through the fusion of more than two pronuclei in
conjugation (52). Chromosomal variations also are produced by matings
between diploid and polypoid strains, as well as between micronucleate
and amicronucleate races. In the latter case, each exconjugant contains a
single haploid nucleus which undergoes three divisions and probably
produces a new nuclear apparatus (53).

Nuclear behavior varies also in Parameciinn trichium (72, 73). Micro-
nuclei are sometimes transferred just after the second or even the first
pregamic division. Occasionally only one of the migratory pronuclei ac-
tually migrates, so that conjugants sometimes contain one and three pro-
nuclei. There also may be no exchange of pronuclei, with resulting
autogamy in each conjugant. After the second pregamic division, three
haploid nuclei sometimes degenerate and the fourth, without dividing
again, migrates into the other conjugant. Each exconjugant thus contains
a haploid nucleus which undergoes postzygotic divisions. Heteroploidy
occurs frequently in P. trichium and has been noted also in P. aiirelia and
P. caudatum (71). The exchange of macronuclear fragments has been ob-
served in P. trichium (72) — but not in other species of Paramecium — and
also in several species of Chilodonella (166, 167).

Factors inducing conjugation

The possible causes of conjugation have been discussed for many
years. Diverse ancestry was one of the prerequisites suggested by Maupas
(176) and the more recent discovery of mating types has proven that
apparently hereditary differentiation of potential conjugants does exist
in certain species. However, conjugation has been observed within single
clones, and also among the descendants of a single exconjugant after only
a few fissions. Some of these matings between closely related conjugants

Reproduction and Life-Cycles 91

— as reported in Paramecium (4, 30, 94, 123), Spatliidiiwi (253), Urolep-
tus (34), and Euplotes (149) — have not yet been correlated with the basic
concepts of mating types. Autogamy might bring about differentiation
within clones of Paramechim, but such an explanation is of uncertain
validity for other ciliates in which autogamy is unknown.

Sexual maturity as a requirement for conjugation also was suggested
by Maupas, who believed that strains of ciliates are immature when first
established in cultures and must complete a certain number of genera-
tions before they can conjugate. In contrast to this view, conjugation has
occurred at intervals of only a few days in Paramecium aurelia (217) and
P. caudatum (4). Jennings (130) has suggested that the duration of "im-
maturity" in P. bursaria varies inversely with the food supply.

Starvation is the third factor which Maupas considered essential. More
recently, conjugation of Paramecium multimicronucleatum (93), Spathi-
dium spathula (253) and Uroleptus mobilis (34), among others, has been
found to follow exhaustion of the food supply. On the other hand, con-
jugation has occurred in P. aurelia (123) just as a rich food supply was
beginning to decline, and also in P. caudatum (4), shortly before the pop-
ulations reached the maximum. The nature of the significant changes
which accompany or precede starvation is not yet known. However, the
physiological condition of individual ciliates seems to be an important
factor, since Boell and Woodruff (24) observed successful conjugation of
P. calkinsi only between ciliates with subnormal respiratory rates. A mat-
ing reaction between a normal ciliate and one with a low respiratory rate
sometimes occurred but conjugation was never completed. Ciliates with
high respiratory rates failed to show any mating reactions.

Various environmental factors also have been correlated with conjuga-
tion. Darkness apparently favors and light suppresses conjugation in P.
aurelia (219), although light shows no comparable effect on P. caudatum
(97) or Euplotes patella (148). Temperature also influences conjugation,
and different optima have been noted for different varieties of P. aurelia
(223). In one variety the frequency of conjugation has ranged from zero
at 24.5° to 68 per cent at 17.6° (219). Conjugation in ConchopJtthirius
lamellidens, parasitic on the gills of a fresh-water mussel, has been ob-
served most frequently on the day following the new moon (208). Dilu-
tion of the medium with weak solutions of aluminum and iron chlorides
is said to have induced conjugation of Paramecium caudatum (258), but
Ball (4) obtained negative results with several clones of P. aurelia and
P. caudatum. One clone of P. caudatum did respond to such treatment
but distilled water was just as effective as the salt solutions. Conjugation
of Glaucoma scintillans has been stimulated by decreasing the salt con-
tent of the medium or increasing the concentration of glucose (43), and
also by adding pyruvic acid to the medium (45).

The bacterial flora of cultures also may influence the incidence of con-

92 Reproduction and Life-Cycles

jugation. Chatton and Chatton (44) found that Glaucoma scintillans
conjugated when fed on Escherichia coli, Proteus vulgaris, Shigella dysen-
teriae, or Staphylococcus aureus, but not on Pseiidomonas aeruginosa, P.
fluorescens or any one of several other bacterial species. Conjugation of
P. caudatum was observed in cultures containing only a gram-negative
bacillus, but not in other cultures containing at least three kinds of bac-
teria (46). Accordingly, it was suggested that so-called conjugating and
non-conjugating races of ciliates may be determined by the bacterial flora.
This conclusion was not supported by Sonneborn and Cohen (222) who
induced conjugation invariably in a Johns Hopkins strain and never in
Woodruff's strain of P. aurelia when both strains were maintained on the
same bacterial types.

Mating types in ciliates^

Following the observations of Sonneborn (218, 219) on Para-
mecium aurelia, P. calkinsi, and P. trichium and those of Jennings (124,
125) on P. bursaria, mating types have been demonstrated also in P.
caudatum (95, 96, 97, 98, 98a), P. midtimicronucleatum (94, 95), and
Euplotes patella (148,149).

The situation in P. bursaria may be illustrated as follows. Two strains,
A and B, have been established in pure lines. Conjugation does not occur
among ciliates of strain A or among those of strain B, although mixtures
of the two do show conjugation. Therefore strains A and B seem to be-
long to different sexes. A third strain, C, tested in the same way with
strain A, behaves like strain B, and consequently might be expected to
have the same sex. However, conjugation occurs also in mixtures with
strains B and C. A fourth strain, D, is found to conjugate with any of the
other three. At this point, conjugation in P. bursaria begins to strain
basic concepts of bisexuality in animals, and confusion in terminology
has been avoided by the substitution of "mating type" for "sex." Further
investigation has demonstrated additional groups of mating types. A
second group, or variety, contains eight mating types (E, F, G, H, J, K,
L, M) which will not conjugate with the four types (A-D) in variety I.
Mating types N, O, P, and O have been assigned to a third variety, since
they will not conjugate with types belonging to varieties I and II. Variety
IV contains types R and S, which do not mate with members of varieties
I, II or III. Variety V is represented by mating type T, composed of
strains obtained from Russia, and will not mate with members of the
other varieties (132). A more recently recognized variety VI, including
strains from Czechoslovakia, England and Ireland, contains mating types
U, V, W and X (55).

In Paramecium aurelia seven varieties have been recognized (224).
Six of these contain two mating types, and one type has been assigned to

^This subject has been reviewed by Kimball (150).

Reproduction and Life-Cycles 93

variety 7. Normal conjugation occurs between the two mating types of
each variety, but not between strains belonging to different varieties.
Thirteen varieties, each with two mating types, have been identified in
P. caudatum (98a).

At first, it was believed that conjugation never occurred between mem-
bers of different varieties in P. aurelia and P. hursaria, but exceptions
have been reported more recently. Type R of variety IV occasionally
conjugates with four types of variety II in P. hursaria, althovigh the par-
ticipants die during or shortly after conjugation (]?)2). Similar cases have
been observed in P. aurelia (224). Mating type I will conjugate occasion-
ally with type X, and mating type II with types V, IX, and XIII. Mating
reactions in these intervarietal crosses of P. aurelia are always less intense
than those within the same variety — only 1-40 per cent as many conjugant
pairs in different combinations. In P. caudatum (98) intervarietal matings
have occurred between variety 10 (type XX) and varieties 8 (type XV)

TABLE 2. 2. INDUCTION OF CONJUGATION IN EUPLOTES
PATELLA BY FLUIDS FROM CULTURES

Mating types

of treated ciliates

Culture
fluids

III

—

III

—

■ 4-

—

and 9 (type XVII), and also betAveen variety 2 (type IV) and variety 8
(type XV).

The situation in Euplotes patella (148, 149) resembles that in P. hur-
saria. Six mating types have been recognized in one variety, and there
may be additional varieties. The mating reactions of E. patella are espe-
cially interesting because specific mating-type substances are released into
the culture medium. Fluid from cultures of one mating type will induce
conjugation among the ciliates of a single mating type in certain cases
(Table 2. 2). The nature of this effect is uncertain. Kimball apparently
favors the view that conjugation is induced in animals which are all of
the same mating type, rather than that the mating type is changed in
some of the treated ciliates and not in others. A particular mating-type
substance induces conjugation only in a type which does not produce that
substance, and these effects have been correlated with the inheritance of
mating types in E. patella (Chapter IX).

Certain analogous effects of culture fluid have been observed in Para-

94 Reproduction and Life-Cycles

meciiim bursaria (54). Fluid from cultures of several Russian strains (type
T) induces conjugation within individual mating types of varieties II,
III, IV and VI, although the effect is usually limited to a small percentage
of the ciliates in a culture.

The recognition of mating types in certain ciliates has shown that con-
jugating pairs, in these species at least, are composed of physiologically
different organisms. However, the relation of mating types to the concept
of bisexuality in animals remains uncertain in Paramecium bursaria and
Euplotes patella. On the other hand, P. aurelia and P. caiiclatum might
possibly be interpreted as species composed of "bisexual" varieties which
interbreed with difficulty or not at all.

Nuclear phenomena of uncertain significance

Endomixis (250) was originally described in Paramecium aurelia
as a complete nuclear reorganization occurring in individual ciliates
(251). Macronuclear disintegration and two micronuclear divisions occur
without the usual third pregamic division of conjugation. Only two of
these eight micronuclear derivatives persist, so that the first fission leaves
each ciliate with one functional nucleus. Two nuclear divisions occur.
Two of the products then differentiate into macronuclei, while the others
divide to form four micronuclei. A second fission completes the reorgan-
ization.

The significance of endomixis in the life-cycle is still unknown. Wood-
ruff believed that meiosis does not occur — although the second pregamic
division is reductional in conjugation of P. aurelia — and he suggested
that endomixis might be analogous to diploid parthenogenesis. The dis-
covery of autogamy in P. aurelia (70) and the accumulation of genetic
data have thrown doubt upon the occurrence of endomixis in P. aurelia.

Hemixis involves unusual behavior of the macronucleus only. The
process has been observed in Paramecium aurelia, P. caudatum, and P.
multimicronucleatum. (70). In one type of hemixis there is a precocious
division of the macronucleus and the normal nuclear situation is restored
in the next fission. In another type, the macronucleus extrudes one or
more densely staining masses and then behaves normally in subsequent
fissions. A third type of hemixis combines the elimination of chromatic
material with precocious division of the macronucleus.

THE PHYSIOLOGICAL LIFE-CYCLE

The description of conjugation by O. F, Miiller in 1786 stimulated
much interest in the sexual activities of Protozoa. For many years, it was
believed that the "ovary" (macronucleus) of ciliates gave rise to "ova"
(products of macronuclear disintegration), while the "testis" (micro-
nucleus) produced "spermatozoa" (chromosomes). In conjugation, two
hermaphroditic ciliates were supposed to exchange spermatozoa. In cer-

Reproduction and Life-Cycles 95

tain cases, small organisms (probably parasites) within the conjugants
were interpreted as "embryos" developing within viviparous parents.
These interpretations were overthrown by Biitschlii (26, 27) and Engel-
mann (80), who showed that the supposed ovary and testis are nuclei
and suggested that products of the micronuclei might be exchanged in
conjugation. The fusion of pronuclei in conjugation was reported a few
years later (133).

Once conjugation was found to involve nuclear reorganization, and
occasionally the reorganization of locomotor structures, the process was
interpreted as a sort of rejuvenation. Engelmann (80) suggested that it
was unnecessary to suspect any other effect. Biitschli (27) supported a
physiological interpretation — ciliates become senescent during continued
fission and as a result reproduce less and less frequently until conjuga-
tion rejuvenates them and restores the normal reproductive rate.

This question was first considered experimentally by Maupas, whose
isolation-culture technique (175, 176) involved tracing single ciliates from
one generation to the next in order to detect possible senescence. Since
all his strains died eventually, Maupas suggested that ciliates, like higher
animals, pass through a cycle of youth, maturity, and old age, ending in
death. The characteristic feature of maturity was assumed to be an ability
to conjugate normally. Conjugation was believed to rejuvenate ciliates
only during the phase of maturity, and therefore was a prophylactic
rather than a therapeutic measure.

Biitschli (28) maintained that conjugation increased fission-rate after
a gradual decline. Hertwig's (113) observations on split-pairs — conjugants
separated at the beginning of conjugation and used for starting parallel
clones — indicated that fission-rates were usually higher in non-conjugant
than in exconjugant lines. As a result, he concluded that conjugation
merely regulates metabolism so as to prevent physiological exhaustion.
Later investigations were designed to test the theories of Biitschli, Hert-
wig, and Maupas.

Joukowsky (137), after studying exconjugant and non-conjugant lines
of Paramecium caudatum and Pleurotricha lanceolata, concluded that the
degenerative changes described by Maupas were the result of unsatis-
factory conditions in cultures. There were no characteristic differences
between exconjugant and non-conjugant lines, neither type showed a
decreasing fission-rate, and there appeared to be no physiological cycle.

The next important papers were those of Calkins (30, 31, 38) who
started isolation-cultures of Paramecium, caudatum on February 1, 1901,
Four lines were started from each of two ciliates and transfers were made
daily or every other day. After a time, recurrent "depressions* developed.
The early depressions, believed to represent the senescence reported by
Maupas, were cured by measures other than conjugation. The depression
of May, 1901, apparently was cured by jolting during a train ride to

96 Reproduction and Life-Cycles

Woods Hole; that of August, 1901, by extract of raw beef; that of De-
cember, 1901, by beef extract; that of March, 1902, by a slight rise in
temperature; that of June, 1902, by brain extract. Since the lines were re-
juvenated by artificial means, the results were considered analogous to
artificial parthenogenesis (31).

In these early papers. Calkins suggested that ciliates have the "poten-
tial of endless existence" without conjugation. Later on, however, the
depressions became more severe. The "B" lines became extinct after 16
months, the "A" lines in December, 1902. Attempts to rejuvenate the
ciliates — treatments with beef extract, pancreas, brain, mutton broth,
lecithin, pineapple extract, apple juice, several acids and salts, dried
Paramecium, the electric current and nitroglycerin — were all unsuccessful.
As a result, Calkins (33) was convinced that the final depressions arose
from "germinal exhaustion" which could not be prevented by external
stimulation. Therefore, strains of P. caudatum must pass through a cycle
of youth, maturity, and old age unless vitality is renewed by conjugation.
A gradual decrease in fission-rate accompanied senescence and the re-
juvenation by conjugation was believed to include an increase in fission-
rate.

The observations of Enriques soon questioned the inevitability of
senescence. The first important demonstration (81) was that excessive
bacterial growth may lead to effects simulating senescence. Later results
(82, 83) included the maintenance of Glaucoma scintiUans without con-
jugation for almost 700 generations. The ciliates remained healthy so
long as fresh medium was supplied; the use of old medium induced
depressions. At this point, Enriques suggested that exhaustion of the in-
vestigators patience is a more important factor than senescence of the
ciliates in such investigations.

On May 1, 1907, Woodruff started the line of Paratnecium aurelia
which was to deal a more serious blow to the physiological life-cycle. In
May, 1908, the strain had passed 490 generations (246), and at the end
of four years (247), had survived for 2,121 generations without conjuga-
tion. By this time, the evidence indicated that P. aurelia might reproduce
indefinitely without conjugation, or else that the "cycle" must be longer
than that of any ciliate investigated previously.

The conclusion suggested by Woodruff's strain of P. aurelia did not
remain unchallenged. Calkins and Gregory (37) maintained that some
strains of Paramecium are conjugating races while others are non-con-
jugating, and it was argued that Woodruff's strain was a non-conjugating
race which should not be compared with the conjugating strain of
Calkins. Woodruff (248) met this objection by reporting conjugation in
mass-cultures started from his strain at the end of 4,102 generations. The
completion of 25 years without conjugation was reported in 1932 (249).

Evidence against the physiological cycle gradually accumulated from

Reproduction and Life-Cycles 97

other sources. Glaucoma scintillans showed no senescence after 2,700
generations (84). Lines of Paramecium caudatum lived for ten years
without conjugation or decrease in vitality (178, 179). The colonial flagel-
late, Eudorina elegans, was maintained for eight years without syngamy
or indications of senescence (110, 111). Actinophrys sol passed more than
1,200 generations without syngamy (19). Spathidium spathula, previously
credited with a cycle, survived for a thousand generations without con-
jugation or endomixis (252). Didinium nasutum was maintained by Beers
without conjugation or a decrease in vitality so long as the food supply
was adequate (8). However, depressions were readily induced by an
inadequate diet (9).

In contrast to various other ciliates, Uroleptus mobilis failed to follow
the prevailing pattern. Instead, a physiological cycle was reported, with
the strains living an average of 350 generations (34, 35). Attempts to
prolong the cycle by varying the environmental conditions were unsuc-
cessful (2), and this species remains one in which the cycle has not been
eliminated. More recently, Jennings (127, 131) concluded that his experi-
ence with Paramecium bursaria also supports the concept of a physiologi-
cal cycle, although some clones were maintained for eight years before
their health began to decline.

In spite of the fact that strains of various ciliates could be grown in
the laboratory for long periods without conjugation — and perhaps they
could be maintained indefinitely — one question remained unanswered.
Does conjugation really have any stimulatory or rejuvenating effect on
ciliates?

The early investigations had produced little information. A few ob-
servations by Hertwig (114) on Dileptus gigas and some inconclusive data
cited by Calkins (33) represented the available evidence. Some years later,
the first convincing experiments were reported by Calkins (34). Several
strains of Uroleptus mobilis, which were entering depressions, showed a
higher fission-rate and greater longevity after conjugation than the non-
conjugant parental stocks. Comparable effects of conjugation were re-
ported subsequently in Spathidium spathula (253) and Paramecium
bursaria (131).

At present it seems clear that conjugation, whether or not it is essential,
can produce a physiological stimulation in at least certain strains. How-
ever, it is equally evident that conjugation is no universal remedy for
senescent ciliates. In fact, the odds are slightly against survival after con-
jugation in Paramecium bursaria. Records kept for 20,478 exconjugants
show that under conditions in which all non-conjugant lines remained
vigorous, 29.7 per cent of the conjugating ciliates died before the first
post-conjugant fission, and only 47.3 per cent survived for more than four
fissions (127). Conjugation between inbred lines is even more dangerous,
and mortality often reaches 90-100 per cent in such cases in P. bursaria

98 Reproduction and Life-Cycles

(129). Conjugation between old stocks which are not closely related also
may be almost 100 per cent lethal, although unrelated young stocks may
show little or no mortality after conjugation (148).

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Reproduction and Life-Cycles 101

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Ill

The Classification of Protozoa

Taxonomy prior to 1900 Prospective sources of taxonomic data

Taxonomic systems of the twentieth cen- The identification of Protozoa
^"■^y Literature cited

HE CLASSIFICATION of Protozoa scrvcs various useful purposes
in addition to furnishing a system for filing species in appropriate cata-
logs. A sound taxonomy favors progress in comparative morphology and
physiology since it facilitates correlation of the information bearing on
related organisms. The projection of experimental and observational data
on a taxonomic background also is helpful in planning investigations to
extend or limit the application of preliminary findings. In fact, without
some knowledge of taxonomic relationships, the choice of material for
certain types of research would be analogous to "wildcat" drilling for oil.
Although a certain amount of "wildcatting" is always needed, the orderly
development of a field often depends extensively upon systematically
directed efforts. As more is learned about the interrelationships of Pro-
tozoa, the benefits derived from the field of taxonomy will become increas-
ingly important.

A major aim of taxonomy^ is the assignment of organisms to species
and larger groups on the basis of degree of kinship. If the available data
are extensive enough and have been interpreted correctly, such a tax-
onomic system not only indicates degrees of relationship among existing
species, but also furnishes sound clues to phylogenetic relationships. Un-
fortunately, this taxonomic ideal has not yet been realized for the Phylum
Protozoa as a whole.

The limitations of current systems are numerous. In the first place,
the boundaries of the phylum are subject to debate, particularly in the
case of phytoflagellates. In studying the Phytomastigophorea and their

^ General problems of zoological classification and conventional taxonomic procedures
have been reviewed in a compact monograph by Caiman (6).

103

104 The Classification of Protozoa

relatives, the taxonomist encounters organisms which range from typical
flagellates (of which many are apochlorotic and some are holozoic) to
filamentous algae with temporary flagellate stages. In assigning algal
flagellates to the Phylum Protozoa and leaving their close relatives with
the botanists, protozoologists obviously have made arbitrary decisions
which are more indicative of taxonomic convenience than of biological
relationships. The dual taxonomic role of the slime-molds as Sarcodina
and Fungi indicates another point at which the boundaries of the Phylum
Protozoa are obscure. Comparable uncertainty exists at the lower levels
of protozoan taxonomy, and there are instances in which orders appar-
ently overlap to such a degree that the exact positions of certain genera
are still uncertain. In modern taxonomic practice, it is no novelty for a
particular genus or family to be moved from one subphylum, class, or
order to another. Old orders have sometimes disappeared completely, in
suppressions or amalgamations, and new orders have been carved out of
older groups. The continued erection of new genera and species is paral-
leled to some extent by the suppression of old names. In other words, a
certain amount of taxonomic confusion extends throughout much of the
Phylum Protozoa. This confusion does not indicate chaos. Instead, it is
the result of continued activity in a field still seriously handicapped by
the lack of adequate information.

TAXONOMY PRIOR TO 1900

Although current classifications leave much room for improvement,
there has been tren;iendous progress since Gesner described one of the
Foraminiferida as a mollusc in 1565. Protozoa apparently were first sep-
arated from other animalcules in 1752, when John Hill placed some of
them in his group of Gymnia (animalcules without external organs). In
1786, O. F. Miiller (17) erected the Infusoria (including about 150 species
of Protozoa) as a subdivision of the worms, and divided the group into
species with, and those without, visible locomotor organelles.

Ehrenberg's (10) more extensive monograph included descriptions of
about 350 species from original observations, but an important part of
his taxonomic system was based upon a liberal interpretation of the
Infusoria as complete organisms. On the basis of feeding experiments with
pigments, Ehrenberg concluded that a digestive system is characteristic
of ciliates. "Polygastric" types were believed to have a mouth, oesophagus,
many stomachs, a spiral intestine, an anus, and possibly a pancreas. The
Infusoria were separated into Anentera (without a digestive tract) and
Enter odela (with a digestive tract). The Anentera were subdivided into
Gymnia (no visible appendages), including about 30 genera of flagellates;
Pseudopoda (with pseudopodia), including Amoeba, Arcella, and certain
Suctorea; and Epitricha (with cilia), including a few ciliates and several
dinoflagellates. Additional ciliates were placed in the Enterodela which

The Classification of Protozoa 105

were subdivided, on the basis of number and position of openings to the
supposed digestive tract, into Anopisthia (with one terminal opening),
Enantiatreta (with an opening at each end of the body), Allotreta (with
a lateral opening), and Catotreta (with a ventral opening).

Ehrenberg's basic misinterpretation of protozoan morphology was soon
corrected by Dujardin (9) who reached the conclusion that Infusoria are
simple organisms composed of a fundamental living substance, sarcode.
Repetition of Ehrenberg's feeding experiments indicated that the sup-
posedly fixed stomachs of ciliates are merely food vacuoles. Dujardin
divided the Infusoria into Asymmetrica and Symmetrica. The former
included species without visible locomotor organelles (bacteria), those
with pseudopodia (mostly Sarcodina, in the modern sense), those with
flagella, and those with cilia (about 50 genera of ciliates). The Sym.metrica
included the ciliate genus Coleps.

In 1845, von Siebold (20) redefined the "Protozoa," in which Goldfuss
(11) had included certain coelenterates with the "Infusoria," and char-
acterized them as unicellular animals. Although such a characterization
is inadequate by modern standards, von Siebold's definition served a
useful purpose in stressing morphological differences between Protozoa
and higher animals. The Protozoa now included the Class Infusoria — the
Astoma, without a mouth {Opalina and the flagellates), and the Stoma-
toda, with a mouth (about 30 genera of ciliates) — and the Class Rhizo-
poda with (pseudopodia).

Further investigation brought more recruits to the Protozoa. In 1845,
von Kolliker concluded that gregarines are Protozoa instead of trema-
todes, and this interpretation was supported by Stein in 1848. Increased
interest in these organisms finally led to Leuckart's erection of the
Sporozoa in 1879. Preliminary observations of Meyen, and the extensive
work of Huxley on Thalassicolla led J. Muller, in 1858, to establish the
Radiolaria as a subdivision of the Rhizopoda. The group Ciliata was set
up by Perty in 1852; the Flagellata, by Cohn in 1853; and extensive in-
vestigations on both groups were reported by Claparede and Lachmann
in 1858-1861. By the time Stein's (21) monograph was completed, the
Flagellata were divided into 15 families, some of which are now con-
sidered orders; the Ciliata, into the orders Holotricha, Heterotricha,
Hypotricha, and Peritricha. Stein's classification of ciliates on the basis
of distribution of cilia has been carried on, with modifications, into later
systems.

Contemporary contributions included Haeckel's separation of the Heli-
ozoa from the Radiolaria, erection of the Mastigophora by Diesing,
the Sporozoa by Leuckart, the Myxosporidia and the Dinoflagellata by
Biitschli, and the Sarcosporidia by Balbiani. As a result, the classification
of Protozoa began to resemble more modern systems.

Kent's monograph (14) covered the following groups:

106 The Classification of Protozoa

Class 1. Rhizopoda

Order 6. Choano-Flagellata

Order 1. Amoebina

Order 7. Spongida

Order 2. Gregarinida

Order 8. Flagellata-F.ustomata

Order 3. Arcellinida

Order 9. Cilio-Flagellata

Order 4. Foraminifera

Class 3. Ciliata

Order 5. Labyrinthulida

Order 1. Holotricha

Order 6. Radiolaria

Order 2. Heterotricha

Class 2. Flagellata

Order 3. Hypotricha

Order 1. Mycetozoa

Order 4. Peritricha

Order 2. Trypanosomata

Class 4. Tentaculifera

Order 3. Rhizo-Flagellata

Order 1. Actinaria

Order 4. Radio-Flagellata

Order 2. Suctoria

Order 5. Flagellata-Pantostomata

Kent's system differed from more recent ones in several respects— assign-
ment of the Mycetozoa and the Spongida (sponges) to the Flagellata;
inclusion of the gregarines in the Rhizopoda; recognition of a Class Ten-
taculifera to include the Suctoria and Actinaria.

Biitschli (2) recognized the Class Sporozoa, although some of the mod-
ern Coccidia were grouped with gregarines. The Microsporidia were listed
as an appendix to the Sporozoa, with exact relationships to be determined.
Among the Mastigophora, the Euglenoidina included several of the
modern Chloromonadida, while such types as Bodo (now in the Proto-
mastigida) and Ejitosiphon (one of the Euglenida) were assigned to the
Heteromastigoda. Butschli's Isomastigoda included the Chrysomonadida,
Cryptomonadida and Phytomonadida of current systems, as well as cer-
tain Polymastigida and the dinoflagellate Oxyrrhis. The Trichonym-
phidae (now in the Hypermastigida) were listed as an appendix to the
ciliates.

Class 1. Sarkodina
Subclass 1. Rhizopoda
Order 1. Rhizopoda
Suborder 1. Amoebaea
Suborder 2. Testacea
Suborder 3. Perforata
Subclass 2. Heliozoa
Subclass 3. Radiolaria
Class 2. Sporozoa

Subclass 1. Gregarinida
Order 1. Monocystidea
Order 2. Polycystidea
Subclass 2. Myxosporidia
Subclass 3. Sarcosporidia
Class 3. Mastigophora
Order 1. Flagellata
Suborder 1. Monadina
Suborder 2. Euglenoidina

TAXONOMIC SYSTEMS OF THE
TWENTIETH CENTURY

Suborder 3. Heteromastigoda
Suborder 4. Isomastigoda
Order 2. Choanoflagellata
Order 3. Dinoflagellata
Suborder 1. Adinida
Suborder 2. Dinifera
Order 4. Cystoflagellata
Class 4. Infusoria
Subclass 1. Ciliata

Order 1. Gymnostomata
Order 2. Trichostomata
Suborder 1. Aspirotricha
Suborder 2. Spirotricha
Section 1. Heterotricha
Section 2. Oligotricha
Section 3. Hypotricha
Section 4. Peritricha
Subclass 2. Suctoria

The system proposed by Calkins (3) showed several changes. The
Suborder Perforata (Foraminifera) became the Order Reticulariida. The

The Classification of Protozoa 107

silicoflagellates, now considered a subdivision of the Chrysomonadida,
appeared as a separate order. The Order Phytoflagellida included groups
now separated as the Orders Phytomonadida and Chloromonadida. The
gregarines, coccidians, and haemosporidians were assigned to separate
orders in the Telosporidia.

Class 1. Sarcodina

Subclass 1. Rhizopoda
Order 1. Amoebida
Suborder 1. Gymnamoebina
Suborder 2. Thecanioebina
Order 2. Reticular! ida
Suborder 1. Nuda
Suborder 2. Imperforina
Suborder 3. Perforina
Suborder 4. Tinoporinae
Subclass 2. Heliozoa

Order 1. Aphrothoracida
Order 2. Chlamydophorida
Order 3. Chalarathoracida
Order 4. Desmothoracida
Subclass 3. Radiolaria
(20 orders)
Class 2. Mastigophora
Subclass 1. Flagellida
Order 1. Monadida
Order 2. Choanoflagellida
Order 3. Heteromastigida
Order 4. Polymastigida
Order 5. Euglenida
Order 6. Phytoflagellida
Suborder 1. Chloromonadina
Suborder 2. Chronionadina

Suborder 3. Chlamydomonadina
Suborder 4. Volvocina
Order 7. Silicoflagellida
Subclass 2. Dinoflagellida
Order 1. Adinida
Order 2. Dinoferida
Order 3. Polydinida
Subclass 3. Cystoflagellidia
Class 3. Sporozoa

Subclass 1. Telosporidia
Order 1. Grcgarinida
Order 2. Coccidia
Order 3. Hacmosporidiida
Subclass 2. Neosporidia
Order 1. Myxosporidiida
Order 2. Sarcosporidiida
Class 4. Infusoria
Subclass 1. Ciliata

Order 1. Holotrichida

Suborder 1. Gymuostomina
Suborder 2. Trichostomlna
Order 2. Heterotrichida
Suborder 1. Poly trichina
Suborder 2. Oligotrichina
Order 3. Hypotrichida
Order 4. Peritrichida
Subclass 2. Suctoria

The system of Dofiein (7) differed in several respects from that of
Calkins. The phylum was divided into two subphyla, Plasmodroma and
Ciliophora. The "Infusoria" disappeared, the Ciliata and Suctoria being
advanced to classes of Ciliophora. In addition, the Foraminifera and
Mycetozoa were recognized as orders of the Rhizopoda, and the Tricho-
nymphidae were listed as an appendix to the Mastigophora.

Subphylum 1. Plasmodroma
Class 1. Rhizopoda
Order 1. Amoebina
Order 2. Heliozoa
Order 3. Radiolaria
Order 4. Foraminifera
Order 5. Mycetozoa
Class 2. Mastigophora
Subclass 1. Flagellata
Order 1. Protomonadina
Order 2. Polymastigina
Order 3. Euglenoidina
Order 4. Chromomonadina

Order 5,
Subclass 2.

Order 1.

Order 2.

Subclass 3.

Class 3. Spor

Subclass I.

Order 1

Order 2
Subclass 2.

Order 1.

Order 2.
Subphylum 2.

, Phytomonadina

Dinoflagellata

Adinida

Dinifera

Cystoflagellata
ozoa

Telosporidia
. Coccidiomorpha
. Gregarinida
, Neosporidia

Cnidosporidia

Sarcosporidia
Ciliophora

108 The Classification of Protozoa

Class 1. Ciliata

Order 1. Holotricha
Order 2. Heterotricha
Order 3. Oligotricha

Order 4. Hypotricha
Order 5. Peritricha
Class 2. Suctoria

Hartmann (12) recognized five orders of Neosporidia among the Spo-
rozoa. To the Subclass Flagellata, was added the Order Binucleata to in-
clude some of the Trypanosomidae and Haemosporidia as supposedly
binucleate organisms. Since the binucleate nature of these organisms has
never been established (21), the Order Binucleata has not been accepted
by later workers.

Subphylum 1. Plasmodroma
Class 1. Rhizopoda

Order 1. Amoebina

Order 2. Mycetozoa

Order 3. Foraminifera

Order 4. Heliozoa

Order 5. Radiolaria
Class 2. Mastigophora

Subclass I. Flagellata
Order 1. Protomonadina
Order 2. Polymastigina
Order 3. Binucleata
Order 4. Euglenoidea
Order 5. Chromomonadina
Order 6. Phytomonadina

Subclass 2. Dinoflagellata
Order 1. Adinida
Order 2. Dinifera

Subclass 3. Cystoflagellata

Class 3. Telosporidia
Order 1. Coccidia
Order 2. Gregarinida

Class 4. Neosporidia
Order 1. Myxosporidia
Order 2. Microsporidia
Order 3. Sarcosporidia
Order 4. Actinomyxidia
Order 5. Haplosporidia
Subphylum 2. Ciliophora

Class 1. Ciliata

Order 1. Holotricha
Order 2. Heterotricha
Order 3. Oligotricha
Order 4. Hypotricha
Order 5. Peritricha

Class 2. Suctoria

In the system of Minchin (16) the subphyla Plasmodroma and Cilio-
phora were dropped and the Class Infusoria restored. The Heliozoa and
Radiolaria were recognized as subdivisions of the Actinopoda. In the
Ciliata, erection of the Sections Aspirigera and Spirigera stressed dif-
ferences in adoral ciliation.

Class 1. Mastigophora
Subclass 1. Flagellata
Order 1. Pantastomatina
Order 2. Protomonadina
Order 3. Polymastigina
Order 4. Euglenoidina
Order 5. Chromomonadina
Suborder 1. Chrysomonadina
Suborder 2. Cryptomonadina
Order 6. Phytomonadina
Subclass 2. Dinoflagellata
Order 1. Adinidia
Order 2. Dinifera
Subclass 3. Cystoflagellata
Class 2. Sarcodina

Subclass 1. Rhizopoda
Order 1. Amoebaea

Suborder 1. Reticulosa
Suborder 2. Lobosa
Order 2. Foraminifera
Order 3. Xenophyophora
Order 4. Mycetozoa
Subclass 2. Actinopoda
Order 1. Heliozoa
Order 2. Radiolaria
Class 3. Sporozoa

Subclass 1. Telosporidia
Order 1. Gregarinoidea
Suborder 1. Eugregarinae
Suborder 2. Schizogregarinae
Order 2. Coccidia
Order 3. Haemosporidia
Subclass 2. Neosporidia
Division 1. Cnidosporidia

The Classification of Protozoa 109

Order 1. Myxosporidia
Order 2. Actinomyxidia
Order 3. Microsporidia
Order 4. Sarcosporidia

Division 2. Haplosporidia
Order 1. Haplosporidia
Class 4. Infusoria
Subclass 1. Ciliata

Section 1. Aspirigera
Order I. Holotricha

Suborder 1. Astomata
Suborder 2. Gymnostomata
Suborder 3. Hymenostomata
Section 2. Spirigera

Order 1. Heterotricha
Suborder 1. Polytricha
Suborder 2. Oligotricha

Order 2. Hypotricha

Order 3. Peritricha
Subclass 2. Acinetaria (Suctoria)

In 1926 two new systems, proposed by Calkins (4) and Wenyon (23),
reflected several diff^erences of opinion in treatment of the Sarcodina,
Mastigophora, and Sporozoa. Wenyon's separation of the Cnidosporidia
from other Sporozoa as a group of equal rank apparently represents a
more realistic appraisal than that reflected in most classifications. In both
systems, the Chrysomonadida and Cryptomonadida appeared as separate
orders, and the Chloromonadida also in that of Calkins. ^Venyon's trans-
fer of the Cystoflagellata to the Zoomastigina is not generally favored. In
the Ciliata, Wenyon followed Minchin in stressing differences in ciliature
of the Holotrichida and the other orders. Wenyon retained the subphyla
Plasmodroma and Ciliophora, whereas Calkins advanced the Mastigo-
phora, Sarcodina, Sporozoa, and Infusoria to subphyla.

The system of Calkins (4):

Subphylum 1. Mastigophora

Class 1. Phytomastigoda
Order 1. Chrysomonadida
Order 2. Cryptomonadida
Order 3. Dinoflagellida
Order 4. Phyloinonadida
Order 5. Euglcnida
Order 6. Chloromonadida

Class 2. Zoomastigoda
Order 1. Pantastomatida
Order 2. Protomastigida
Order 3. Polymastigida
Order 4. Hypermastigida
Subphylum 2. Sarcodina

Class 1. Actinopoda
Subclass 1. Heliozoa
Subclass 2. Radiolaria

Class 2. Rhizopoda
Subclass 1. Proteomyxa
Subclass 2. Mycetozoa

The system of Wenyon (23):

Subclass 3. Foraminifera
Subclass 4. Amoebaea
Subphvium 3. Infusoria
Class 1. Ciliata

Order 1. Holotrichida
Order 2. Hcttrotrichida
Order 3. Oligotrichida
Order 4. Hypotrichida
Order 5. Peritrichida
Class 2. Suctoria
Subphylum 4. Sporozoa
Class 1. Telosporidia
Subclass 1. Gregarinida
Subclass 2. Coccidiomorpha
Order 1. Coccidia
Order 2. Haemosporidia
Class 2. Neosporidia

Subclass 1. Cnidosporidia
Subclass 2. Sarcosporidia

Subphylum 1. Plasmodroma

Class 1. Rhizopoda

Order I. Amoebida

Order 2. Heliozoa

Order 3. Radiolaria

Order 4. Foraminifera
Order 5. Mycetozoa
Class 2. Mastigophora

Subclass 1. Phytomastigina
Order 1. Chrysomonadina

no The Classification of Protozoa

Order 2. Chryptomonadina
Order 3. Dinoflagellata
Order 4. Euglenoidida
Order 5. Phytomonadida

Subclass 2. Zoomastigina
Order 1. Protomonadida
Order 2. Hypermastigida
Order 3. Cystoflagellata
Order 4. Diplomonadida
Order 5. Polymonadida
Class 3. Cnidosporidia

Order 1. Myxosporidiida

Order 2. Microsporidia
Class 4. Sporozoa

Subclass 1. Coccidiomorpha
Order 1. Coccidiida
Order 2. Adeleida

Subclass 2. Gregarinina
Order 1. Schizogregarinida
Order 2. Eugregarinida
Subphylum 2. Ciliophora
Group 1. Protociliata

Class 1. Opalinata
Group 2. Euciliata
Class 1. Ciliata

Subclass 1. Aspirigera

Order 1. Holotrichida
Subclass 2. Spirigera

Order 1. Heterotrichida
Order 2. Oligotrichida
Order 3. Hypotrichida
Order 4. Peritrichida
Class 2. Suctoria

In the later system of Doflein and Reichenow (8) the Heterochlorida
were added to the orders of Mastigophora, although the Phytomastigoda
(Phytomastigina) and Zoomastigoda (Zoomastigina) were not recognized
as subclasses. Addition of the Testacea increased the orders of Rhizopoda
to six. Several new groups of ciliates were recognized and the Order Spiro-
tricha was rescued, with modifications, from Biitschli's (2) system.

Subphylum 1. Plasmodroma
Class 1. Mastigophora

Order 1. Chrysomonadina
Order 2. Heterochloridina
Order 3. Cryptomonadina
Order 4. Dinoflagellata
Order 5. Euglenoidina
Order 6. Chloromonadina
Order 7. Phytomonadina
Order 8. Polymastigina
Order 9. Rhizomastigina
Class 2. Rhizopoda
Order 1. Amoebina
Order 2. Testacea
Order 3. Foraminifera
Order 4. Heliozoa
Order 5. Radiolaria
Order 6. Mycetozoa
Class 3. Sporozoa

Subclass 1. Telosporidia
Order 1. Gregarinae

Order 2. Coccidia
Order 3. Haemosporidia
Subclass 2. Cnidosporidia
Order 1. Myxosporidia
Order 2. Microsporidia
Subclass 3. Sarcosporidia
Subclass 4. Haplosporidia
Subphylum 2. Ciliophora
Class 1. Ciliata

Subclass 1. Protociliata
Subclass 2. Euciliata
Order 1. Holotricha
Order 2. Spirotricha

Suborder I. Heterotricha
Suborder 2. Oligotricha
Suborder 3. Entodiniomorpha
Suborder 4. Ctenostomata
Suborder 5. Hypotricha
Order 3. Peritricha
Order 4. Chonotricha
Class 2. Suctoria

The system of Kudo (15) suggested progressive changes in treatment
of the Sporozoa.

Subphylum 1. Plasmodroma
Class 1. Mastigophora

Subclass 1. Phytomastigina
Order 1. Chrysomonadida

Order 2. Cryptomonadida
Order 3. Dinoflagellida
Order 4. Phytomonadida
Order 5. Euglenoidida

The Classification of Protozoa HI

Order 6. Chloromonadida

Subclass 2. Zoomastigina
Order 1. Pantastomatida
Order 2. Protomonadida
Order 3. Polymastigida
Order 4. Hypermastigida
Class 2. Sarcodina

Subclass 1. Rhizopoda
Order 1. Proteomyxa
Order 2. Mycetozoa
Order 3. Foraminifera
Order 4. Amoebaea
Order 5. Testacea

Subclass 2. Actinopoda
Order I. Heliozoa
Order 2. Radiolaria
Class 3. Sporozoa

Subclass 1. Telosporidia
Order 1. Coccidia
Order 2. Haemosporidia

Order 3. Giegarinida
Subclass 2. Cnidosporidia
Order 1. Myxosporidia
Order 2. Actinomyxidia
Order 3. Microsporidia
Order 4. Helicosporidia
Subclass 3. Acnidosporidia
Order 1. Sarcosporidia
Order 2. Haplosporidia
Subphylum 2. Ciliophora
Class 1. Ciliata

Subclass 1. Protociliata
Subclass 2. Euciliata
Order 1. Holotrichida
Order 2. Heterotrichida
Order 3. Oligotrichida
Order 4. Hypotrichida
Order 5. Peritrichida
Class 2. Suctoria

In a later classification Calkins (5) omitted the Phytomastigophora, as
a group, from the Mastigophora, However, the Peranemidae, a family of
Euglenida, was transferred to the Protomonadida to contain Peranema
and several related genera. Other Peranemidae (such as Heteronema,
Anisonema, Dinema, and Entosiphon) were placed in the family Bodoni-
dae of the Protomonadida. The Mastigophora were divided into two
classes, Protomastigota (the Order Protomonadida) and Metamastigota.
The opalinid ciliates were reduced from a separate subclass (Protociliata)
to a family in the Astomida.

Subphylum 1. Mastigophora
Class 1. Protomastigota

Older 1. Protomonadida
Class 2. Metamastigota
Order 1. Hypermastigida
Order 2. Polymastigida

Suborder 1. Monokaryomastigina
Suborder 2. Diplokaryomastigina
Suborder 3. Polykaryomastigina
Subphylum 2. Sarcodina
Class 1. Actinopoda
Subclass 1. Heliozoa
Subclass 2. Radiolaria
Class 2. Rhizopoda

Subclass 1. Proteomyxa
Subclass 2. Mycetozoa
Subclass 3. Foraminifera
Subclass 4. Amoebaea
Order 1. Amoebida
Order 2. Testacea
Subphylum 3. Infusoria
Class 1. Ciliata

Subclass 1. Holotricha
Order 1. Astomida
Order g. Gymnostoniida

Subclass 2. Spirotricha
Order 1. Heterotrichida
Order 2. Oligotrichida
Order 3. Ctenostomida
Order 4. Hypotrichida
Subclass 3. Peritricha
Subclass 4. Chonotricha
Class 2. Suctoria
Subphylum 4. Sporozoa
Class 1. Telosporidia
Subclass 1. Gregarinina
Order 1. Eugregarinida
Order 2. Schizogregarinida
Subclass 2. Coccidiomorpha
Order 1. Coccidiida
Suborder 1. Eimeriina
Suborder 2. Haemosporidiina
Suborder 3. Babesiina
Order 2. Adeleida
Class 2. Cnidosporidia
Order 1. Myxosporidia
Order 2. Actinomyxidia
Order 3. Microsporidia
Class 3. Acnidosporidia

112 The Classification of Protozoa

In 1936 a list of subdivisions of the Protozoa, as generally favored
by a number of American protozoologists, was prepared for the American
Association for the Advancement of Science (18). This list of names, with
their authors, illustrates the multiple origins of current systems.

Phylum Protozoa Goldfuss 1820 em. von Siebold 1845
Subphylum 1. Plasmodroma Dofiein 1901
Class 1. Mastigophoia Diesing 18(55.

Subclass 1. Phytomastigophora Calkins 1909
Order 1. Chrysomonadida Stein 1878
Order 2. Heterochlorida Pascher 1912
Order 3. Cryptomonadida Stein 1878
Order 4. Dinoflagellida Butschli 1885
Order 5. Euglenida Blochmann 1895
Order 6. Chloromonadida Klebs 1892
Order 7. Phytomonadida Blochmann 1895
Subclass 2. Zoomastigophora Calkins 1909
Order 1. Pantastomatida Minchin 1912
Order 2. Protomastigida Klebs 1893
Order 3. Polymastigida Klebs 1893
Order 4. Hypermastigida Grassi 1911
Class 2. Sarcodina Hertwig and Lesser 1874 em. Biitschli 1880
Subclass 1. Rhizopoda von Siebold 1845

Order 1. Amoebida ClaparMe and Lachmann 1858
Order 2. Proteomyxa Lankester 1885
Order 3. Testacea Schultze 1854
Order 4. Foraminifera d'Orbigny 1826
Order 5. Mycetozoa de Bary 1859
Subclass 2. Actinopoda Calkins 1909
Order 1. Heliozoa Haeckel 1866
Order 2. Radiolaria Haeckel 1866
Class 3. Sporozoa Leuckart 1879

Subclass 1. Telosporidia Schaudinn 1900
Order 1. Gregarinida Lankester 1866
Order 2. Coccidiomorpha Doflein 1901
Suborder 1. Coccidia Leuckart 1879
Suborder 2. Haemosporidia Danilewsky 1886
Subclass 2. Cnidosporidia Dofiein 1901
Order 1. Myxosporidia Biitschli 1881
Order 2. Actinomyxidia Stole 1899
Order 3. Microsporidia Balbiani 1883
Subclass 3. Sarcosporidia Balbiani 1882
Order 1. Sarcosporidia Balbiani 1882
Order 2. Globidia Badudieri 1932
Subclass 4. Haplosporidia Caullery and Mesnil 1899
Subphylum 2. Ciliophora Doflein 1901
Class 1. Ciliata Perty 1852

Subclass 1. Protociliata Metcalf 1918

Order 1. Opalinata Stein 1867
Subclass 2. Euciliata Metcalf 1918
Order 1. Holotrichida Stein 1859

Suborder 1. Gymnostomina Biitschli 1889
Suborder 2. Trichostomina Biitschli 1889
Suborder 3. Astomina Minchin 1912
Order 2. Spirotrichida Biitschli 1889
Suborder 1. Heterotrichina Stein 1859

The Classification of Protozoa 113

Suborder 2. Oligotrichina Biitschli 1887

Suborder 3. Tintinnoina Claparede and Lachmann 1858

Suborder 4. Entodinioinorphina Reichenow 1929

Suborder 5. Hypotrichina Stein 1859
Order 3. Peritrichida Stein 1859
Order 4. Chonotrichida ^^'alIengren 1896
Class 2. Suctoria ClaparMe and Lachmann 1858

Pearse's report (18), in which the preceding names were listed,
strongly advocated adoption of the following endings for names of tax-
onomic groups: phylum, -a; subphylum, -a; class, -ea; subclass, -ia; order,
-ida; suborder, -ina. The obvious advantages of such imiformity, both to
professional taxonomists and to students, far outweigh any potential re-
strictions on creative license in formulating new taxonomic names. This
system of uniform spelling has been adopted in one recent classification
(13), and will be adhered to in the following chapters on taxonomy of the
Protozoa.

The writer will follow the system outlined below; this is similar to the
classification adopted by Jahn and Jahn (13).

Subphylum 1. Mastigophora

Class 1. Phytomastigophorea
Order 1. Chrysomonadida
Order 2. Heterochlorida
Order 3. Cryptomonadida
Order 4. Dinoflagellida
Order 5. Phytomonadida
Order 6. Euglenida
Order 7. Chloromonadida

Class 2. Zoomastigophorea
Order 1. Rhizomastigida
Order 2. Protomastigida
Order 3. Polymastigida
Order 4. Trichoraonadida
Order 5. Hypermastigida
Subphylum 2. Sarcodina

Class 1. Actinopodea
Order 1. Helioflagellida
Order 2. Heliozoida
Order 3. Radiolarida

Class 2. Rhizopodea
Order 1. Proteomyxida
Order 2. Mycetozoida
Order 3. Amoebida
Order 4. Testacida
Order 5. Foraminiferida
Subphylum 3. Sporozoa

Class 1. Telosporidea
Subclass I. Gregarinidia
Order 1. Eugregarinida
Order 2. Schizogregarinida
Subclass 2. Coccidia

Subclass 3. Haemosporidia
Class 2. Cnidosporidea
Order 1. Myxosporida
Order 2. Actinomyxida
Order 3. Microsporida
Order 4. Helicosporida
Class 3. Acnidosporidea
Subclass 1. Sarcosporidia
Subclass 2. Haplosporidia
Subphylum 4. Ciliophora
Class 1. Ciliatea

Subclass 1. Protociliatia

Order 1. Opalinida
Subclass 2. Euciliatia
Order 1. Holotrichida
Suborder 1. Astomina
Suborder 2. Gymnostomina
Suborder 3. Trichostomina
Suborder 4. Hymenostomina
Suborder 5. Thigmotrichina
Suborder 6. Aposloniina
Order 2. Spirotrichida

Suborder 1. Heterotrichina
Suborder 2. Tintinnina
Suborder 3. Oligotrichina
Suborder 4. Eiitodiniomorphina
Suborder 5. Hypotrichina
Suborder 6. Ctenostomina
Order 3. Peritrichida
Order 4. Chonotrichida
Class 2. Suctorea

114 The Classification of Protozoa

PROSPECTIVE SOURCES OF
TAXONOMIC DATA

As will be noted in Chapters 4-7, there are still many taxonomic
areas in which inadequate information makes disagreements unavoidable.
Since it becomes increasingly evident that superficial characteristics form
an inadequate foundation for a natural classification of Protozoa, present
differences of opinion cannot be reconciled completely until more is
known about the morphology, biochemistry, physiology, and life-cycles
of many species. Therefore, future progress will depend largely upon the
contributions of specialists working in different fields. Such details as
the finer structure of flagella, the organization of ciliary patterns and
peristomial areas in ciliates, distribution of the various types of chloro-
phyll and other pigments in flagellates, the composition of stored foods,
the structure of endoplasmic organelles, the organization of nuclei, and
the basic details of mitosis should all contribute to the development of
a less imperfect taxonomic system. The bearing of biochemical data on
taxonomic questions may prove to be very important. The determination
of minimal food requirements and the analysis of synthetic potentialities,
which are possible for species established in chemically defined bacteria-
free media, may yield clues to relationships now obscured by morphologi-
cal specializations. Taxonomists may even become concerned with such
matters as comparative data on digestive enzymes. For instance, the ob-
servation that Amoeba proteus (Chaos diffluens) and Pelomyxa caroUnen-
sis (Chaos chaos) are similar in their content of peptidase and catheptic
proteinase and are both quite different from Pelomyxa palustris (1), is
especially interesting in view of the disputes concerning their generic
status. And finally, a more thorough analysis of life-cycles is probably
essential for the satisfactory classification of various genera and families
whose taxonomic status is uncertain at present.

THE IDENTIFICATION OF
PROTOZOA

In beginning a study of the Protozoa, the student is often interested
in identifying species as they are encountered in the laboratory. Un-
fortunately, such identifications are not always easy, and are occasionally
impossible with the more readily available library facilities. There is no
comprehensive determinative manual for the Protozoa as a whole. Nor
is there available a complete manual for any of the four major groups
of Protozoa. As a result, the identification of a particular species some-
times becomes a problem for the specialist with extensive knowledge of
a certain taxonomic group. In some cases, as pointed out by Pringsheim
(19), the establishment of pure-line cultures from single organisms may
be a desirable, or even an essential step.

However, the existence of such difficulties does not mean that the
student should consider the task of identification a hopeless one. Many

The Classification of Protozoa 115

of the better known species are described recognizably in general taxa-
nomic works that are widely accessible. In addition, there are increasing
numbers of monographs dealing with single genera or families. It is only
in the areas not adequately covered by general monographs and not yet
touched by special surveys, that the protozoologist encounters major diffi-
culties. In such cases, identification of a species may involve a laborious
search through isolated and sometimes numerous papers dealing with
members of the genus in question.

For those who are beginning to cultivate an acquaintance with the
Protozoa, an illustrated key written by Jahn and Jahn (13) will prove
to be very helpful. The authors have explained the use of taxonomic keys
and have included instructive discussions of the criteria to be considered
in identifying members of the major groups. This key also will be useful
to the advanced student who has not specialized in taxonomy of the
Protozoa. For species not listed by Jahn and Jahn, more extensive taxo-
nomic works must be consulted. A number of these special monographs
are listed in Chapters IV-VII.

LITERATURE CITED

1. Andresen, N. and H. Holter 1949. Science 110: 114.

2. Biitschli, O. 1880-1889. "Protozoa" in Bronn's Klassen und Ordnungen des Thier-

reiclis (Leipzig).

3. Calkins, G. N. 1901. The Protozoa (New York: Columbia Press).

4. 1926. The Biology of the Protozoa (Philadelphia: Lea & Febiger).

5. 1933. The Biology of the Protozoa, 2d ed. (Philadelphia: Lea & Febiger).

6. Caiman, W. T. 1949. The Classification of Animals: an Introduction to Zoological

Taxonomy (New York: J. Wiley & Sons).

7. Doflein, F. 1902. Arch. f. Protistenk. 2: 169.

8. and E. Reichenow 1927-1929. Lehrbuch der Protozoenkunde (Jena: G.

Fischer).

9. Dujardin, F. 1841. Histoire naturelle des zoophytes (Paris).

10. Ehrenberg, C. G. 1838. Die Infusionsthierchen als volkommene Organismen

(Leipzig).

11. Goldfuss, G. A. 1820. Handbuch der Zoologie (Niirnberg).

12. Hartmann. M. 1907. Arch. f. Protistenk. 10: 139.

13. Jahn, T. L. and F. F. Jahn 1949. How to Knoiv the Protozoa (Dubuque: W. C.

Brown Co.).

14. Kent, W. S. 1880-1882. A Manual of the Infusoria; including a description of all

known flagellate, ciliate and tentaculiferous Protozoa, British and foreign, and
an account of the organization and affinities of the sponges (London).

15. Kudo, R. R. 1931. Handbook of Protozoology (Springfield: Thomas).

16. Minchin, E. A. 1912. An Introduction to the Study of the Protozoa (London:

Arnold).

17. Miiller, O. F. 1786. Animalcula infusoria fiuviatilia et marina (Havniae et Leipzig).

18. Pearse, A. S. 1936. Zoological Names. A List of Phyla, Classes and Orders (Durham:

Duke University Press).

19. Pringsheim, E. G. 1949. Pure Cultures of Algae. Their Preparation and Maintenance

(Cambridge).

20. Siebold, C. T. E. and H. Stannius v. 1845. Lehrbuch der vergleichende Anatomic,

H. 1.

21. Stein, S. N. F. v. 1859-1883. Der Organismus der Infusionsthiere (Leipzig).

22. Swezy, O. 1916. Univ. Calif. Publ. Zool. 16: 185.

23. Wenyon, C. M, 1926. Protozoology (London: Balli^re, Tindall & Cox),

The Mastigophora

Class 1. Phytomastigophorea
Order 1. Chrysomonadida

Suborder 1. Euchrysomonadina
Family I. Chromulinidae
Family 2. Syncryptidae
Family 3. Ochromonadidae
Family 4. Prymnesiidae
Suborder 2. Silicoflagellina
Suborder 3. Coccolithina
Suborder 4. Rhizochrysodina
Family 1. Rhizochrysidae
Family 2. Myxochrysidae
Suborder 5. Chrysocapsina
Family 1. Chrysocapsidae
Family 2. Celloniellidae
Family 3. Hydruridae
Family 4. Nageliellidae
Order 2. Heterochlorida

Suborder 1. Euheterochlorina
Suborder 2. Rhizochloridina
Suborder 3. Heterocapsina
Order 3. Cryptomonadida
Family 1. Cryptochrysidae
Family 2. Cryptomonadidae
Family 3. Nephroselmidae
Order 4. Dinoflagellida
Suborder 1. Prorocentrina
Suborder 2. Gymnodinina
Family 1. Protonoctilucidae
Family 2. Gymnodiniidae
Family 3. Polykrikidae
Family 4. Noctilucidae
Suborder 3. Peridinina
Family 1. Glenodiniidae
Family 2. Gonyaulacidae
Family 3. Peridiniidae
Family 4. Ceratiidae
Family 5. Dinophysidae
Family G. Heterodiniidae
Suborder 4. Dinocapsina
Suborder 5. Dinococcina
Family 1. Phytodiniidae
Family 2. Blastodiniidae
Family 3. Ellobiopsidae
Order 5. Phytomouadida
Family 1. Polyblepharidae

Family 2. Chlamydomonadidae
Family 3. Haematococcidae
Family 4. Phacotidae
Family 5. Spondylomoridae
Family 6. \'olvocidae

Order 6. Euglenida

Suboider 1. Euglenoidina
Suborder 2. Peranemoidina
Suborder 3. Petalomonadoidina

Order 7. Chloromonadida

Class 2. Zoomastigophorea
Order 1. Rhizomastigida
Order 2. Proiomastigida
Family 1. Codosigidae
Family 2. Phalansteriidae
Family 3. Trypanosomidae
Family 4. Cryptobiidae
Family 5. Amphimonadidae
Family 6. Bodonidae
Order 3. Polymastigida
Family 1. Trimastigidae
Family 2. Tetramitidae
Family 3. Streblomastigidae
Family 4. Retortomonadidae
Family 5. Callimastigidae
Family 6. Polymastigidae
Family 7. Pyrsonymphidae
Family 8. Hexamitidae
Order 4. Trichomonadida

Family 1. Monocercomonadidae
Family 2. Dcvescovinidae
Family 3. Calonymphidae
Family 4. Trichomonadidae
Order 5. Hypermastigida
Suborder 1. Lophomonadina
Family 1. Lophomonadidae
Family 2. Joeniidae
Family 3. Kofoidiidae
Suborder 2. Trichonymphina
Family I. Hoplonymphidae
Family 2. Staurojoeninidae
Family 3. Holomastigotidae
Family 4. Trichonymphidae
Family 5. Teratonymphidae

Literature cited

116

The Mastigophora 117

T„

HE Mastigophora possess flagella at some stage of the life-
cycle, although many develop pseudopodia and show amoeboid activity.
The group may be divided into two classes, Phytomastigophorea and
Zoomastigophorea.

CLASS 1. PHYTOMASTIGOPHOREA

The phytoflagellates range from typical plants to forms whose
affinities with animals are more apparent, and some genera have even
occupied positions in both the Phytomastigophorea and the Zoomasti-
gophorea in different systematic treatises. The majority possess chroma-
tophores which contain chlorophyll, although the green color may be
masked to some extent by other pigments. The rest of the phytoflagellates
are colorless. Some differ from their pigmented homologues mainly in the
lack of chromatophores, and in certain instances, both colorless and pig-
mented species belong to the same genus. At the other extreme, certain
predominantly holozoic species have developed new organelles which
assist in feeding.

Life-cycles may involve dimorphism, sometimes with alternation of
amoeboid and flagellate stages, or flagellate and palmella stages. Sexual
phenomena are well known in Phytomonadida and have been reported
occasionally in certain other orders (Chapter II).

The Phytomastigophorea^ may be divided into the following orders:

(1) Chrysumonadida: usually one or two flagella, sometimes three; typ-
ically with one or two, biu sometimes more chromatophores ranging from
golden-yellow to greenish-yellow or brown; a few genera lack chromato-
phores: no cytopharynx or "reservoir" is present; the cyst wall is typically
siliceous and contains a pore; encystment is endogenous; stored reserves
include leucosin and lipids, but no starch; many species are naked, some
secrete a lorica or test, others are enclosed in a membrane to which silice-
ous scales or calcareous elements (coccoliths) are added; the majority are
solitary, but some genera develop arboroid or spheroid colonies.

(2) Heterochlorida: typically naked, with two unequal flagella; one
to a dozen or more chromatophores, pale yellow-green, or sometimes pale
yellow; no cytopharynx; reserves include leucosin and lipids, but no
starch; the cyst wall, which may contain two layers, lacks a pore; encyst-
ment is endogenous, as in Chrysomonadida.

(3) Cryptomonadido: biflagellate; pellicle usually restricts changes in

^ From the botanical standpoint, the Class Phytomastigophorea is a somewhat artifi-
cial arrangement of certain algal groups. As considered in the present chapter, the
Chrysomonadida represent part of the algal Class Chrysophyceae, the Phytomonadida
correspond to the Order Volvocales of the Class Chlorophyceae, and the Dinoflagellida
to the Class Dinophyceae. A modern discussion of the phytoflagellates as algae has been
published by Smith (260).

118 The Mastigophora

form of the body, which often shows dorso- ventral differentiation; some
genera have an open ventral "pharyngeal" groove; in others the groove is
closed, posteriorly or throughout its length, to form a pouch; refractile
granules ("trichocysts") usually lie just beneath the wall of the groove
or pouch; there may be a single bilobed chromatophore or two or more
chromatophores which are usually brown, less commonly red, blue, blue-
green, or green; starch and lipids are stored.

(4) Dinoflagellida: biflagellate forms, typically with two grooves, a
transverse girdle and a longitudinal sulcus in the body wall or theca; one
of the flagella typically lies in the girdle; chromatophores, when present,
are usually golden-brown to dark-brown, sometimes green or bluish-green;
starch and lipids are stored.

(5) Phytomonadida: except in one family, there is a distinct membrane
of cellulose or pectins, or a test impregnated with calcium or iron salts;
usually two or four, sometimes eight flagella; there is often a single cup-
shaped chromatophore; one or more pyrenoids are usually present; chloro-
phyll typically is not masked by other pigments; starch and lipids are
stored; red haematochrome accumulates in some species.

(6) Euglenida: relatively large forms, usually with one or two flagella
arising from an anterior reservoir ("gullet"); pellicle may be flexible or
relatively rigid; green chromatophores, usually numerous and equipped
with pyrenoids; reserves include paramylum and lipids; some species
accumulate red haematochrome.

(7) Chloromonodida: typically biflagellate, one flagellum trailing; the
body is often flattened dorso-ventrally, with a shallow groove on the
ventral surface; presence of a cytopharynx, reported for some species, has
been denied (232); chromatophores, when present, are typically numerous
and grass-green (or "meadow-green"); no stigma is reported; lipids are
stored.

Order 1. Chrysomonadida

This group, represented by fossils from Upper Cretaceous to recent
deposits, is widely distributed in salt, brackish and fresh water. Although
chromatophores occur in the majority, colorless holozoic species are com-
mon and there is a marked trend toward holozoic nutrition in many pig-
mented types. Formation of pseudopodia is fairly common. Some species
possess delicate pseudopodia which superficially resemble the myxopodia
of Foraminiferida and capture food in comparable fashion. Others form
lobopodia. Non-flagellated amoeboid and palmella stages are not unusual
and have become the dominant phase in some life-cycles. Most species
measure less than 50[j, and the majority probably less than half as much,
although some fossils exceed 100[x. One, two, or three flagella may be
present; if two, they may be equal or unequal in length. Mastigonemes

The Mastigophora H9

(pantonematic pattern) have been reported on the flagellum, or on one of
two flagella (200).

In the simpler species a thin periplast permits moderate amoeboid
activity. Cortical specialization has followed several trends: (1) the de-
position of a secreted layer just outside the periplast; (2) development
of a lorica (Fig. 4. 2, A, F) or a test (Fig. 4. 2, C, D); (3) development of

Fig. 4. 1. A. Ochromoyias granularis Doflein, showing nucleus and
stored lipids; chromatophore omitted; x2100 (after D.). B. Chromulina
annulata Conrad; ribbon-like chromatophore, mass of leucosin; x3000
(after C). C. Ochromonas reptans Conrad; two chromatophores, leucosin
granules; x2250 (after C). D. O. granularis, typical chromatophore, mod-
erate leucosin; x2025 (after Doflein). E. O. granularis, chromatophore dis-
placed by large mass of leucosin; xl875 (after Doflein). F. Chromulina
commutata Pascher, narrow chromatophore, leucosin granules; xl400
(after P.). G. Unusually large chromatophore in Ochromonas sp.; schematic
(after Pascher). H, I. Chrysapsis fenestrata Pascher, posterior and lateral
views of net-like chromatophore; x2100 (after P.). J. Ochromonas pinguis,
large chromatophore, peripheral zone of lipoid globules; x2500 (after
Conrad).

120 The Mastigophora

a siliceous skeleton (Fig. 4. 9). The simplest type of secreted covering is
represented by the layer of "mucus" in Monas (Fig. 4. 3, E, F). Secreted
membranes may be thin, or they may be quite thick as in some of the
Coccolithina (Fig. 4. 2, K). Siliceous scales (Fig. 4. 2, G) or calcareous
coccoliths (Fig. 4, 10) are added to the membrane in various genera. In

Fig. 4. 2. A. Dmobryon utriculus Stein, single loricate flagellate; x700
(after Pascher). B. Hyalobryon voigtii Lemmermann, a single flagellate
(colony shown in Fig. 1. 2, C); xlSOO (after Pascher). C. Cfnysococcus umbo-
natus with test; xl845 (after Conrad). D. Pseudokephyrion mmutissimum
Conrad, test only; x3000 (after C). E. Dinobryon stokesii Lemmermann,
single lorlca; x960 (after Pascher). F. Single lorica of Hyalobiyon lauterbornii
Lemmermann; x810 (after Pascher). G. Mallomonas dentata Conrad, chroma-
tophore, covering of siliceous scales (some bearing spines); x2500 (after C).
H. Stokesiella lepteca (Stokes) Lemmermann; xl045 (after S.). L Kephyrion
spirale (Lackey) Conrad, test only; x4500 (after C). J. Derepyxis amphora
Stokes; x880 (after S.). K. Syracosphaera mediterranea Lohmann, shell mem-
brane after dissolution of coccoliths in acid; single chromatophore; basal por-
tions of the two equal flagella; x2100 (after L.).

The Mastigophora 121

some cases the inorganic elements apparently are adherent to the "shell-
membrane"; in others, they are embedded in the membrane (46, 186).

Chromatophores (Fig. 4. 1, B-J) range from the network of Chrysapsis
fenestrata to a broad plate or a narrow ribbon. In addition to the usual
colors — golden-yellow to greenish-brown or brown — blue chromatophores
have been reported (173). Pigments include chlorophyll a, lutein (a
xanthophyll) and ^-carotene. Supposed pyrenoids have been noted in
some species but not in many others. A stigma may or may not be present
in chlorophyll-bearing forms. Species within a genus, such as Chromulina,

Fig. 4. 3. A-D. Ochromonas granulans (after Doflein). A. Specimen with
three food vacuoles, chromatophore, leucosin; xl650. B. Temporarilv at-
tached form just after ingestion of food; x2100. C. An amoeboid form just
after ingesting a bacillus; x2025. D. Nucleo-flagellar connections; x2100. E, F.
Monas vestita, during and after ingestion of food; note stigma and outer layer
of "mucus" with radiating strands; xl800 approx. (after Reynolds). G. Oiko-
monas termo (Ehrbg.) Kent, ingestion of a bacterium just completed; xl600
(after Lemmermann).

122 The Mastigophora

apparently may differ in this respect. Some colorless species (Fig. 4. 3, E)
also have a stigma. Scattered granules, similar in color to the stigma, have
been reported in Dinobryon, Mallomonas, and other genera (247).

Solid food is ingested by certain pigmented species as well as colorless
types (Fig. 4. 3), and ingestion often involves formation of a food-cup
in a particular region. Refractile granules of leucosin (Fig. 4. 1, B-F) and

Fig. 4. 4, Apochlorotic colonial types. A, B. Cladonema pauperum
Pascher; portion of colony and a single flagellate; schematic (after P.). C, D.
Codoiwdendrnn ocellatum Pascher; portion of the Diuobryon-\i\^e colony,
and a single flagellate showing stigma and ingested food; schematic (after
P.). E, F. Monadodendrnn distans Pascher; portion of a colony and a single
flagellate; schematic ('after P.).

globules of oil or fat (Fig. 4. 1, A) are stored. Leucosin is sometimes con-
sidered a polysaccharide but its chemical nature has not been determined.
Colonial organization is fairly common. Arboroid types include Hyalo-
bryon (Fig. 1. 2), Dinobryon, Codonodendron (Fig. 4. 4, C) and certain
other loricate genera and also such naked forms as M onadodendron (Fig.
4. 4, E) and Cladonema (Fig. 4. 4, A). Spheroid colonies are developed in
Synura (Fig. 4. 5, C), Cyclonexis (Fig. 4. 5, A, B), Syncrypta (Fig. 1. 2, F)
and ChrysosphaereJla (Fig. 4. 5, D), among others.

The Mastigophora 128

Life-cycles often include palmella or amoeboid stages. Species such as
Ochromnnas granularis (66) may become amoeboid (Fig. 4. 3, C) without
losing the fiagella. Amoeboid and flagellate phases occur in Chrysamoeba
radians (66) and Myxochrysis paradoxa (203); in the latter (Fig. 4. 6,
A-C), the amoeboid stage develops into a large plasmodium. A palmella is
dominant in life-cycles of the Chrysocapsina; an amoeboid phase, in the
Rhizochrysodina.

Endogenous formation of a siliceous cyst wall is characteristic (247). As

Fig. 4. 5. A, B. CycJonexis annularis Stokes, lateral and surface views; x720
(after S.). C. Synura uvella Ehrbg.; x310 (after Stein). D. Chrysosphaerella
longispina Laiiterborn; x540 (after L.).

encystment begins in Uroglena sonaica (Fig. 4. 7, H-J) the fiagella are
resorbed and the organism, packed with fat globules, becomes approxi-
mately spherical. Within the cytoplasm, a thin membrane is laid down.
This membrane gradually increases in thickness, a pore is differentiated,
and surface decorations are added. The development of a plug finally
closes the pore, separating the endocystic from the subsequently discarded
ectocystic protoplasm (50). The plug may or may not be siliceous in dif-
ferent species. In either case, the ping is either dislodged or dissolved in
excystment. Encystment in Ochromo7ias granularis (66) resembles that in
Uroglena. In certain other types, such as Chromulina (67), part or all

124 The Mastigophora

of the external cytoplasm is drawn into the cyst before the pore is plugged.
Binucleate cysts, described in Dinobryon divergens (Fig. 4. 7, D-F), appar-
ently are the result of nuclear division just before encystment (92). The
mature cyst (Fig. 4. 7) is approximately spherical, but the external appear-

Fig. 4.6. A-C. Myxochrysis pnradoxa; flagellate phase (B) and stages in
development of the plasniodiuni; xI6()0 (after Pascher). D-F. Kremastochrysis
pendens Pascher; non-flagellated forms suspended from the umbrella-like
float, and a flagellate stage; schematic (after P.) G. Formation of slender
pseudopodia in Dinobryon sertularia; diagrammatic (after Pascher). H. Gleo-
cystis-stage in D. sertularia; diagrainmatic (after Pascher).

ance varies with the presence or absence of surface decorations and a
collar around the pore.

Following suggestions of Pascher (214), five suborders may be recog-
nized: Euchrysomonadina, with a dominant flagellate stage; Silicoflagel-
lina, with a siliceous skeleton; Coccolithina, with a peripheral zone of
coccoliths; Rhizochrysodina, with a dominant amoeboid or plasmodial
stage; Chrysocapsina, with a dominant palmella.

The Mastigophora 125

Fig. 4. 7. A. Cyst of Cladonema pauper urn; diagrammatic (after
Pascher). B. Cvst of Ochromouas reptans; x2250 (after Conrad). C. Cyst of
Cellionella palensis; diagrammatic (after Pascher). D-F. Dinohryon diver-
gens; completion of nuclear division (D) is sometimes followed li\ encvst-
ment (E) to produce a binucleate cyst (F); xl2I0 (after Geitler). G. Cvst of
Ochromonas ludibunda: xl500 (after Conrad). H-J. Stages in development
of the cyst wall in Uragleiia snniara; diagrammatic (after Conrad).

Suborder 1. Euchrysomonadina. On the basis of flagellar equipm-nt
four families have been erected: Chromulinidae, with one flagelltun.
Syncryptidae, with two equal flagella; Ochromonadidae, with one long
and one short fiagellum; and Prymnesiidae, with three flagella.

Family 1. Chromulinidae. This group includes solitary and colonial
types. The type genus, Chromulina Cienkowski (67), contains small
naked flagellates with one band-like chromatophore or two smaller ones
(Fig. 4. 1, B, F). Amoeboid changes in form are observed in some species.

Solitary types without a lorica or test are assigned to Chromulina and several addi-
tional genera: Amphichrysis Korshikoff (165); Chrysapsis Pascher (202; Fig. 4. 1, H, I);

126 The Mastigophora

Clirysogleyia Wislouch (207); and the colorless Oikomonas Kent (181; Fig. 4. 3, G).
Cyrtophora Pascher and Pedinella AV^ysotzki contain stalked sessile forms (202). In
Epicysiis Pascher (211), there is an epiphytic non-flagellated phase and a Chromulina-
like stage. In Pyrainidochrysis Pascher, the firm membrane is decorated with three
longitudinal flanges, while that of Mihroglena Ehrenberg contains numerous granules
(202). These granules may be analogous to the cortical inclusions of Ochromonas
pingitis (Fig. 4. 1, J), or possibly represent primitive coccoliths.

Solitary loricate forms include: Bicoeca Clark, without chromatophores (181); Chryso-
coccocystis Conrad (47); LepochromuUna Scherffel (202); Histiona Voigt, colorless forms
with a stalked lorica (225); and Palatinella Lauterborn, with several slender pseudopodia
("tentacles") sunounding the flagellum (202).

A test (or "shell") is present in the following: Chrysococcus Klebs (174; Fig. 4. 2,
C), swimming types with a spheroid to ovoid test; Kepfiyrion Pascher (Fig. 4. 2, I),
tests with a recognizable neck (51).

Siliceous plates cover much or all of the body in Mallomouas Perty and Chryso-
sphaercUa Lauterborn. Mallomonas (Fig. 4. 2, G) includes about sixty species (45, 48),
differing in shape and arrangement of the scales, and in the presence or absence of
spines. Chrysosphaerella (Fig. 4. 5, D) includes .spheroid colonial forms. "Pseudomallo-
monas Chodat" apparently falls within the limits of the genus Mallomonas (48).

Loricate colonial types without chlorophyll are included in Codonodendron Pascher
(Fig. 4. 4, C, D) and Stephauocodon Pascher (224). In the latter, the simple four- or
eight-rayed colonies are formed by the adherence of loricae near their basal ends. Also,
Poteriodendron Stein may belong in this group (93).

Family 2. Syncryptidae. Two flagella of equal length are characteristic.
Syncrypta Ehrenberg (Fig. 1. 2, F) includes spherical colonies with the
flagellates embedded in a granular matrix (202). In Chlorodesmiis Phil-
lips (202), pairs of flagellates, adherent basally, are aligned in simple
band-like colonies. The cortex is decorated with spines, perhaps similar
to the siliceous scales of Synura. Derepyxis Stokes (Fig. 4. 2, J) includes
solitary loricate types (202, 207).

Family 3. Ochromonadidae. This group, like the Chromulinidae, in-
cludes both solitary and colonial forms. Ochromonas Wysotzki (Fig. 4. 1,
A, C, D-F) contains flagellates with a flexible periplast permitting changes
in shape and sometimes the formation of a temporary protoplasmic
"stalk" (Fig. 4. 3, B). A detailed cytological description is available for
Ochromonas graniilaris Doflein (66). The colorless homologue of Ochro-
monas, the genus Monas Miiller (including Sterromonas Kent and
Physomonas Kent) contains at least 13 species (243), some of which have
a stigma. The periplast of Monas vestita (Stokes) Reynolds is enclosed
in a mucous envelope from which radiate slender mucotis threads (Fig.
4. 3, E, F).

Additional solitary non-loricate types are assigned to the following genera: Ochry-
ostylon Pascher, usually sessile with, or sometimes without, a delicate stalk (222);
StomatocJione Pascher, colorless, usually sessile with a short protoplasmic stalk (222);
Kremastochrysis Pascher (Fig. 4. 6, D-F), with an Ochromoiias-Mke flagellate stage and
a dominant non-flagellated form attached to a float which suspends the organism from
the surface of the water (223).

Solitary loricate types are included in several genera. The lorica of the sessile
Epipyxis Ehrenberg resembles that in the colonial Dinobryon (Fig. 4. 2, A). A stalked
Dmobryon-type lorica is characteristic of Stylnpyxis Bolochonzew (202) and the color-

The Mastigophora 127

less Stokesiella (210; Fig. 4. 2, H). A cup-shaped to spheroid lorica bears a slender
stalk in Arthrochrysis Pascher (222) and the colorless Arthropyxis (222). The stalked
lorica of Poteriochromonas Scherffel (202) is funnel-shaped; that of Stenocodon Pascher
(222) is compressed laterally, with an oval mouth. The stalkless lorica of Pseudo-
kephyrion Pascher (Fig. 4. 2, D) — including "Kcphyriopsis Pascher and Ruttner" — is
cup-shaped.

Arboroid colonies of loricate flagellates arc included in Dinobryon Ehrenberg (1,
202; Fig. 4. 2, A) and its colorless homologue, Hyalobryon Lauterborn (202; Fig. 4. 2,
B). In the apochlorotic Codonobotrys Pascher (222). a cluster of individually stalked
loricate flagellates is attached to a heavy common stalk. Stylobrynn Fromentel (181),
another colorless type, probably belongs to the Ochromonadidae.

Non-loricate arboroid colonies are assigned to several genera. In Anthophysis Bory
and Ceplialothamnion Stein, both colorless genera, the flagellates are attached in
clusters at the ends of branching stalks (181). In the colorless Cladonema Kent em.
Pascher (Fig. 4. 4, A), MonadodendroJi Pascher (Fig. 4. 4, E, F), and Dendromonas
Stein (181), as well as in the pigmented Chrysodendron Pascher (207) which often
forms small colonies, the flagellates are attached singly to branches of the stalk.

More or less spherical colonies are characteristic of several genera. In Uroglena
Ehrenberg, including Uroglenopsis (181), the flagellates are embedded in a gelatinous
matrix (50). No matrix is evident in Cyclonexis Stokes (Fig. 4. 5, A, B), Skadovskiella
KorshikofE (163. 165), Synochromonas Korshikoff (165), or Synura Ehrenberg (165;
Fig. 4. 5, C).

Fig. 4. 8. Prymnesiidae. A. Prymnesium parvum Carter;
x2360 (after C). B, C. Platychrysis pigra Geitler, flagellate and
amoeboid stages; xl850 (after Carter). D. ChrysochromiiUna
parva Lackey; x3200 approx. (after L.).

Family •/. Prymnesiidae. These flagellates have three flagella and a
rather plastic body. Prymyiesiiwi Massart (Fig. 4. 8, A) has a short in-
active median flagellum and two long ones, and usually two yellow-green
to brown chromatophores. Platychrysis Geitler (Fig. 4. 8, B, C) shows both
amoeboid and flagellate stages. The flagella of the latter resemble those of
Prymnesium, but are coiled and apparently inactive in the amoeboid
stage (31). In Chrysochromulina Lackey (174; Fig. 4. 8, D) the median
flagellum is longer than the other two, which are usually trailed in
swimming.

128 The Mastigophora

An interesting occurrence of Prymnesium pai-viim has been reported
in brackish fish-ponds in Palestine. Populations of the flagellates reached
500,000/ml. or more, changing the color of the water to a yellowish-
brown and resulting in death of many fish (241).

Suborder 2. Silicoflagellina. These widely distributed marine flagellates
occur also as fossils from Upper Cretaceous to recent deposits. Their
taxonomic position was in doubt until Borgert (124) discovered the
flagellum and assigned them to a new group, the Silicoflagellata. Their

Fig. 4. 9. Silicoflagellina. A-H. Skeletons of various species (after De-
flandre). A. Dictyocha crux, x560. B. D. octonaria, x560. C. D. triacantha
Ehrbg. from Tertiary, x345. D. D. navinda Ehrbg. from Tertiary, x345.
E. Vallacerta hortoni Hanna from Upper Cretaceous, x540. F. Dictyocha
speculum Ehrbg. from Tertiary, x630. G. D. polyactis Ehrbg. from Ter-
tiary, x630. H. D. fibula Ehrbg., x560. I. D. speculum, showing nucleus,
chromatophores, and internal skeleton; xl320 (after Deflandre).

The Mastigophora 129

characteristic siliceous skeleton varies in complexity in difierent species
and has been interpreted as an external structure and as an internal
one (Fig. 4. 9, I) by different workers (63). A single flagellum, numerous
greenish-brown chromatophores, and stored granules of leucosin appear
to be typical.

Generic boundaries have been disputed to some extent. However,

^J^

Fig. 4. 10. Coccolithina. A. Discosphaera tubifer (Murray and Black-
man) Lohmann: schematic optical section showing rhabdoliths and two
chromatophores; x2800 (after L.). B. Single rhabdolith from Rhabdo-
sphaera claviger M. and B.; schematic (after Murray and Blackman). C.
Rhabdosphaera stylifer Lohmann, showing nucleus and two chromato-
phores; x2100 (after L.). D. Surface view of placolith from Coccolithus
wallichi; x2700 (after Lohmann). E. Optical section of coccolith from Coc-
colithus pelagica; schematic (after Murrav and Blackman). F. Coccolithus
ivallichi showing arrangement of coccoliths; xl800 (after Lohmann). G.
Hymenomonas roseola Stein; xT'jO (after Pascher). H. Longitudinal section
of rhabdolith from Discosphaera tubifer; schematic (after Lohmann). L
Syracosphaera pulchra. anterior coccoliths with spines; xl900 (after Loh-
mann).

130 The Mastigophora

Lyramula Hanna and Vallacerta Hanna (Fig. 4. 9, E) seem to be limited
to fossils from the Upper Cretaceous (101), while Dictyocha Ehrenberg
(Fig. 4. 9, AD, F-I) includes both living and fossil types. According to
Deflandre (63), three generic names — Cannophilus, Distephanus, and
Mesocena — have been applied to forms which fall within the genus
Dictyocha.

Suborder 3. Coccolithina. These flagellates occur mostly in salt and
brackish water; only a few are known from fresh water (46). Collections
in the Mediterranean (186) have yielded specimens from depths of 400
meters, but the flagellates are most abundant in the zone above 100
meters. Two flagella of equal length have been observed most frequently.
However, a few supposedly uniflagellate types have been reported; also
flagella have not yet been seen in some described species. Either two or
four chromatophores may be present (179). Little is known about the
life-cycles. Schiller (250) has noted in two species of Calyptrosphaera
stages which suggest fission within the theca, and he has pointed out
the need for study of the Coccolithina in cultures.

The diagnostic feature of the group is the possession of calcareous
coccoUths which may be deposited at the surface of, or embedded within,
a secreted membrane (Fig. 4. 2, K). Several types of coccoliths (186, 250)
are known. Solid platelets (discoliths), with or without spines, are found
in Syracosphaera (Fig. 4. 10, I), Pontosphaera, and related genera. Per-
forated coccoliths {tremaliths) are of various kinds. Elongated trema-
liths containing a long canal are known as rhabdoUths (Fig. 4. 10, A, B),
while simple perforated plates or double discs joined by a short canal
are called placoliths (Fig. 4. 10, D). Structme of the coccoliths has been
used as a basis for differentiating several families (127).

Representative genera include Acantltoica Lohinann (46, 250), Calyptrosphaera Loh-
mann (250), Coccolithus Schwarz (Fig. 4. 10, F), Deutschlandia Lohmann (250), Dis-
cosphaera Haeckel (250; Fig. 4. 10, A), Halopappus Lohmann (250), Hymenomonas
Stein (127, 174; Fig. 4. 10, G), Pontosphaera Lohmann (186, 260), Rhabciosphaera
Haeckel (250; Fig. 4. 10, C), Syracosphaera Lohmann (31, 186, 250), and Umbilicosphaera
Lohmann (186).

It is uncertain whether Hymenomonas actually belongs in this group. A pitted "shell"
has been reported in H. roseola Stein (174), although discrete coccoliths have been
described in H. dariubiensis (127).

Suborder 4. Rhizochrysodina. The amoeboid phase is dominant. In
the Rhizochrysidae, the amoeboid stages are solitary or else form loose
aggregates with pseudopodial attachments. Genera which develop true
Plasmodia are placed in the Myxochrysidae.

Family 1. Rhizochrysidae. Net-like aggregates of naked organisms (221)
are produced in Rhizochrysis Pascher (Fig. 4. 11, E) Chrysarachnion
Pascher (Fig. 4. 11, G), and the apochlorotic Lenkapsis Pascher. Similar
aggregates of thecate organisms (221) are formed in Heliapsis Pascher

The Mastigophora 131

7\ \ >v /

\:'-;\ \r:^>/ /,:^'Nvi V,^;?

^^^^i

^'T-

,^^*

w ..•>'^""

'•;

^ w

A ''

Fig. 4. 11. Rhizochrysodina. A. Heliapsis mutabilis Pascher, x550 approx.
(after P.). B. Cl>r\'sidiastntm catenatutti Lauterborn, x810 (after Pascher).
C. Heliochrysis erodians Pascher, xl380 approx. (after P.). D. H. sphagnicola
Pascher, parasitic stage; x610 approx. (after P.). E. Rhizorhrysis planktonica,
xl400 (after Pascher). F. Hetcrolag\uion oedogonii Pascher, x3300 (after P.).
G. Chrysarachnion insidians Pascher; diagrammatic (after P.). H. Lagynion
subovatiim Prescott and Croasdale; x665 (after P. &: C).

(Fig. 4. 11, A). Chrysidiastrum Lauterborn (Fig. 4. 11, B) shows a strong
tendency to produce chains instead of definite nets.

The family also contains a number of solitary types. Chrysamoeba Klebs inchidcs
naked amoeboid forms with a Chromulina-like flagellate stage, as in C. radians (66).
Thecate species are assigned to several genera. Hcterolagynion Pascher (Fig. 4. 11, F)
includes epiphytic forms, the lorica of which lacks a neck like that in Lagynion (201,
211; Fig. 4. 11, H). Eleuthcropyxis Scherffel (248), Plagiorhiza Vascher, Platytheca
Stein, Kybotion Pascher, and the colorless Leuknpyxis Pascher resemble Lagynion and
Hetcrolagynion in that pseudopodia emerge through a single opening in the lorica

132 The Mastigophora

(221). In Diporidion Pascher and Porostylon Pascher, there are two pores in the lorica,
which bears a stalk in the latter genus (220). In Heliochrysis Pascher (Fig. 4. II, C,
D), intracellular parasites of Sphagnum, as well as in the similar Heliaktis Pascher,
Chrysocriniis Pascher, and Stephanoporos Pascher. the spheroid to ovoid theca contains
a number of pores through which slender pseudopodia extend (220).

Family 2. Myxochrysidae. In the life-cycle of Myxochrysis Pascher
(203), a Chromuli)ia-\ike flagellate becomes an amoeboid stage which

Fig. 4. 12. Chrysocapsina. A-E. Celloniella pale?isis (after Pascher). A.
Branching palmella in flowing water, xlO. B. Bladder-like stage attached to
stones in dripping water. C. Tip of branch of palmella, highly magnified. D.
Amoeboid stage. E. Flagellate stage (C-E, diagrammatic). F. NagelieUa natam
Scherffel, x4o6 approx. (after S.).

develops into a plasmodium (Fig. 4. 6, A-C). The mature plasmodium
secretes a thick brownish membrane, within which many uninucleate
naked stages or cysts are produced. Cysts hatch into the flagellate or
amoeboid forms.

Suborder 5. Chrysocapsina. The dominant stage is a palmella which
may grow to a fairly large size in some genera.

Family 1. Chrysocapsidae. The organisms are distributed throughout
the matrix which is not highly differentiated, and fission may occur in
any region of the palmella. The matrix of Chrysocapsa Pascher (202) is

The Mastigophora 133

spheroid; that of Phaeoplaca Chodat (90), discoid; and the matrix of
Phaeosphaera West and West (202) is cylindrical and sometimes
branched.

Family 2. Celloniellidae. Fission does not occur throughout the pal-
mella. Instead, growth of the palmella depends upon fission in particular
groups of cells which produce new points of growth. In the sessile
Celloniella Pascher (Fig. 4. 12, A-E), the form of the palmella varies
with rate of flow of the water in which the mass is suspended (209).

Family 3. Hydruridae. Hy drums Agardh (152, 202) resembles Celloni-
ella, but the palmella is profusely branched and sometimes reaches a
length of 25-30 cm. Furthermore, fission apparently is limited to the
apical flagellates in each branch.

Family 4. NagelieUidae. Species of Nageliella Correns (202, 248),
usually epiphytic on algae, develop a somewhat discoid palmella from
the free surface of which extends a bundle of gelatinous filaments. In
N. natans (Fig. 4. 12, F) each filament contains an axial fibril which arises
from the apical end of each flagellate (248).

Order 2. Heterochlorida

These flagellates have a flexible periplast, typically two unequal
flagella, and one or more pale yellow-green or pale yellow chromato-
phores. The occurrence of pyrenoids is doubtful. Leucosin and lipids
are stored.

Endogenous encystment (Fig. 4. 13, A-D) is known in Chloromeson
and Myxochloris (216). Unlike that in Chrysomonadida, the cyst wall
is composed of two unequal valves and lacks a pore. In addition to the
encystment of uninucleate stages, an entire plasmodium of Myxochloris
sphagnicola may become enclosed in a membrane apparently containing
pectins (213).

On the basis of life-histories, the Heterochlorida may be divided into
three suborders: Euheterochlorina, with a dominant flagellate stage;
Rhizochloridina, with a dominant non-flagellated or plasmodial stage;
Heterocapsina, with a dominant palmella.

Suborder 1. Euheterochlorina. Representative types (Fig. 4. 13) in-
clude Chloromeson Pascher (31, 212, 216), Nephrochloris Geitler and
Gemisi (31), and Olithodiscus Carter (31).

Suborder 2. Rhizochloridina. Myxochloris Pascher (Fig. 4. 14, D-G),
Rhizochloris Pascher (Fig. 4. 14, A-C) and the loricate Stipitococcus
West and West (Fig. 4. 14, H) are included. The dominant stage in
Rhizochloris arachnoides is a small amoeba with slender pseudopodia
and a number of chromatophores. An amoeboid plasmodium also has
been observed. The flagellate stage apparently has only one flagellum.
The vegetative stage of Myxochloris sphagnicola (213) is a plasmodium

134 The Mastigophora

Fig. 4. 13. Euheterochlorina. AD. Chloromeson agile Pascher; successive
sta^^es in encystment; schematic (after P.). E, F. Nephrochloris salina Carter,
different aspects; E, x2430; F, x2250 (after C). G. Chloromeson parva Carter;
stigma, one chromatophore; x2360 (after C). H-J. Olithodiscus liiteus
Carter; dorsal, ventral and ventro-lateral views; xl340 (after Carter). K.
Cyst of O. hi tens; xl275 (after C).

endoparasitic in Sphagnum. Encystment of the plasmodium is followed
by division into smaller plasmodia or into uninucleate flagellate or amoe-
boid stages.

Suborder 3. Heterocapsina. A palmella is the dominant stage (218)
in C hlorosaccus L.uther, Gleochloris Pascher, and Malleodendroyi Pascher.

Order 3. Cryptomonadida

These typically biflagellate forms are widely distributed in fresh
water and fairly common in salt and brackish waters. Some marine types
have been reported as parasites (symbiotes?) in Radiolarida. A rather
constant body form, often with dorso-ventral differentiation, is charac-
teristic. A ventral groove, or "pharynx," is commonly present. The
"pharynx" of Chilomonas, which may represent the primitive condition,
is an open groove extending almost to the middle of the body (Fig. 4.

The Mastigophora 135

,ro

Fig. 4, 14. A-C. Rhizochloris arachnoidcs Carter (after C). A. Flagellate
stage, x2180. B. Amoeboid stage, x2080. C. Amoeboid stage, xl540. D-G. Myxo-
chloris spliagnicola, diagrammatic (after Pascher): flagellate stage (D), Plas-
modium endoparasitic in Sphagiiiim (E), developing cyst (F) and mature cyst
(G). H. Stipitococcus capense Prescott and Croasdale, x665 (after P. & C.).

15, L). In Cryptomonas (Fig. 4. 15, I-K) the posterior part of the pharynx
is closed ventrally to form a pouch, leaving the anterior portion an open
furrow. The wall of the pharynx and the groove is lined with refractile
granules ("trichocysts"), usually visible in the living organism. These
inclusions disappear in old cysts of Cryptomonas (110). The pharynx of
the holozoic genus Cyathomonas is a pouch extending posteriorly and
ventrally from the anterior end of the body, and partly encircled an-
teriorly by an incomplete ring of trichocysts.

One, two, or more chromatophores have been reported. The single
chromatophore of Cryptomonas (Fig. 4. 15, 1) and similar types is bilobed,
a condition interpreted occasionally as two separate chromatophores.
The chromatophore is usually brown, less commonly green, blue-green,
or red. Storage of starch and lipids is characteristic. The colorless Chilo-
monas Paramecium synthesizes amylopectin and ^-amylose (117), and
pectins have been reported in the endocyst in Cryptomonas (110).

Three families have been recognized: Cryptochrysidae, Cryptomo-
nadidae, and Nephroselmidae.

Family 1. Cryptochrysidae. The pharyngeal groove, along which rows

136 The Mastigophora

Fig. 4. 15. Cryptochrysidae and Cryptomonadidae. A. Chroomonas vectinsis
Carter, xl845 (after C). B. Cyst of Cryptomonas ovata, xl770 approx. (after
Hollande). C. Rhodomonas bnltica Karsten, xl440 (after Carter). D. Crypto-
chrysis commutata Pascher, xl250 (after P.). E. Rhodomoyias lacustris Pascher
and Ruttner, x2400 (after Pascher). F, G. Cryptochrysis atlantica Lackey, ven-
tral and lateral views; xl450 approx. (after L.). H. Chroomonas baltica
(Biittner) Carter, xl560 (after C). I. Cryptomonas similis Hollande, showing
gidlet, contractile vacuole, chromatophore and nucleus; diagiammatic (after
H.). J, K. Cryptomonas ovata, diagrammatic cross-sections anterior to, and at
the level of the nucleus; note gullet, "trichocysts" and chromatophore (after
Hollande.) L. Chilomonas Paramecium, showing "pharyngeal" groove and
contractile vacuole; diagrammatic (after Hollande).

of refractile granules are usually visible, is not closed ventrally. The
flagella arise near the anterior end of the groove.

Chlorophyll-bearing types include: Chroomonas Hansgirg (31, 202; Fig. 4. 15, A, H),
with one or two bluish chromatophores; Cryptochrysis Pascher (175, 202; Fig. 4. 15, D,

The Mastigophora 13'

F, G); Cyanomonas Oltmanns (173, 202), with several blue-green chromatophores);
Rhodomonas Karsten (31, 202; Fig. 4. 15, C, E), with reddish-brown chromatophores.
Chromatophores are lacking in Chilomonas Ehrenberg (110; 202; Fig. 4. 15, L).

Family 2. Cryptomonadidae. In these flagellates, the pharyngeal groove
is closed ventrally, through part or all of its length, to form a pouch.
The genus Cryptomonas Ehrenberg (110, 202; Fig. 4. 15, I-K) includes
chlorophyll-bearing forms; Cyathomonas Fromentel (110, 202), color-

a:)

Fig. 4. 16. A. Protochrysis phaeophycearum Pascher, xl500
(after P.). B. Cross-section of same. C. Nephroselmis olivacea Stein,
xl300 (after S.). D. Cross-section of jV. oUxmcea.

less holozoic types in which the pharyngeal pouch is a functional gullet.
Family 3. Nephroselmidae. As compared with the other families, the
Nephroselmidae show a modification of the primitive cryptomonad or-
ganization. If the origin of the flagella is considered anterior, these
flagellates have become shortened along the anterior-posterior axis and
correspondingly elongated along the transverse axis. The result is a
more or less bean-shaped body, with the pharyngeal-groove and bases of
the flagella lying near the equatorial plane. The two genera, Nephro-
selmis Stein (202; Fig. 4. 16, C, D) and Protochrysis Pascher (202; Fig. 4.
16, A, B) differ in shape of the body in cross-section.

Order 4. Dinoflagellida

This order includes many living species and a variety of fossil
types (75). Most dinofiagellates are marine, forming an important part
of the plankton. Under conditions not yet fidly understood, the popu-
lations of certain dinofiagellates in localized areas may increase tre-
mendously, sometimes to densities above 5,000,000 per liter. The result
is discoloration of the water — "red water" or "red tide" — and lumi-
nescence at night, and occasionally the death of fish in large num-
bers (42).

138 The Mastigophora

The flagellate stages of many species fall within the range, 10-200[ji,.
However, certain parasitic types grow to diameters of 600-700[jl, while
Noctiluca scintillans sometimes measures 1.0-1.5 mm. A girdle and a
sulcus are characteristic of flagellate stages (Fig. 4. 17, A). The girdle
(or annulus) is a groove which usually encircles the body in a descending,
or sometimes in an ascending, left-hand spiral, although the two ends
may meet in the same plane. In extreme cases, the girdle may trace more
than one complete spiral, or it may be rudimentary as in Protonocfiliiai

Fig. 4. 17. A. Gymnodinlum dorsum Kofoid and Swezy; pusules opening
into flagellar pores; x940 (after K. & S.). B. Cochliodinium lebourae Kofoid
and Swezy, spirally twisted sulcus; x525 (after K. & S.). C. Amphidiniopsis
kofoidi Woloszynska, dorsal view showing intercalary bands; x630 (after
W.). D. Gymnodinium racemosus .Cofoid and Swezy, showing chromato-
phores; x475 (after K. & S.). E. Protonoctiluca (Protoditiifer) tentaculatum
(K. & S.), showing tentacle arising from sulcus; x700 approx. (after Kofoid
and Swezy). F. Cross-section of apical horn, Ceratium hirundinella; diagram-
matic (after Entz). G. Erythropsis extrudens Kofoid and Swezy, showing
prod and outline of ocellus; x450 (after K. & S.). Key: b, intercalary band;
e, epicone; //?, flagellar pore; g, girdle; /;, hypocone; //, longitudinal flagel-
lum; o, ocellus; p, pusule; pr, prod; 5, sulcus; t, transverse flagellum; te,
tentacle.

The Mastigophora 139

(Fig. 4. 17, E). The epicone and hypocone, the anterior and posterior
regions of the body, are marked off by the girdle. The sulcus (longi-
tudinal furrow) is usually a straight groove intersecting the girdle, al-
though it may undergo spiral torsion (Fig. 4. 17, B), or may be expanded
into a "ventral area." From the sulcus arise the tentacle of Protodinifer
(Fig. 4. 17, E) and the prod of Erythropsis (Fig. 4. 17, G). The two
flagella of typical species also emerge through one or two flagellar pores
in the sulcus (Fig. 4. 17, A). The transverse flagellum is often ribbon-
like (81, 159). Occasionally, as in Peridinium, species within a genus
may differ in this respect (81).

A theca, composed of a cellulose-like substance and sometimes im-
pregnated with calcium salts, is present in many dinoflagellates. The
typical theca is composed of plates, the margins of which may be sepa-
rated by intercalary bands (Fig. 4. 17, C) in some species and particularly
in older specimens. The theca covering the epicone is known as the
epitheca; the posterior portion, as the hypotheca. The two are joined
by the girdle hand, composed of one or more girdle plates. The theca
may, as in Ceratium hirundinella (Fig. 4. 17, F), contain pores through
which extend cytoplasmic papillae.

There are commonly two vacuoles, or pusules (Fig. 4. 17, A), usually
containing a pink fluid. A slender c^jial extends directly from each
pusule to a flagellar pore or else joins a common canal which opens
externally. Intake of fluid has been observed in pusules of Peridinium
steini (155), and it has been suggested that a pusule may function as a
pharynx for intake of liquids and possibly solid particles (159).

The nucleus usually contains one or more nucleoli and many long
chromosomes whose beaded structure may persist through mitosis.
Chromatophores, present in many species, are often golden-brown to
dark-brown, although sometimes yellow, orange, green, or bluish-green.
In addition, various cytoplasmic pigments — either diffuse or forming
granules or globules — occur in many species (159). The known pigments
include chlorophyll a, chlorophyll r, peridinin, /^-carotene, dinoxanthin,
diadinoxanthin, and neodinoxanthin (Chapter I). Stored reserves in-
clude starch and lipids. A simple stigma, composed of red granules,
occurs in various fresh-water species. At the other extreme, the Pou-
chetiidae possess a complex ocellus (Fig. 1. 17, H, M) composed of a
lens and a mass of pigment.

The group shows a strong trend toward holozoic feeding, as indicated
by inclusions which are obviously ingested food in many species and
apparently such in others. In the chlorophyll-free Cochliodinium rosa-
ceum (159), Oxyrrhis marina (Fig. 4. 18, C), Noctiluca milaris, and
others, holozoic nutrition is undoubtedly important. Ingested food also
appears in various chlorophyll-bearing species of Gymnodinium, Gyro-
dinium, and Amphidinium (159). Furthermore, such thecate types as

140 The Mastigophora

Ceratium (106) may capture and presumably digest microorganisms
outside the theca by means of pseudopodial nets (Fig. 4. 18, A, B). These
pseudopodia apparently arise from cytoplasmic papillae extending
through pores in the theca (Fig. 4. 17, F).

The life-cycle may be apparently simple, or may show dimorphism or
polymorphism (159). Sexual phenomena have been reported in several
species (Chapter II). Fission is typically oblique (Fig. 2. 2, A-H) and,
in armored species, involves regeneration of different portions of the
theca by the daughter organisms. In contrast, as represented by certain
species of Glenodinium, fission may occur within the theca and the

'-nv%'^

Fig 4. 18. A, B. Capture of microorganisms by pseudopodial networks
in Ceratium 'hirundinella; x425 (after Hofender). C. Oxyrrhis marina, four
food vacuoles, nucleus in outline; x885 (after Hall).

daughter organisms are liberated as naked forms which later secrete a
theca. In other cases, each daughter organism develops a new theca
before it emerges from the parental one. Incomplete separation after
fission may result in chains (Fig. 4. 19, I), which are characteristic of
certain species but not of others. Reproductive cysts, known in a number
of species, may be more or less spherical ("pyrocystis" type) or some-
times crescentic ("crescent-cysts"). In Gymnodinium lunula (159), cres-
cent-cysts are developed within a pyrocystis-stage. Fresh-water species
may produce a thick-walled protective cyst, as in Ceratium hirundinella
(Fig. 2. 15, A). A palmella stage in which fission occurs is known in
some species, and is the dominant phase in Gleodinium (Fig. 4. 24, C,
D). In Amyloodinium ocellatum (Fig. 4. 19, A-C) the dominant phase

The Mastigophora 141

is a large ovoid stage, attached by means of a hold-fast to the gill-fila-
ments of a marine fish. At maturity, the parasite drops off the host,
the hold-fast is retracted, and the corresponding gap in the cellulose-
membrane is closed. Fission then results in many gymnodinioid flagel-
lates which seek a new host (26, 199).

The Dinoflagellida may be divided into five suborders: Prorocentrina,
with a bivalve theca but no distinct girdle or sulcus; Gymnodinina,

Fig. 4. 19. A-C. Amyloodinium ocellatum, X475 (after Nigrelli); para-
sitic stage (A), palmella stage after several fissions (B), and flagellate stage
(C). D, E. Exuviella perforata Gran, valve view and ventral view; xl230
approx. (after Lebour). F, G. Oxyrrhis marina Dujardin, ventral and dorsal
views; x875 (after Hall). H. Oxyrrhis teiitaculifera Conrad, ventral view
showing long tentacle; xl890 (after C.). I. Gymnodinium catenatum, chain
formation; x350 (after Graham).

142 The Mastigophora

athecate types with a girdle and sulcus; Peridinina, with a theca com-
posed of separate plates; Dinocapsina, with a dominant palmella and
a gymnodinioid flagellate stage; Dinococcina, in which the life-cycle may
include a dominant "pyrocystis" or crescent-cyst stage, a floating or at-
tached palmella, and a gymnodinioid flagellate stage.

Fig. 4. 20. A. Gyrodinium melo Kofoid and Swezy, x475 (after K. & S.).
B. Gyrodinium submarinum Kofoid and Swezy, x425 (after K. & S.). C.
Torodinium teredo Kofoid and Swezy, x300 (after K. & S.). D. Gymnodi-
nium dissimile Kofoid and Swezy, x475 (after K. & S.). E. Amphidinium
dentatum Kofoid and Swezy, x575 (after K. & S.). F. Cochliodinium pul-
chellum Lebour, x720 (after K. & S.). G. Polykrikos scfnvartzi Biitschli,
x250 (after K. & S.). H. Pavillardia tentaculijera Kofoid and Swezy, x475
(after K. & S.). I. Noctiluca scintiUans, x60 approx. (after K. & S.). Key:
/, longitudinal flagellum; p. oral pouch; t, tentacle.

Suborder 1. Prorocentrina. This group includes Exuviella Cienkowski
(178, 249, 252), Porella Schiller (252) and Prorocentrum Ehrenberg
(249, 252). Exuviella perforata (Fig. 4. 19, D, E) is a small marine flagel-
late with a thick bivalved theca. Each somewhat flattened valve is ap-
proximately circular in outline and shows a central conical invagination.
The flagella, emerging anteriorly through pores in one valve, show a
differentiation into longitudinal and transverse types. The two chroma-
tophores are yellowish-brown to yellow.

The Mastigophora 143

Suborder 2. Gymnodinina. These are the unarmored dinoflagellates
(159) which, except for some of the Gymnodiniidae, are limited to salt
water.

Family 1. ProtoJioctilucidae. The girdle and sulcus are rudimentary
and the transverse flagellum is not appreciably flattened. A tentacle is
characteristic. There are no chromatophores and the organisms are holo-
zoic. The family includes Oxyrrhis Dujardin (Fig. 4. 19, F-H) and Proto-
noctiluca Fabre-Domergue (Protodinifer Kofoid and Swezy). In the latter
(Fig. 4. 17, E), the shallow girdle extends about one-fourth the circum-
ference of the body, and the tentacle, arising from the sulcus, is more
pronounced than in Oxyrrhis. Only one pusule is present. The longitu-
dinal flagellum is vestigial in Protonoctiliica but is well developed in
Oxyrrhis.

Family 2. Gymnodiniidae. Both girdle and sulcus are well developed
and the transverse flagellum is typically flattened. Neither a tentacle nor
an ocellus is present. Some species lack chromatophores and a number
are holozoic. The family, represented in both fresh and salt water, in-
cludes the following genera: Amphidinium Claparede and Lachmann,
Cochliodiniuni Schiitt, Gyinnodiniiim Stein em. Kofoid and Swezy, Gyro-
diniiim Kofoid and Swezy {Spirodinium Schiitt), Massartia Conrad (10,
31, 44), and Torodininm Kofoid and Swezy (159).

The genera may be distinguished largely on the basis of their girdles. The girdle
forms one complete turn in Amphidinium, Gymnndinium, Massartia, and Torodinium.
In Amphidinium (Fig. 4. 20, E) the girdle is anterior, so that the epicone is small. The
girdle of Gymnodinium (Figs. 4. 17, D, 4. 20, D) lies nearer the equator and the ends
are displaced less than one-fifth the body length. Massartia differs from Gymnodinium
in having a larger and broader epicone. The girdle of Torodinium (Fig. 4. 20, C) is
posterior and the epicone is several times as long as the hypocone. Posteriorly, the
sulcus forms a half-turn around the body before intersecting the girdle. In Gyrodinium
(Fig. 4. 20, A, B) the girdle makes 1.0 to less than 1.5 turns and the ends are dis-
placed not less than one-fifth the body length. The girdle of Cochliodiniuni (Fig. 4.
20, F) makes 1.5 or more turns around the body.

Family 3. Polykrikidae. The single genus, Polykrikos Biitschli (Fig. 4.
20, G), contains permanent linear somatellae composed of two, four, or
eight zooids as a rule, although chains of sixteen have been observed,
Nematocysts are present. All species are marine.

Family 4. Noctihicidae. The diagnostic feature is a mobile tentacle
which arises in the sulcal area and extends posteriorly. The known spe-
cies are marine. Two genera, Noctihica Suriray and Pavillardia Kofoid
and Swezy, are assigned to the family. Pavillardia (Fig. 4, 20, H) shows a
body and girdle of the Gymnodinium-type, but a longitudinal flagellum
is absent and a tentacle arises from the posterior end of the sulcus. In
Noctihica (Fig. 4. 20, I), the mature organism is a highly vacuolated
spheroidal stage ranging from 200 to 2,000[j, in diameter. A short longitu-
dinal flagelJum is present. The girdle is vestigial, and the posterior por-

144 The Mastigophora

tion of the sulcus is expanded into a deep pouch extending to the base
of the tentacle.

Suborder 3. Peridinina. A theca composed of separate plates is charac-
teristic. Such features as relative size of the epicone and hypocone, extent
and torsion of the girdle and sulcus, and the number and arrangement
of thecal plates are important in taxonomy. Three small families differ
from the rest with respect to development of the girdle. In the Sino-
diniidae, the girdle is an irregular belt, instead of a groove, and shows

Fig. 4. 21. A-D. Glenodinium cinctum Ehrbg., x580 approx. (after Eddy);
ventral, apical, dorsal, and antapical views. E. Hemidinium nasiitum Stein,
ventral view showing girdle, sulcus, and thecal plates; schematic (after Wolo-
szynska). F. Palmella stage of H. nasutum; schematic (after Baumeister). G-J.
Diplopsalis lenticulata Bergh; x700 (after Lebour); ventral, lateral, antapi-
cal, and apical views. K, L. Heterodinium scrippsi Kofoid, dorsal and ventral
views; plates numbered; x350 (after K.).

The Mastigophora 145

no marginal ridges (lists), whereas the Lissodiniidae and Podolampidae
have undergone apparently complete suppression of the girdle (197).

The thecal plates are usually differentiated into several circular series
(Fig. 4. 21, K, L), and those in each series are conventionally numbered in
order, beginning at the left of the sulcal plane or the mid-ventral suture
(155). The apical plates (numbered V, 2', 3' . . .) extend to the apical
pore, or sometimes to a closing plate if the pore is closed. Anterior inter-
calary plates (la, 2a, . . .) lie between the apical and precingular plates.
Precingular plates (1", 2", . . .) extend from the apical or intercalary
plates to the girdle. The girdle plates (1, 2, 3 . . .) line the girdle. Post-
cingular plates {V", 2' ", . . .) lie in the hypotheca between the girdle
and the antapical plates (or posterior intercalary plates, if present). Pos-
terior intercalary plates (Ip, 2p, . . .) lie between the postcingular and
antapical plates. The a7itapical plates {\"",2"", . . .) cover the posterior
end. According to Kofoid's (155) system, the plate formula for Diplopsalis
lenticulata (Fig. 4. 21, G-J) would be written as 3'la6"5'"r'" (omitting
the girdle).

Fainily 1. Glenodiniidae. These flagellates have a thin theca, with plates
which are not easily distinguished, and were at one time assigned to the
Gymnodinina. Most species are known from fresh water (74, 254). Gleno-
dinium (Ehrbg.) Stein (Fig. 4. 21, A-D) differs from Glenodiniopsis
VVoloszynska (Fig. 4. 22, M-O) in number of postcingular plates and in
a sulcus limited mostly or entirely to the hypocone. In Hemidinium Stein
(9, 11; Fig. 4. 21, E), the girdle extends only about a half turn. A palmella
stage (Fig. 4. 21, F), resembling that of Gleodinium, has been reported for
H. nasutum (11).

Family 2. Gonyaulacidae. The thecal plates are distinct and one antapi-
cal plate is characteristic. Several species are known from fresh water (74,
254), but most are marine. Species of Gonyaiilax have attracted attention
as the source of mussel poisoning on the Pacific Coast (Chapter X) and
as a component of "red tide."

The family includes Chalubinskia Woloszynska (Fig. 4. 22, E-H), Dinosphaera Kofoid
and Michener (157), Diplopsalis Bergh (178), Entzia Lebour (178), and Gonyaulax
Diesing em. Kofoid (156). Gonyaulax (Fig. 1. 5, A, B) has a plate formula of
l-6'0-3a6"66' "Ipl" ", while Dinosphaera (Fig. 4. 22, A-D) has 5 postcingulars and no
posterior intercalary. Diplopsalis (Fig. 4. 21, G-J) has the plate formula, 3'la6"5' "1" ";
Entzia, 4'l-2a7"5' "1" ", but otherwise similar to Diplopsalis. Chalubinskia (Fig. 4. 22,
E-H) has 3 postcingular and 1 antapical plates.

Family 3. Peridiniidae. The thecal plates are distinct as in the Gonyaul-
acidae, but there are two antapical plates. Many species occur in fresh
water (74, 254); others are marine.

The family includes Peridiniurn Ehrenberg (64; Fig. 4. 22, I-K), Amphidiniopsis
Woloszynska (Figs. 4. 17, C, 4. 22, L), Glenodiniopsis Woloszynska (Fig. 4. 22, M-O),

146 The Mastigophora

Fig. 4. 22. AD. DiuospJmera palustris, ventral, dorsal, apical, and anta-
pical views; x575 approx. (after Eddy). E-H. Chalubinskia tatrica Wolo-
szynska, ventral, left lateral, apical, and antapical views; x675 (after W.).
I-K. Peridiniufn kulczynskii Wolosz)nska, \entral, apical, and antapical
views; x835 (after W.). L. Amphidiniopsis kofoidi Woloszynska, ventral view;
x630 (after W.). M-O. Glenodiniopsis steinii W'olos/ynska, ventral view;
x850 (after W.). P, Q. Sphaerodinium limneticuui Woloszynska, x800 ap-
prox. (after W.). R. Staszicella dinobryonis Woloszynska, x720 (after W.).

Sphaerodinium Woloszynska (Fig. 4. 22, P, Q), and Staszicella AVoloszynska (Fig. 4. 22,
R). Tfie epitheca is distinctly smaller than the hypotheca in Amphidiniopsis and
Staszicella. The two genera are distinguished by the sulcus, which extends to the apex
in Amphidiniopsis, but only a short distance into the epitheca in Staszicella.

Family 4. Ceratiidae. The epitheca is prolonged into an apical horn,
the hypotheca into two or three posterior horns (Fig. 4. 23, A). The genus
Ceratium Schrank is represented by many marine and several fresh-water
species (74, 80). Species differ in number of posterior horns, in form and
length of the horns, and in the sculpturing and detailed pattern of the
thecal plates. Of the posterior horns, the accessory may be vestigial or

The Mastigophora 147

Fig. 4. 23. A. Ceratium hirundinella O. F. M., ventral view; diagram-
matic (after Entz). B-D. Dinophysis diegensis Kofoid, ventral, dorsal, and
right lateral views; B, C, x445; D, x505 (after K.). E, F. Dolichodinmm
lineatum (Kofoid and Michener) Kofoid and Adamson, dorsal and ventral
views; x700 (after K. & A.). Key: g, gullet; s, sulcal area.

lacking, as in some strains of C. furcoides (Levander) Langhans; in addi-
tion, the antapical and postequatorial horns may be reduced in length,
as in C. brachyceros Daday. The apical horn shows differences in length
and is curved instead of straight in C. cornutiim Schrank and C. ciir-
virostre Kaas.

Family 5. Dinophysidae. The elongated body is laterally compressed,
with a minute epitheca, and the girdle is bordered by prominent flanges
("collars"). The theca consists of right and left valves, joined in a median
suture. Known species are marine. Dinophysis Ehrenberg (252; Fig. 4. 23,
B-D), Phalacronia Schiller (252) and Oxyphysis Kofoid are included in
the family.

Family 6. Heterodiniidae. The precingular ledge (or list) is well de-
veloped but the postcingular ledge is reduced or absent. A ventral pore
lies between the apical pore and the single flagellar pore. The plate
formula is 3-4'0-la6"6 6-7' "3"". The family includes Heterodinium
Kofoid (Fig. 4. 21, K, L) and Dolichodinium Kofoid and Adamson (Fig.
4. 23, E, F).

Suborder 4. Dinocapsina. A dominant palmella and a gymnodinioid

148 The Mastigophora

flagellate stage are the diagnostic features. In the palmella, a pectic sheath
may enclose the usual cellulose membrane. The suborder includes only
the family Gleodiniidae. The type genus is Gleodinium Klebs (132, 270;
Fig. 4. 24, C, D). Structure and division of the nucleus in G. montanum,
as noted in material from cultures (242), conform to the dinoflagellate

Fig. 4. 24. A, B. Cystoclnnum iners Gcitler, crescent-cysts containing one
and two organisms, the latter showing gymnodinioid featmes; x500 approx.
(after Thompson). C, D. Gleodinium montanittn Klebs. showing fission in
palmella stage; x625 approx. (after Thompson). E. Hypnodinium spliae-
ricum Klebs; stigma (beneath sulcus), chromatophores, large reddish oil
globule; xl95 approx. (after Thompson). F, G. Dinopodiella phaseolus
Pascher, sessile and flagellate stages; xl250 approx. (after P.). H, I. Tetra-
dinium javanicum Klebs, stalked and unstalked forms; schematic (after
Thompson). J, K. Phytodinedria procubans Pascher, sessile and flagellate
stages; xl250 approx. (after P.). L. Stylodinium sphaera Pascher, x940 ap-
prox. (afler P.).

The Mastigophora 149

pattern. Urococcus Kiitzing, in which the pahnella shows a very thick and
stratified sheath, has been referred to the family (259).

Suborder 5. Dinococcina. The dominant phase is a "pyrocystis" or a
"crescent-cyst" stage and the flagellate stages are typically gymnodinioid.
The non-flagellated stage, which may be floating or sessile, has a cellulose
membrane and is enclosed in a sheath composed of pectin.

Family 1. Phytodiniidae. This group, as the most typical family, shows
the characteristics of the suborder. A number of American species have
been described by Thompson (270).

The family includes: Cystodinedria Pascher (226); Cystodinium Klebs (11, 91, 205,
208, 225; Fig. 4. 24, A, B); Dinastridium Pascher (205); D'mopodiella Pascher (226; Fig.
4. 24, F, G); Dissodinium Klebs (205); Hylmodinhun Klebs (205; Fig. 4. 24, E); Phyto-
dinedria Pascher (226; Fig. 4. 24, J, K); Phytodinium Klebs (205); Rnciborskia Wolo-
szynska (219); Stylodinium Klebs (11, 205, 226; Fig. 4. 24, L); and Tetradinium Klebs
(91, 205; Fig. 4. 24, H, I). According to Baumeister (11), the flagellate stage of
"Stylodinium tarnuni" has a theca composed of discrete plates.

Fig. 4. 25. A-C. Blastodinium spiuulosum Chatton; undivided parasitic
stage (A), x210; trophocyte and four small sporocytes (B), x2I0; flagellate
stage (C), X1840 (after C). D-F. Haplozoon dogieli, x325 (after Shumway);
young parasite (D), trophocyte and gonocvte (E), gonocytes and sporocytes
(F). G. Flagellate stage of Haplozoon clymenellae, xl520 (after Shumway).
FI-L. Coccodinium duboscqi Chatton and Biecheler, parasitic in Peridinium
sp.; growth and nuclear division preceding merogony (H-K); g>mnodinioid
stage (L); schematic (after C. & B.).

150 The Mastigophora

Family 2. Blastodiniidae. This group (36) includes intestinal parasites
of copepods and sessile polychaetes; ectoparasites of copepods, annelids,
and salpids; intracellular parasites of Siphonophora, tintinnioid ciliates,
Radiolarida, and eggs of copepods; and parasites of the body cavity in
copepods. Amyloodinium (Fig. 4. 19, A-C) parasitizes the gills of marine
fish (26, 199), and Oodinium limneticum (118) has been described from
the same location in fresh-water fish. Chromatophores are present in some
Blastodiniidae and absent in others.

In a representative life-cycle (Fig. 4. 25, D-F), the young parasite divides
into two cells, a "trophocyte" and a "gonocyte." The latter undergoes a
number of divisions to produce "sporocytes" which develop into gymno-
dinioid flagellates. In the meantime, the trophocyte may divide into a
second gonocyte and a trophocyte. The second gonocyte produces another
generation of sporocytes, and the procedure may be repeated several
times. This pattern is not followed in Amyloodinium, which apparently
does not produce differentiated trophocytes and gonocytes.

The family includes Amyloodinium Hovasse and Brown (115; Fig. 4. 19, A-C),
Apodinium Chatton (36), Atelodinium Chatton (36), Blastodinium Chatton (36; Fig. 4.
25, A-C), Chytriodinium Chatton (36), Duboscquella Chatton (36), Endodinium Hovasse
(111), Haplozoon Dogiel (258; Fig. 4. 25, D-G), Merodinium Chatton (37) from Radio-
larida, Oodinium Chatton (36, 112), Paradinium Chatton (37), Protoodinium Hovasse
(112), Syndi7iium Chatton (36), and Trypanoditiium Chatton (36). Coccodiuium Chat-
ton and Biecheler (Fig. 4. 25, H-L), parasitic in other dinoflagellates, possibly should
be referred to this family.

Family 3. Ellohiopsidae. These ectoparasites of Crustacea resemble the
Blastodiniidae in their parasitic stages, but the known free-living stages
show no obvious relationships to dinoflagellates. Therefore, the tax-
onomic position of the group is uncertain. Ellobiopsis Caullery (32) is
the type genus.

Order 5. Phytomonadida

These flagellates are mostly ovoid to spherical, but various spindle-
shaped, hemispherical, flattened, and spirally twisted types are known.
Medusochloris phi ale, one of the more unusual forms, is a medusa-like
flagellate which swims mainly by contractions of the body (204). Except
in the Polyblepharidae, the body is enclosed in a distinct membrane, com-
posed at least partly of cellulose. In the Phacotidae the membrane is im-
pregnated with calcium salts to form a "shell." One to eight, but usually
two or four flagella are present. The flagella of membrane-covered species
may emerge through one opening or through individual flagellar pores
(Fig. 4. 26, A-C). In the first case the flagella may or may not arise from a
cytoplasmic papilla. Contractile vacuoles vary in number and position,
but there are often two near the bases of the flagella. A single large green
chromatophore is typical, although two or more smaller ones occur in

The Mastigophora 151

some species. The usual single chromatophore is cup-shaped (Fig. 4. 26,
B). However, lobed, "H-shaped," and other variations are known (Fig. 4.
26, D-J). The nucleus lies in the inner zone of cytoplasm. One or more
pyrenoids, typically spherical or ellipsoidal, but sometimes U-shaped (Fig.
4. 26, K, L), are characteristic of green species. A single pyrenoid usually

Fig. 4. 26. AC. Flagellar insertions, schematic: A, Chlamydomonas na-
suta (after Kater); B. C. longirubra (after Pascher); C. C. ignova (after
KorshikofF). D-J. \'arious types of chromatophores; pyrenoids indicated as
clear areas; schematic: D. Chlamydomonas obversa (after Pascher); E. Chlo-
rogonium elongatiim (after Dangeard); F, G. Chlamydomonas ovata (after
Dangeard); H. C. basistellata (after Pascher); I. C. korschikoffia (after
Pascher); J. Gigaiitochloris permaxima (after Pascher). K, L. Unusual U-
shaped pyrenoid; optical cross-section and lateral view of flagellate; starch
granules surround the pyrenoid; diagrammatic (after Vlk).

lies in the posterior portion of the cup-shaped chromatophore; if several
pyrenoids are present, distribution is variable. A stigma, when present,
is a rounded or discoid structure, usually anterior in position but some-
times near the equator. Starch, stored both in the cytoplasm and around
the pyrenoid of chlorophyll-bearing species, occurs also in colorless types.
Lipids, although usually not abundant, are stored by many phytoinonads.
A reddish pigment (red haematochrome) also may be accumulated in the

152 The Mastigophora

cytoplasm. In the "haematocyst" of Haematococcus phivialis (76), the
pigment may completely mask the chromatophore.

The order may be divided into four families of solitary types and two
of colonial genera. Among the solitary types, a typical cellulose mem-
brane is lacking in the Polyblepharidae, present in the Chlamydomonadi-
dae, and is replaced by a calcified bivalve "shell" in the Phacotidae. In
the Haematococcidae cytoplasmic processes extend into the thick mem-
brane. Colonial genera with a well developed matrix are assigned to the
Volvocidae; those without a matrix, to the Spondylomoridae. The order
has been surveyed by Pascher (206).

Family 1. Polyblepharidae. These are typically solitary types with
somewhat flexible bodies. The genus Raciborskiella (Fig. 4. 27, C) is ex-
ceptional in that 4-8 flagellates may remain attached posteriorly to form
simple aggregates (colonies?). Flagellar numbers of 1, 3, 4, 5, 6, and 8
have been reported, but there are usually two or four. Binary fission
occurs in the flagellate stage, and the old flagella are usually inherited
by the daughter organisms.

Chlorophyll-bearing species are included in the following genera: BipecUnomonas
Carter (31), Diuialiella Teodoresco (227), Hetcromastix Korshikov (31), Korschikoffia
Pascher (206), Mesostiginn Laiiterborn (206; Fig. 4. 27, D), Pedinomoiias Korshikov
(206), Phyllocardium Korshikov (162), Pocillomonas Steinecke (206). Polyblepharides
Dangeard {2d&), Pyramimouas Schm^rda. (Pyramidomonas Stein) (23,31,89,217; Fig. 4.

27, J), Raciborskiella Wislouch (206; Fig. 4. 27, C), Spennatozopsis Korshikov (2^)6).
TrichJoris ScherfFel and Pascher (206; Fig. 4. 27, K), and Tetrachlnris Pascher and
Jahoda (227) with four flagella. Chromatophores are lacking in Furcilla Stokes (206)
and Polytomella Aragao (128, 206; Fig. 4. 27, F-I). Cytological descriptions are avail-
able for Pyramimonas (25, 89) and Polytomella (128).

Collodictyon Carter (244; Fig. 4. 27, A, B) is sometimes included in this family. How-
ever, the plastic body, the longitudinal groove, the development of pseudopodia, and
the lack of information on stored reserves cast doubt upon the validity of such an
assignment.

Family JC Chlamydomonadidae. There is a well-developed membrane,
within which fission results in two or more daughter organisms (Fig. 4.

28, A, B). In Chlamydomonas nasuta (129), the plane of the first fission
is perpendicular to the long axis of the body. Prior to fission, the or-
ganism either rotates within its membrane through an arc of 90°, or else
the chromatophore and nucleus change their positions accordingly (Fig.
4. 28, C, D). The plane of the second fission is perpendicular to that of
the first. In various species, adhesion of the membranes of adjacent or-
ganisms often produces large palmellar aggregates or sheets, especially
during growth on a solid medium.

The following genera contain chlorophyll-bearing species: Apiococcus Korshikov
(206), Brachiomonas Bohlin (206; Fig. 4. 28, F), Carteria Diesing (23, 206, 217; Fig. 4.
28, O), Characiochloris Pascher (206), Chlawydonwnas Ehrenberg (94, 206, 217; Fig.
4. 28, C, D, G). Chlorobrachis Korshikov (206.' 256), Chloroceras Schiller (207), Chloro-
goniuin Ehrenberg (206; Fig. 4. 26, E), Clilornphysema Pascher (206), Diplostauron

The Mastigophora 153

Fig. 4. 27. A. B. Collodictyon triciliatiim Carter, basal portions of flagella,
longitudinal groove, development of pseiidopodia; x500 (after Rhodes). C.
Raciborskiella uroglenoides Swirenko, cluster of four flagellates; xlOOO ap-
prox. (after S.). D. Mesostigma viride Lauterhorn: x2100 approx. (after
Pascher). E. Ped'niomonas minor Korshikoff, x3100 approx. (after K.). F-I.
Polytomella citri Kater; living specimen showing stored food and contractile
vacuoles (F): a variation in form, nucleus stained (G); young (H) and older
(I) cysts; x2250 (after K.). J. Pyramijnojias tetrarhynchus Schmarda; large
chromatophore indicated as transparent to show positions of pyrenoid, an-
terior nucleus and contractile ^acuoles; xl425 (after Geitler). K. Trichloris
paradoxa ScherfEel; xllOO approx. (after S.).

Korshikov (217), Fortiella Pascher (206). Gigautochloris Pascher (206; Fig. 4. 26, J),
Gleomonas Rlebs (206), Hypnomonas Korshikov (206). Lobomonas Dangeard (206; Fig.
4. 28, E), Malleochloris Pascher (206), Nautococcus Korshikov (161; Fig. 4. 28, I, J),
Phyllomonas Korshikov (206), Platychloris Pascher (206), Platymonas West (31; Fig. 4.
28, N), Scourfieldia West (206; Fig. 4. 28. K, L), Selenochloris Pascher (207, 217),
Sphaerellopsis Korshikov (206), Sphenochloris Pascher (206), Spirogonium Pascher (206),
and Stylosphaeridium Geitler (206).

Colorless types are included in the following genera: Chlamydoblepharis France
(206), Hyalogonium Pascher (206; Fig. 4. 28, M), Parapolytoma Jameson (121), Poly-
tnma Ehrenberg (206), Tetrablepharis Senn (206), and Tussetia Pascher (206).

154 The Mastigophora

Four flagella are present in Carteria, Chlorobrachis, Fortiella, Malleochloris, Platy-
monas, Spirogonium, and Tetrablepharis; one flagellum in Chloroceras and Seleno-
chloris: two flagella in other genera. In some cases, a knowledge of life-cycles is
essential for assignments to genera. In Nautococcus, for example, there is a typical
flagellate stage in addition to the floating stage without flagella (Fig. 4. 28, I, J); in
Stylosphaeridium, the corresponding non-flagellated stage is epiphytic on filamentous
algae. Cytological descriptions are available for Chlamydomonas (129), Chlorogonium
(102), Parapolytoma (121), and Polytoma (78, 110).

Fig. 4. 28. A, B. Fission in Chlamydomonas seriata Pascher (schematic,
after P.). C, D. Rotation of the chromatophore at the beginning of fission
in Chlamydomonas nasuta; schematic (after Kater). E. Lobomonas roslrata
Hazen; xl750 approx. (after H.). F. Brachiomonas ivestiana Pascher; .x690
approx. (after P.). G. Chlamydomonas umbonata Pascher, xl330 approx.
(after P.). H. Tussetia polytomoides Pascher, xl400 approx. (after P.). I, J.
Nautococcus mammilatus Korshikofl^; stage with umbrella-like float, xl250;
flagellate stage, x2500 (after K.). K, L. Scourfieldia complanata West, views
of l)road and narrow surfaces; xl725 approx. (after W.). M. Hyalogonium
klebsii Pascher, x500 approx. (after P.). N. Platymonas tetrathele West,
xl430 (after Carter). O. Carteria coccifera Pascher, x960 (after P.).

The Mastigophora 155

Family 3. Haematococcidae. The outer membrane is separated from
the periplast by a thick layer of "gelatinous material" into which extend
cytoplasmic processes. These features have been considered adequate
grounds for separating the family from the Chlamydomonadidae (260).
The Haematococcidae include Haematococcus Agardh (76; Fig. 4, 29,
H) and Stephanosphaera Cohn (256; Fig. 4. 29, F, G).

Family 4. Phacotidae. The rather rigid membrane is often impregnated

Fig. 4. 29. A, B. Dysinorphococcus variabilis Takeda, surface view and
median optical section; xl200 (after Bold). C-E. Pteromonas anguJosa Lem-
mermann, edge view, broad side, and outline in cross-section; xlOOO approx.
(after Pascher). F, G. Stephanosphaera pluvialis Cohn, colony and young stage;
diameter of colony reaches 50-60/x; diagrammatic (after Pascher). H. Haeyna-
tococcus pluvialis Flotow em. Wille, large flagellate stage; xl500 (after
Elliott).

with calcium or iron compounds and possibly contains little or no cellu-
lose. A bivalve membrane (or "shell"), which does not fit the enclosed
organism very closely, is present in at least some genera. Fission occurs
within the membrane.

The family includes the following genera: Cephalomonas Higinbotham (104), Coc-
comonas Stein (206), Dysinorphococcus (23; Fig. 4. 29, A, B), Pedinopera Pascher (206),
Phacotus Perty (206, 207), Pteromonas Seligo (174, 206; Fig. 4. 29, C-E), Thoracomonas
Skvortzow (206, 217), Wislouchiella (207).

Family 5. Spondylomoridae. The membranes of the individual flagel-
lates are thin and the colony is not held together by a matrix. The larger

156 The Mastigophora

colonies are composed of two or four circlets of flagellates so arranged
that one organism does not lie directly above another. Individual flagel-
lates have two or four flagella. Daughter colonies are produced by fission
of any member of a colony within its original membrane. In contrast to
the Volvocidae, a plakea stage is not formed in development.

The family includes the following genera: Pascheriella Korshikov (164; Fig. 4. 30, B),
Pyrobotrys Arnoldi (Chlamydobotrys Korshikov) (256; Fig. 4. 30, A), Spotidylotnorum
Ehrenberg (206. 207; Fig. 4. 30, D).

In Corone Fott (Fig. 4. 30, C), the widely separated flagellates are joined by tough
strands. Since this type of organization differs from that of typical Spondylomoridae,
perhaps a new family Coronidae should be recognized, as suggested by Fott (84).

Fig. 4. 30. A. Pyrobotrys (Chlamydobotrys) squarrosa (Korshikofl), xl050
(after K.). B. Pasclieriella tetras Korshikotf, xl575 (after K.). C. Corone bo-
hemica Fott; length of colony (without flagella), 35-50^; flagella (one pair
shown full length) measure 35-40^ (after F.). D. Spondylomorum quater-
narium Ehrbg. (after Stein); colonies reach lengths of 50-70yn.

Family 6. Volvocidae. This group differs from the Spondylomoridae in
two major features: colonial organization is maintained by a matrix, and
a plakea stage (Fig. 4. 32, C) appears in the development of a young
colony.

The Mastigophora 157

The following genera are included: Eudorina Ehrenberg (103, 206); Go7iium Miiller
(103, 206; Fig. 4. 31, D); Pandorina Bory (206; Fig. 4. 31, A); Platydorina Kofoid (154,
268; Fig. 4. 31, B, C); Pleodorina Shaw (206); Stephanoon Schewiakoff (206); Volvox
Linnaeus (259); Volvuima Playfair (95).

Life-histories show basic similarities throughout the group, btit certain
genera are less specialized than others. In Gotiiiim, Pandorina, and
Platydorina, daughter colonies may be produced by any member of the
parental colony. This is not the case in certain other genera. Reproduc-
tion is limited to flagellates of the posterior four rows in Eudorina, to

Fig. 4. 31. A. Patidorina morum (Miiller) Bory (after Smith); colonies
may reach 250^ in diameter. B, C. Platydorina caudata Kofoid; surface
view, x225; lateral view, x260 (after K.). D. Gonium pectorale Miiller; colo-
nies reach diameters of 60-70/^; diagrammatic.

those in the posterior half of the colony in Pleodorina, and to a few
flagellates ("gonidia") in the posterior half of the Volvox colony.

In development of a daughter colony, continued fission within the
original membrane produces a hollow spherical or hemispherical stage,
the plakea (Fig. 4. 32, C), in which the anterior ends of the flagellates
are directed centrally. Later development in Gonium pectorale (103) in-
volves a flattening of the plakea, and then further inversion, so that the
young colony becomes slightly convex on the anterior, or flagellated,
surface. In Platydorina (268) the plakea is a hollow sphere with a single
opening (phialopore). After the 32-cell stage is reached, inversion occurs
through the phialopore and the inverted daughter colony becomes a
hollow sphere (Fig. 4. 32, Q). Further development involves collapse of

158 The Mastigophora

the sphere, with intercalation of flagellates from opposite sides so that
flagella are present on both surfaces (Fig. 4. 32, R). As secretion of the
matrix begins, the young colony approaches the adult form at about
the time cUssolution of the parental matrix occurs.

Fig. 4. 32. A-G. Development of a daughter colony in Volvox aureus;
pyrenoids are indicated as black dots; diagrammatic (after Zimmermann).
A-C. Fission results in a plakea, in which the anterior ends of the flagellates
are directed centrally. D-G. The plakea undergoes inversion to produce the
young colony. H-K. Mature zygotes, x615 (after Smith): Volvox perglobator
(H), V. globator (I), V. aureus (J), V. weismanni (K), L-P. The mature
macrogamete (L) of Platydorina caudata emerges from the parent colony
(M, N); a microgamete (O) then penetrates the macrogamete (P); diagram-
matic (after Taft). Q. Platydorina caudata, optical section of young colony
after inversion and tlevelopment of flagella; diagrammatic (after Taft). R.
Young plate-like colony (lateral view) derived from the earlier spherical
stage (Q); diagrammatic (after Taft).

The Mastigophora 159

Development of the Volvox colony (170, 234, 287) also involves the
formation of a spherical plakea with a phialopore and the inversion
("extroversion") of the plakea through the phialopore to produce a
young colony (Fig. 4. 32, AG). This process of inversion in Volvox is of
some general interest in its similarity to a process which the "stomatoblas-
tida" undergoes in certain species of Grant ia and Sycon (73). In general,
the young colonies of Volvox escape separately after rupturing the sur-
rounding membranes, but those of V. aureus may emerge through a com-
mon pore in the wall of the colony.

The details of sexual reproduction vary somewhat in different genera
and species. The gametes are similar in Gonium, but anisogamy is obvi-
ous in Eudorina, Payidorina, Platydorina, Pleodorina, and Volvox. Some
species of Eudorina and Volvox are heterothallic and others are homo-
thallic, althotigh the homothallic species of Volvox are protandrous. Some
of the heterothallic species of Volvox show sexual dimorphism involving
dwarf male colonies and large female colonies (259). Pleodorina is usually
heterothallic, with occasional homothallic variants. Such variation is
known also in the typically heterothallic Volvox aureus. Pandorina,
Platydorina, and at least some species of Gonium (255) are heterothallic.

Sexual reproduction is preceded by differentiation of gametes. The de-
veloping macrogametes in Platydorina caudata (268) show no significant
increase in volume but they become denser in appearance and acquire a
yellowish tinge as they approach maturity. The flagella are retained and
the mature macrogamete emerges from the colony as an active flagellate
(Fig. 4. 32, L-N). The macrogametes escape from the female colony in
Pandorina also, whereas those of Eudorina, Pandorina, and Volvox
remain in place and are fertilized there. The development of micro-
gametes in Platydorina is similar to that of a daughter colony. Fission, at
the 32-cell stage, results in a curved plakea which soon undergoes inver-
sion and develops into a sphere. Flagella are developed and the spheroid
packets escape intact from the colonial matrix. Upon contact with macro-
gametes, the packet dissociates into its component gametes and fertiliza-
tion occurs (Fig. 4. 32, O, P).

Microgametes of Volvox develop from enlarged cells resembling the
"gonidia." Development of microgametes is precocious in Volvox sperma-
tosphaera, V. weismannia, and several other species in that packets of
gametes reach maturity while young male colonies are still within the
parental colony. In other heterothallic species, mature packets develop
only after the male colonies emerge from the parent and grow to about
the size of female and asexual colonies. Volvox spermatosphaera differs
from other species in that every flagellate in the male colony may develop
into a packet of gametes. The mature packet is discoid in Volvox aureus,
V. spermatosphaera, and V. weismannia, while spheroid packets are de-
veloped in V. globator, V. perglobator, and several others (259), The

160 The Mastigophora

spheroid packet results when fission produces 256 or more cells; the in-
verted plakea remains plate-like when the number is only 16-128.

The developing macrogamete of Volvox, early in the life of the young
colony, grows into a large spheroidal cell containing much stored food.
Microgametes enter female colonies, sometimes before the ova are fully
grown, and finally penetrate the ova as they approach or reach maturity.
After fertilization, the zygote encysts. The ectocyst may show characteristic
decorations (Fig. 4. 32, H-K). After disintegration of the female colony
the cyst sinks to the bottom, where it remains dormant until the follow-
ing spring. Under natural conditions, colonial forms may occur only
during two or three months of the year, so that the encysted zygote
is the predominant phase of the cycle (259). In laboratory cultures, how-
ever, repeated generations of asexual colonies have been obtained over a
period of a year or more (275).

Order 6. Euglenida

The Euglenidai are rather large flagellates, mostly with one or
two flagella. The body is generally elongated and often spindle-shaped,
with some degree of spiral torsion, but modifications occur in such genera
as PJiacus (Fig. 4. 34, I-L). The reservoir (Fig. 4. 33, A-D), or "gullet,"
from which the flagella arise, is a characteristic feature. Flagellates as-
signed to two genera, Chlorachne and Ottonia, apparently lack reservoirs,
but Schiller's (251) descriptions do not supply conclusive evidence that
these are Euglenida. One or two contractile vacuoles empty into the
reservoir, and each flagellum is inserted in the posterior or postero-dorsal
wall of this cavity. The pellicle permits euglenoid movement (metaboly)
in many species but it may be only slightly flexible, as in Euglena acus,
or rather rigid in such genera as Menoidium and Phacus. As reported for
Euglena viridis (228), this membrane gives negative tests for cellulose,
but is completely digested by trypsin and presumably contains proteins.
According to Chadefaud (34), the pellicle (Fig. 4. 33, E) consists of a thin
epicuticle and a deeper and thicker cuticle. Only the epicuticle extends
into the reservoir. The usually noticeable spiral striations seem to be
cuticular ridges (34); presumably the rows of papillae in Euglena
spirogyra are comparable decorations. The distribution of peripheral
inclusions, and sometimes that of the chromatophores, may follow the
spiral decorations of the pellicle. In addition to the pellicle, a lorica oc-
curs in Ascoglena and Klebsiella (Fig. 4. 33, K, L); a shell, or test, in
Trachelomonas (Fig. 4. 33, J).

Perhaps the majority of Euglenida are chlorophyll-bearing, although
there are many colorless species. The chromatophores range from one to
many and also vary in size and form (Fig. 4. 33, F-J) in different species.
The green color of chlorophyll is not masked by other pigments. How-

^ The literature on Euglenida has been reviewed by Jahn (119).

The Mastigophora 161

Fig. 4. 33. A-D. Flagella and reservoir: Euglena mutabilis (A), Euglena-
morpha, green form (B), Eutreptia (C), Distigma (D); diagrammatic (after
Hollande). E. Plasmolyzed specimen of Eugleua archaeoplastidiata, pellicle
separated from body, two pyrcnoids shown; schematic (after Chadefand).
F-I. Various types of chromatophores in Euglena: E. geniculata (F), E. ana-
baena (G), E. viridis (H), E. variabilis (I); schematic (after Pringsheim).
J. Trachelomonas volvocina Ehrbg., showing test, chromatophores, stigma,
nucleus (outline); x720 (after Deflandre). K, L. Klebsiella alligata, external
view of lorica and optical section through posterior end; xlOOO approx.
(after Pascher). M. Euglena gracilis, palmella; x455 (after Krichenbauer).
N. E. gracilis, somatella with six luiclei; chromatophores not shown; x675
(after Krichenbauer). O. Phacus caudata, four daughter flagellates being
produced from a somatella; x850 (after Krichenbauer).

162 The Mastigophora

ever, red haematochrome may accumulate in the cytoplasm in large
amounts, as in Euglena rubra (125). Pyrenoids are usually attached to
chromatophores or to non-pigmented "pyrenophores" (34). A typical
pyrenoid consists of two pyrenosomes, each covered with a paramylum
shell and applied to a surface of the chromatophore (Fig. 1. 17, L). The
inner pyrenosome may be reduced in size, and is lacking in some cases
(34). In such types as Euglena gracilis (Fig. 4. 34, A), there are many
chromatophores, each of which probably bears a pyrenoid. At the other
extreme, represented by Euglena archaeoplastidiata (Fig. 4. 33, E), there
is one chromatophore equipped with two pyrenoids (34). Bleaching of
the chromatophores in Euglena gracilis apparently is accompanied by
resorption of the pyrenoids, which reappear if the flagellates are returned
to the light and develop chlorophyll (240). A stigma, lying on the wall
of the reservoir near the paraflagellar body (Fig. 4. 33, A-C), is charac-
teristic of green species and also of certain colorless types (120, 237, 240).
The stigma may divide in fission (8), or may undergo dispersal and re-
aggregation of the piginent granules (96). Flagellar number and struc-
ture vary. The bifurcated flagellum of Phacus (Fig. 4. 34, H) and Euglena
has been interpreted as a biflagellate condition (Fig. 4. 33, A) in which
a rudimentary flagellum is often fused distally with a normal flagellum
(110). The bifurcation apparently is absent in Colacium (123) and
Rhabdomo7ias (99) but present in Menoidium (240). The situation in
Astasia has been disputed, some workers reporting a non-bifurcated and
others a bifurcated flagellum. More recent observations (240) indicate
that the flagellum, in at least certain species of Astasia, is much like that
of Euglena and that the rudimentary flagellum may or may not be in-
dependent of the normal flagellum. Such observations support the view
that a biflagellate condition is the primitive one and indicate the desir-
ability of reexamining those species in which a simple flagellum has been
reported. No "bifurcation" has been reported in biflagellate or triflagel-
late species. A paraflagellar body (photoreceptor, or flagellar swelling) is
characteristic of green species (Fig. 4. 33, A-C) but is absent in colorless
forms.

Stored reserves include lipids and paramylum; the latter is an iodine-
negative polysaccharide, insoluble in hot water and yielding glucose on
hydrolysis. Paramylum is deposited as refractile bodies which may show
concentric stratification in dilute solutions of KOH (62). Size and form
may be fairly constant for a species, while the number ranges from typi-
cally one (Phacus longicauda) or two {Euglena spirogyra) large bodies to
many small ones.

The life-cycle may include a flagellate stage, a palmella (Fig. 4. 33, M),
and a cyst. Fission may occur in both palmella and flagellate stages.
Palmella stages are unknown in many species and their distribution
within the order remains uncertain, although they may be absent in

The Mastigophora 163

colorless species (240). A palmella is dominant in the cycle of Eugleno-
capsa ochracea (263), and a sessile non-flagellated stage plays a compa-
rable role in Colacium vesicidosum (Fig. 4. 35, O, P). The sessile stage of
the latter is derived from a flagellate which becomes attached at its flag-
ellar end. The flagellum and reservoir disappear, a sheath and stalk are
secreted, and mitosis may produce as many as eight nuclei. A naked

Fig. 4. 34. A-D. Euglena: E. gracilis Klebs (A), xl200; E. sociabilis Dan-
geard (B), x450; E. pisciformis Klebs (C), xl650; E. tripteris (D), x475 (after
Johnson). E. Eutreptiella marina da Cunha. xl600 approx. (afte; da C). F.
Trachelomonas hystrix, test only; x600 (after Dangeard). G. Euglena oxyu-
ris Schmarda, x350 (after Johnson). H. Phacus p\rum, showing two large
lateral paramylum bodies, small discoid chromatophores, nucleus (in out-
line); xl750 (after Krichenbauer). I, J. Phacus quinquenwrginatus Jahn and
Shawhan, surface and anterior views; length, 35-52/^; schematic (after Allegre
and Jahn). K, L. Phacus torta Lemmermann, broad sinface and anterior
end; length, 80-100/^; schematic (after Allegre and Jahn).

164 The Mastigophora

multinucleate form also has been observed in cultures (123). Either mul-
tinucleate stage may produce flagellate buds. A comparable plasmodium
has been reported in Euglena gracilis (Fig. 4. 33, N) and Phacits caudata
(Fig. 4. 33, O) by Krichenbauer (167); also, in Astasia klehsii. In A.
klebsii no fission occurs in the plasmodial stage, which apparently origi-
nates as a result of increased osmotic pressure in old cultures. Even a
return to a normal medium does not induce fission (57).

Although the Euglenida are mostly fresh-water flagellates, a number
of genera are represented in salt-water and certain fresh-water species
have become adapted to sea water under laboratory conditions (82).
However, Euglena gracilis grows only in a salt concentration less than
that of 40 per cent sea water (185).

Although it is not difficult to recognize Euglenida, in view of their
characteristic features, subdivision of the group into taxonomically sound
suborders and families apparently remains a problem for the future. The
old three-family system — Euglenidae, Astasiidae, and Peranemidae — was
convenient up to a certain point. Green flagellates could be placed in the
Euglenidae, and holozoic types, often with a pharyngeal-rod apparatus.
could be assigned to the Peranemidae. The residue of colorless flagel-
lates could be dropped into the Astasiidae. Various observations have
disturbed this taxonomic tranquillity. The discovery of colorless stigma-
bearing flagellates (good species of Euglena except for the absence of
chromatophores), the recognition of Hyalocephalus as a colorless homo-
logue of Phacus, and recent observations on the loss of chlorophyll in
Euglena make the presence or absence of chromatophores a feature of
doubtful value in separating families. In fact, certain generally recog-
nized species of Astasia are possibly nothing more than colorless strains
of Euglena (239). Furthermore, Pringsheim and others have observed
that growth of Euglena gracilis in darkness, following treatment with
streptomycin, induces loss of the stigma after the chromatophores have
disappeared. This new creation is a genetically stable strain which would
be eliminated automatically from the old family Euglenidae. The old
family Peranemidae also is not homogeneous in that a pharyngeal-rod
apparatus is present in some genera and not in others, holozoic nutrition
has not been demonstrated in certain cases, and differences in flagellar
apparatus are well known.

Hollande (110) has divided the Euglenida into three groups which, in
conformity with the present system, would be recognized as suborders —
Euglenoidina, Peranemoidina, and Petalomonadoidina. These suborders
would be divided into appropriate families as adequate information be-
comes available. Although separation of the Peranemoidina and Petalo-
monadoidina may not be clear cut, if the siphon of Entosiphon (Fig. 4.
37, B) is only a modified rod-apparatus as seen in Peranema (Fig. 4. 36,

The Mastigophora 16!

Fig. 4. 35. A. Distiginopsis grassci HoUande, x2430 approx. (after H.). B.
Eutreptia viridis Perty, x240 (after Lenimennann). C. Astasia comma Pring-
sheim, x835 approx. (after P.). D. Menoidium cultellus Pringsheim, x500 ap-
prox. (after P.). E. Astasia dangeardii Lemmermann, x860 approx. (after
Pringsheim). F. Cryptoglena pigra Ehrl)g., xl300 (after Lemmermann). G.
Rhal)dnmouas incurva Presenilis, xl470 (after Hall). H. Astasia longa Pring-
sheim, x720 approx. (after P.). I. Astasia torta Pringsheim, x835 approx.
(after P.). J. Lepocinclis niarssoni Lemm., showing two lateral paramylum
bodies; xGOO (after L.). K, L. Pliacus pleurouectes (O. F. M.) Dujardin, dor-
sal surface and anterior end; chromatophores not shown; length, 40-lOOju:
schematic (after Allegre and Jahn). M. Menoidium obtusum Pringsheim, xr)00
approx. (after P.). N. Ascoglemi vaginicola Stein, with lorica; x412 (after
Lemmermann). O, P. Colacium vesiculosum Ehrbg., a budding multinucleate
sessile stage and a uninucleate stage; xl955 (after Johnson). Q. Distigma sen-
nii Pringsheim, x900 approx. (after P.).

166 The Mastigophora

C, D), Hollande's system seems to have certain advantages in the present
stage of taxonomic progress.

Suborder 1. Euglenoidina. These flagellates have one or more flagella,
may or may not contain chlorophyll, are not holozoic, may be metabolic,
or may have a rigid pellicle. The flagellar sheath is not swollen at the
base. On the basis of flagellar equipment, Hollande (110) recognized
the families Euglenamorphidae, Eutreptiidae, Distigmidae, Euglenidae,
and Menoidiidae, but the erection of definitive families may require more
information than is now available.

The following genera may be assigned to the suborder: Ascoglena Stein (202; Fig. 4.
33, N); Astasia Dujardin (236, 238, 239, 240; Fig. 4. 35, C, E, H, I); Colacium Ehren-
berg (123, 202; Fig. 4. 35, O, P); Cryptoglena Ehrenberg (202; Fig. 4. 35, F); Distigma
Ehrenberg (108, 172, 236; Fig. 4. 35, Q), without chroraatophores; Distig)nopsis Hol-
lande (110; Fig. 4. 35, A); Euglena Ehrenberg (124, 202; Fig. 4. 34, A-D. G); Euglena-
morpha Wenrich (277;, Fig. 4^ 33, B), from tadpoles; Eiitreptia Perty (202; Fig. 4. 35,
B); Eutreptiella da Cunha (Fig. 4. 34, E); Hyalocephalus Pringsheim (236), a colorless

Fig. 4. 36. A. Peranemopsis striata Lackey; one long anterior flagellum;
no second flagellum like that of Peranerna, and only one pharyngeal-rod;
length, 90-110^ (after L.). B. Urceolus cyclostomus (Stein) Mereschkowski,
showing vestibule, reservoir, pharyngeal-rod apparatus, nucleus, ingested
food; x933 (after Klebs). C. Peranerna trichophorum (Ehrbg.) Stein,
slightly contracted, ventral vieu' showing pharyngeal-rod apparatus and
trailing flagellum adherent to the body; in swimming, the anterior flagel-
lum (shown in part) is extended as in Peranemopsis (A); schematic (after
Chadefaud). D. Pharyngeal-rod apparatus of P. trichophorum, right lateral
aspect; schematic (after Chadefaud). E. Heteronema acus (Ehrbg.) Stein;
ingested Euglena in a food-vacuole not yet separated from the reservoir;
flagella shown leaving cytostome; x2240 (after Loefer).

The Mastigophora 167

"Phacus"; Khawkinea Jahn and McKibben (120), similar to Euglena except for the
absence of chromatophores; Klebsiella Pascher (215; Fig. 4. 33, K, L); Lepocinclis Perty
(49, 202; Fig. 4. 35, J); Menoidium Perty (236, 238; Fig. 4. 35, M); Phacus Dujardin
(3, 202, 230; Fig. 4. 34, H-L); Rhabdoinonas Fresenius (99, 238; Fig. 4. 35, G); Tra-
chelomonas Ehrenberg (60, 202; Figs. 4. 33, J, 4. 34, F). In addition, Euglenocapsa
Steinecke (263), in which a palmella stage is dominant, may be a valid genus.

Suborder 2. Peranemoidina. These are colorless, metabolic types with
two flagella, one of which is trailed. Each flagellum is said to be swollen

Fig. 4. 37. A. Marsupiogaster striata Schewiakoff, x835 (after S.). B. Etito-
siphon sulcatum (Duj.) Stein; length, 20-25/t; siphon, gullet, nucleus and
food vacuoles; schematic (after Lackey). C, D. Triangulomonas rigida
Lackey; 18x15;^; surface and lateral views (after L.). E. Sphenomonas teres;
length, 20-40/i; large retractile inclusion of uncertain nature, smaller para-
mylum bodies (after Hollande). F. Tropidoscyphus octocostalus Stein, show-
ing prominent ridges; x412 (after Lemmermann). G. Anisonema aci^ius Duj.,
showing one "pharyngeal-rod," nucleus, ingested food; x633 (after Lemmer-
mann). H. Notosolenus apocarnptus Stokes; length, S-lO^ii; short trailing
flagellum arises from convex ventral surfaces (after S.). I,J. Petalomonas
dorsalis Stokes, 38-45/n; entire flagellate and optical cross-section (after
Shawhan and Jahn).

168 The Mastigophora

at the base (110). Solid food is usually ingested. The characteristic
pharyngeal-rod apparatus, which lies dorsal to the reservoir, is composed
of two long rods and a shorter falcate rod which extends ventrally at its
anterior end (Fig. 4. 36, C, D).

The conclusion of Tannreuther (268a), that the rod apparatus in
Peranema is a "perforatorium" used for piercing the prey, has been con-
firmed by Chen (38). The identity of the cytostome and gullet in these
holozoic Euglenida has been disputed. Chen (38) and Pitelka (229),
among others, have been convinced that ingestion takes place through a
cytostome and gullet independent of the reservoir and its external open-
ing. Chadefaud (35), on the other hand, maintains that there is no
separate gullet in at least certain members of the group. Previous ob-
servations on the continuity of food vacuoles with the cavity of the reser-
voir (Fig. 4. 36, E) in Heteronema (184) and Peranema (100) support
the latter conclusion.

The following genera are included: Heteronema Stein (184, 202; Fig. 4. 36, E);
Peranema Dujardin (35, 202, 229; Fig. 4. 36, C, D), trailing flagellum adherent to the
pellicle; Peranemopsis Lackey (175; Fig. 4. 36, A); Urceolus Mereschkowsky (202; Fig.
4. 36, B). However, Pitelka (229) has considered Heteronema a synonym of Peranema.

Suborder 3. Petalomonadoidina. The body of these colorless flagellates
is typically compressed and not plastic. There may be one or two flagella
and each flagellum is swollen at the base (110). Some species are definitely
holozoic. A pharyngeal-apparatus, described for several genera, may or
may not be homologous with that of Peranema.

The suborder includes the following genera (110): Anisonema Dujardin (202; Fig. 4.
37, G); Dinema Perty (202); Entosiphon Stein (110, 171, 202; Fig. 4. 37, B); Marsupio-
gaster Schewiakoff (202; Fig. 4. 37, A); Notosolenus Stokes (202; Fig. 4. 37, H); Peta-
lomonas Stein (251; Fig. 4. 37, I, J); Scytomonas Stein (202); Sphenomonas Stein (110,
202); Triangiilomonas Lackey (175; Fig. 4. 37, C, D); Tropidoscyphus Stein (202; Fig.
4. 37, F).

Order 7. Chloromonadida

Little is known about these flagellates. The described species are fairly
large (30-100[jl) forms with somewhat plastic bodies which are usually
dorso-ventrally flattened, and may show a ventral groove arising near the
anterior end. The numerous bright green chromatophores are peripheral
and radially arranged in Chattonella (Fig. 4. 38, B) and Gonyostomum
(Fig. 4. 38, H). The pigments are said to include xanthophylls as well as
chlorophyll; the mixture turns blue-green in dilute acid (69). No stigma
has been reported. Oil droplets are usually stored. Glycogen also occurs
in Gonyostomum semeyi (114), but starch apparently is not formed. There
are typically two flagella, one of which is trailed.

A gullet (Fig. 4. 38, E, H, J) not unlike the reservoir of Euglenida

Fig. 4. 38. A, B. Chattonella subsalsa Biecheler; length, 30-50^; surface
view showing chromatophores, ventral groove, and basal portions of flagella;
optical section showing chromatophores, nucleus, and flagellar connections
(after B.). C. Merotrirhia capitatu Skuja, showing ventral groove, chromato-
phores, and "trichocysts"; x550 (after S.). D. Nuclear cap and flagellar con-
nections in Vacuolaria virescens; schematic (after Poisson and HoUande).
E. Vacuolaria viridis (Dangeard) Senn, longitudinal section of stained speci-
men showing nucleus, "gidlet," and chromatophores; diagrammatic (after
Fott). F. Vacuolaria virescens Cienkowski; length, 50-150^; stained specimen
showing chromatophores, contractile vacuole, nucleus and nuclear cap; sche-
matic (after Poisson and Hollande). G. Dividing nucleus of V. virescens;
diagrammatic (after Poisson and Hollande). H-J. Gonyostomum semen
Diesing, length 40-65;^. H. Diagrammatic optical section showing chromato-
phores, trichocysts, nucleus (in outline), and contractile vacuole lateral to
"reservoir" (after Chadefaud). I. Ventral view, showing groove and flagella.
J. Optical section showing outline of nucleus and reservoir; diagrammatic
(after Drouet and Cohen). K. Fission in palmella stage, Vacuolaria virescens;
schematic (after Poisson and Hollande).

170 The Mastigophora

has been described in some species. However, it has been suggested that
in Vacuolaria (Fig. 4. 38, F) at least, a large contractile vacuole has pre-
viously been misinterpreted as a gullet (232). The lack of such a gullet
would suggest that the Chloromonadida are not closely related to the
Euglenida. The dividing nucleus of Vacuolaria (Fig. 4. 38, G), strikingly
different from the euglenoid type, points to the same conclusion, as does
the insertion of the fiagella (Fig. 4. 38, B, F). The fiagella of Gonyosto-
rnum semen, on the other hand, apparently arise from the base of the
triangular cavity, or "gullet" (70). A peculiar "supranuclear cap" (Fig.
4. 38, D), lying just anterior to the nucleus, occurs in Vacuolaria (232).
Various globular, discoid, or spindle-shaped bodies, subpellicular in dis-
tribution (Fig. 4. 38, C, H), have been interpreted as mucous globules
(15, 232) and as trichocysts (33). Upon discharge, such inclusions give
rise to filaments in Gonyostomum (33). The cytoplasm of Gonyostomum
semen (114) and Chattonella subsala (15) is differentiated into two zones,
apparently separated by a delicate membrane ("central capsule"), per-
haps merely an interface. The outer zone contains the chromatophores,
vacuome, fat globules, and trichocysts. Fission occurs in flagellate stages
of Chattonella (16) and Gonyostommii (69, 114), and in palmella stages
of Vacuolaria (Fig. 4. 38, K). Cysts with a thick membrane have been re-
ported in Gonyostomiun (69).

The Chloromonadida are fresh water types whose ecological distribu-
tion may be somewhat restricted. Gofiyostotnum semen, for instance,
seems to be limited to the rather acid waters of marshes (114),

Tfie following genera have been referred to the order: Chattojiella Biecheler (15,
16; Fig. 4. 38, A, B), Coelomonas Stein (231), Gonyostomum Diesing (33, 69, 70, 114;
Fig. 4. 38, H-J), Merotrichia Mereschkowski (Fig. 4. 38, C), Rhaphidomonas Stein,
Rickertia Conrad (43), Thaumatomastix Lauterborn, Thaumatomonas de Saedeleer
(246), Trentonia Stokes (264), and Vacuolaria Cienkowski (83, 232). Three of these
generic names are said to be invalid, since Rhaphidomonas is a synonym of Gony-
ostomum, and both Coelomonas and Trentonia appear to be synonyms of Vacuolaria
(232). The relationships of Thaumatomastix, Thaumatomonas, and Rickertia to Chat-
tonella, Gonyostomum, and Vacuolaria need further investigation.

CLASS 2. ZOOMASTIGOPHOREA

These flagellates have no chromatophores and they store lipids
and glycogen but apparently no starch or paramylum. Some are sapro-
zoic but there are many holozoic species. The body is generally rather
plastic and no cellulose membrane or test is produced. Many are small
and simple in structure, while others are perhaps as complex as any of
the Protozoa. Zoomastigophorea occur as parasites in various groups of
invertebrates, in all classes of vertebrates, and also in certain plants. As
free-living flagellates, they are found in the soil and in both fresh and
salt water. The life cycle is simple in the majority, but polymorphic

The Mastigophora 171

cycles are known, as in the Trypanosmidae, and sexual phenomena have
been reported in a few instances, most recently by Cleveland (Chapter II).

Present classifications are tentative at best and are based, to an im-
portant extent unfortunately, upon somewhat artificial criteria rather
than upon detailed information which might suggest natural relation-
ships. The recent erection of the order Trichomonadida (147), the result
of a long series of intensive studies, has set a sound pattern for the pos-
sible establishment of additional coherent orders within certain areas of
the class. In the meantime, the remnants of the "Polymastigida" may
be retained, along with the other older orders, for taxonomic convenience.
Accordingly, the Zoomastigophorea may be subdivided as follows:

Order I. Rhizomastigida. This inadequately defined group of amoe-
boid flagellates has served occasionally as a repository for genera of un-
certain taxonomic position, and has also been treated as a family of the
Protomastigida.

Order 2. Protomastigida. These are solitary or colonial types with one
or two fiagella. The body is plastic but does not show the amoeboid
activity of the Rhizomastigida.

Order 3. Polymastigida. The remnants of the old Order Polymastigida
include mostly uninucleate and binucleate species, although there are a
few with a number of nuclei. There are usually 3-8 fiagella.

Order 4. Trichomonadida. These are uninucleate or multinucleate
(but not binucleate) flagellates with an axostyle, a parabasal body, and
a mastigont of 3-6 fiagella. One of the fiagella is typically a trailing fiag-
ellum which may or may not form part of an undulating membrane.

Order 5. Hypermastigida. These are uninucleate flagellates with many
fiagella. The known sjoecies are intestinal parasites of termites, wood
roaches and cockroaches.

Order 1. Rhizomastigida

This order may be limited to flagellates with 1-4 fiagella and
amoeboid bodies which often show considerable pseudopodial activity.
In at least some species, a cytoplasmic fibril ("rhizostyle") of uncertain
significance extends posteriorly from one of the blepharoplasts.

The following genera may be assigned to the order: Heliobodo Valkanov (276; Fig.
4. 39, I); Histomonas Tyzzer (20, 273, 274, 280; Fig. 4. 39, A-F); Mastigamoeba Schulze
(153); Mastigella Frenzel (88, 97, 153; Fig. 4. 39, L); Mastigina Frenzel (12, 13, 88; Fig.
4. 39, J, K); and Rhizomastix Alexeieff (191; Fig. 4. 39, G, H). Tricholimax Frenzel
apparently is a synonym of Mastigina Frenzel (97). Certain other genera, sometimes
included in the Rhizomastigina, probably do not belong here. Pteridomonas Penard
possibly should be referred to the Chrysomonadida, while Actinomonas Kent and
Dimorpha Gruber probably belong in the Helioflagellida (Chapter V). The relationships
of Multicilia Cienkowski (177) are uncertain on the basis of available data. Although
the body is amoeboid, the many fiagella (or axopodia?) and the 1-4 nuclei are not very
strong inducements for retaining this genus in the Rhizomastigida.

172 The Mastigophora

Fig. 4. 39. A-F. Uistomonas nieleagridis Tyzzer: A. Rounded stage showing
nucleo-flagellar connections, x2310 (after Bishop). B. Specimen with four flagella
and rhizostyle, xl866 (after Wenrich). C. Daughter nuclei joined by paradesmose,
x2310 (after Bishop). D. Uniflagellate form with rhizostyle, x2310 (after Bishop).
E. Ingestion of food by means of a "tube," xl866 (after Wenrich). F. Elongated
imiflagellate form, x2310 (after Bishop). G. Rhizomast/x gracilis Alexeieff, nu-
cleus and rhizostyle stained; x2000 (after Mackinnon). H. Cyst of R. gracilis,
two nuclei and two rhizostyles; x2000 (after Mackinnon). I. Heliohndo radians
Wilkanov; x2400 (after V.). J, K. Mastigina hylae (Frenzel) Goldschmidt (after
Becker); specimen showing nucleus, flagellum, rhizostyle extending posteriorly,
and cap-like "cape" fitting over nucleus anteriorly (J), x515; pattern of proto-
plasmic streaming (K), diagrammatic. L. Mastigella polyniastix Frenzel, x400
(after F.).

Species of Mastigatnoeba and Mastigella are similar with respect to the
single flagellum and the development ot slender pseudopodia. However,
the nucleus is approximately central and not connected with the flag-
ellum in Mastigella, while the nucleus in Mastigamoeha is anterior and

The Mastigophora 173

apparently joined to the blepharoplast. In Mastigina the nucleus is an-
terior as in Mastigamoeha and is joined to the blepharoplast, but slender
pseudopodia seem to be lacking. The nucleoflagellar relationships of
Mastigina liylae (Frenzel) Goldschmidt have been described by Becker
(12). In addition to the flagellum, two other structures are joined to the
blepharoplast (Fig. 4. 39, J). A rhizostyle extends posteriorly, and a cap-
shaped "cape" fits over the anterior surface of the nucleus. From the cape,
filaments extend to the anterior end of the body.

Rhizornastix gracilis Alexeieff, recovered from an axolotl and from
crane-fly grubs, shows a rhizostyle, extending almost to the posterior end
of the body (Fig. 4. 39, G), but there is no "cape" as in Mastigina hylae
and the nucleus is central (191). Nuclear division occurs within the cyst
(Fig. 4. 39, H), and a second rhizostyle develops by outgrowth from a
blepharoplast.

HeJiobodo (Fig. 4. 39, I) includes spheroid uninucleate organisms with
two flagella and many slender pseudopodia which apparently are not
axopodia. Whether this genus actually belongs in the Rhizomastigida is
uncertain.

Histomonas meleagridis Tyzzer (Fig. 4. 39, A-F) is associated with
"blackhead" (enterohepatitis) in turkeys and chickens. An interesting
featiue of blackhead in turkeys is that young birds are readily infected
by feeding them embryonated eggs of the cecal worm, Heterakis gallinae.
The flagellates apparently remain viable in such eggs for more than a
year when kept in a refrigerator (189). H. meleagridis is an amoeboid or
slug-like organism which may produce slender pseudopodia and is ca-
pable of changing shape rapidly (20). Some of these slender pseudopodia
may correspond to the tubular protrusions (Fig. 4. 39, E) noted by Wen-
rich (280) in stained preparations. The unusual variability in number of
flagella raises questions concerning the validity of Histomonas meleagridis
as a specific name for all the various strains described from birds. One
flagellum is typical in cultures from chickens (20), although binucleate
forms with two flagella, and tetranucleate forms with four, occur occa-
sionally. In material from ring-neck pheasants (280), flagellate stages
nearly always showed four flagella. Flagellar resorption occurs at an early
stage of nuclear division so that non-flagellated uninucleate and bi-
nucleate forms are common and tetranucleate stages without flagella are
sometimes seen (20). Whether the "rhizostyle" is a normal organelle, or
merely an occasionally observed remnant of the paradesmose is still un-
certain.

Order 2. Protomastigida

These are relatively small organisms with one or two flagella. The
body is typically plastic, but not markedly amoeboid. Nutrition is sapro-
zoic in some types and holozoic in many others. The order includes

174 The Mastigophora

Fig. 4. 40. A. Salpingoeca brunnea Stokes, with theca; x660 (after France).

B. Codonocladium iimbellatum (Tatein) Stein, x325 (after Lemmermann).

C. Desmarella moniliformis Kent, typical linear "colony"; x477 (after Lem-
mermann). D. Lagenoeca globulosa France, free-swimming loricate type; x530
(after Lemmermann). E. Diplosigopsis entzii France, sessile loricate type; x600
(after F.). F. Spliaeroeca volvox Lauterborn, x350 (after Lemmermann). G.
Protospongia hacckelii Kent, x442 (after Lemmermann). H. Codonosigopsis
socialis (France) Lemmermann, with double collar; x500 (after F.). L Diplo-
siga socialis Frenzel, with double collar; xl350 (after F.). J. Monosiga angus-
tata Kent, x2000 (after K.). K. Codosiga botn'tis Ehrbg.; length of body
(excluding collar), 7-16/n; body enclosed in a mucous envelope (outline em-
phasized); schematic (after Lapage).

free-living species and parasites of invertebrates, vertebrates, and certain
plants. The life-cycle is often simple, but is dimorphic to polymorphic in
Trypanosomidae. Interrelationships of the different families are not en-
tirely clear and the limits of the order have been disputed to some extent.
For example, Trimastix Kent and Tricercomonas Wenyon and O'Connor
have been classified both with Protomastigida and the Polymastigida.

The Mastigophora 175

Six families may be assigned to the order: Codosigidae, Phalansteriidae,
Trypanosomidae, Cryptobiidae, Amphimonadidae, and Bodonidae.

Family 1. Codosigidae. This group (30, 181) includes species with a
"collar" (Fig. 4. 40). As described in Codosiga (Fig. 4. 40, K), this collar
is a protoplasmic membrane which can be extended as a hollow cone
surroimding the basal portion of the flagellum (176). The collar can be
retracted completely. The body is enclosed in a thin "mucous envelope"
apparently continuous with the stalk. During feeding, the anterior end
of the body contracts away from the envelope and food particles, driven
by flagellar currents, drop into this space. As the body surges back against
the envelope, the food particles apparently are forced into the body. The
expanded collar evidently directs food into the space between the body
and the envelope. Many choanoflagellates resemble the choanocytes of
sponges to such a degree that Kent (130) included them with the sponges
in his order "Choano-flagellata." The similarity may involve not only
the collar but also a parabasal body, or apical body (Fig. 1. 10, L, M). A
single flagellum is characteristic. An interesting feature of the sessile
Codosiga botrytis is that flagellates which become detached swim stalk-
first (176). Both solitary and colonial forms are known. In addition, tem-
porary clusters of several flagellates, failing to separate after fission, may
remain attached to a stalk, as in Codosiga (176).

The family includes several genera of naked flagellates — Codoiiosigopsis Senn (Fig.
4. 40, H); Codosiga James-Clark (Fig. 4. 40, K); Desmarella Kent (174; Fig. 4. 40, C);
Diplosiga Frenzel (Fig. 4. 40, I); and Monosiga Kent (245; Fig. 4. 40. J). A lorica is
present in several others: Diplosigopsis France (Fig. 4. 40, E); Lagenoeca Kent (Fig.
4. 40, D); and Salpingoeca James-Clark (Fig. 4. 40, A). Spheroid colonies are developed
in Protospongia Kent (Fig. 4. 40, G) and Sphaeroeca Lauterborn (Fig. 4. 40, F).

Poteriodendron Stein and Histiona Voigt, sometimes grouped with the choanoflagel-
lates, probably are Chrysomonadida (93. 224). This is also the case for Bicoeca James-
Clark (222).

Family 2. Phalansteriidae. Little is known about Phalansterium Cien-
kowski (181; Fig. 1. 3, A), although the presence of a simple collar
closely fitting the flagellum suggests a relationship to the Codosigidae.
The genus includes both branching and spheroid or discoid colonies
with a granular matrix.

Family 3. Trypanosomidae. These parasites have a single flagellum
ending in a blepharoplast, near which lies a spheroid or discoid kineto-
plast (Fig. 1. 10, J, K). The flagellum may or may not form part of an
undulating membrane. Life-cycles are dimorphic or polymorphic. Four
different types (Fig. 4. 41) occur in the family — the leishmanial, lepto-
monad, crithidial, and trypanosomal forms. In invertebrate hosts, the
flagellates are often attached to the lining of the digestive tract or to
other surfaces, Such stages are sometimes referred to as haptomonads;

176 The Mastigophora

the unattached flagellates, as nectomonads. Attachment may involve loss
of the distal portion of the flagellum, although the axoneme persists
between the kinetoplast and the tip of the body (Fig. 4. 41, J).

On the basis of life-cycles, six genera have been recognized (285):
Crithidia, Herpetomonas, Leishmania, Leptomonas, Phytomonas, and
Trypanosoma. Only leptomonad and leishmanial forms are found in
Leptomonas, Leishmania, and Pliytomonas.

Fig. 4. 41. A, B. Leptomonas patellae Porter, leptomonad and leish-
manial forms; x3120 (after P.). C, D. Leishmania chamaelonis Wenyon,
leptomonad and leishmanial forms; from cloaca of Chamaeleon vulgaris;
x2750 (after Wenyon). E-G. Crithidia euryophthalmi McCulloch, from
Euryophthahnus coyivivus; leishmanial stage from hind-gut, crithidial stage
(with narrow undulating membrane) from crop, and crithidial haptomonad
from hind-gut; xl875 (after McC). H-K. Trypanosoma lewisi; form from
blood of the rat, small metacyclic trypanosome from hind-gut of flea, two
crithidial haptomonads from the hind-gut, and a stage in intracellular re-
production (stomach of flea); H-J, x2400; K, xl350 (after Wenyon). L. Tryp-
anosoma brucei, xl800 (after Wenyon). M-R. Herpetomonas muscarum,
leptomonad form, two crithidial stages, trypanosomal form, and two leish-
manial stages; xI600 approx. (after Wenyon).

The Mastigophora 177

Leptomonas Kent (Fig. 4. 41, A, B) includes parasites of invertebrates.
However, the type species — Leptomonas bhtschlii Kent from the gut of
a nematode {Trilobiis gracilis) — has not been studied in detail and it is
not yet certain that more recently erected species actually belong in Kent's
genus. According to current concepts of the genus, both haptomonad and
nectomonad leptomonads may occur in the digestive tract and leishmanial
stages are to be expected in the posterior intestine. The leishmanial
forms of L. ctenocephali, which become resistant to desiccation (284),
are voided in the feces and ingested by flea larvae. The infection persists
through development of the flea (68).

Phytomonas Donovan. Members of this genus occur in invertebrates
and plants. Phytomonas davidi (85) is found as leptomonad and leish-
manial forms in the latex of Euphorbia segetalis and in the digestive tract
of a bug, Stenocephalus agilis, which feeds on the plant. After a period
of multiplication in the insect, leptomonad stages appear in the salivary
glands. These are presumably the forms infective for plants. In addition,
transfer of leishmanial stages from insect to insect has been reported.

Leishmania Ross (Fig. 4. 41, C, D). The life-cycle involves a vertebrate
and an invertebrate host. In mammals, the leishmanial form is predom-
inant, or else the only stage found, and occurs primarily in lymphoid-
macrophage cells, and occasionally in mononuclear and polynuclear
leucocytes of the peripheral blood. Leishmanial stages ingested by the
invertebrate hosts (species of Phlebotomus) develop into leptomonad
forms which multiply in the digestive tract. Infective stages are eventu-
ally inoculated into a vertebrate. Leishmania donovani, L. tropica, and
L. brasiliensis, which are parasitic in man, are discussed in Chapter XII.
Leishmania chamaeleonis, in contrast to the typical species of mammals,
occurs both as leptomonad and leishmanial forms in the cloaca of a
chameleon (285).

Crithidia Leger (Fig. 4. 41, E-G). Crithidial, leptomonad, and leish-
manial forms occur in the invertebrate hosts. However, the leptomonad
forms may be mere transitory stages in fission or in development of
crithidial and leishmanial forms. The type species, C. fasciculata, was
described from the intestine of Anopheles maculipennis (180). Leish-
manial stages, produced in the hind-gut, apparently are eliminated and
then ingested by new hosts. The occurrence of infections with C. lepto-
coridis in nymphs of the box-elder bug (188) indicates that insects may
become infected before the adult stage is reached.

Herpetomonas Kent (Fig. 4. 41, M-R) is limited to invertebrates, but
the life-cycle includes trypanosomal forms as well as the other types.
Detailed studies of the type species, H. muscarum (Leidy) Kent — some-
times known as H. muscae-domesticae (Stein) Kent — have shoAvn that
trypanosomal stages occur in flies (283) and in cultures (68). Leishmanial

178 The Mastigophora

stages may arise either from leptomonad or trypanosomal forms in the
natural host, and the crithidial stage typically lacks an undulating mem-
brane.

Trypanosoma Gruby (Fig. 4. 41, H-L). The life-cycle usually involves
both vertebrates and invertebrates (arthropods, leeches). The trypano-
somal stage occurs in the blood of vertebrates, while leptomonad and
crithidial forms are rare, if they are found at all. Intracellular leishmanial
stages may occur, as in T. cruzi. All four stages may occur in the inverte-
brate host. Haptomonads may be expected in insects infected with T.
lewisi (hind-gut of fleas), T. vivax (proboscis of Glossina morsitans), or
T. gamhiense ("salivary glands" of Glossina palpalis), for example. The
stage infective for vertebrates — the metacyclic trypanosome — is typically
an active trypanosomal form often derived from crithidial haptomonads.

Methods of transfer from invertebrate to vertebrate vary with the
species of Trypanosoma. In one group, which includes T. cruzi of man,
T. lewisi of rats, and T. melophagiu7n of sheep, metacyclic forms are
voided from the hind-gut of the vector, and infection of the vertebrate
follows contamination of wounds or mucous membranes. The metacyclic
stages of T. gamhiense, T. rhodesiense, T. evansi, and similar species de-
velop anteriorly in the vector and are transferred to the vertebrate host
by inoculation. A third type, represented by T. equiperdum, is trans-
ferred in vertebrates by coital contact and the vector has dropped out of
the cycle.

Vertebrate hosts of trypanosomes include fishes. Amphibia, aquatic and
terrestrial reptiles, birds, and various groups of mammals. Most species
of Trypanosoma, if not all, are probably non-pathogenic in their natural
hosts, or at least produce no serious damage. In man and domesticated
ungulates, however, several species cause diseases of considerable medical
and economic importance. This is particularly true in the tsetse fly areas
of Africa, where sleeping sickness of man (Chapter XII) and trypanosomi-
asis in cattle, sheep, horses, and goats have been important hindrances
to economic and social progress.

Family 4. Cryptobiidae. These are biflagellate parasites with a kineto-
plast somewhat larger than that of the Trypanosomidae. One of the
flagella extends anteriorly. The other, which is usually adherent to the
body and may or may not form part of an undulating membrane, extends
posteriorly as a free trailing portion.

The genera Cryptobia Leidy (Fig. 4. 42, A) and Trypanoplasma La-
veran and Mesnil are usually included, although some workers believe
that Trypanoplasma is a synonym of Cryptobia. However, this question
needs further study, since an undulating membrane has been described
in various species of Trypanoplasma but is absent in Cryptobia helicis
(166). Furthermore, the aciculum of C. helicis may be lacking in T^ypano-

The Mastigophora 179

\ '

Fig. 4. 42, A. Cryptobia helicis Leidy, sliowing "parabasal body" (at left),
trailing flagellum, aciculum, (at right) and nucleus; x2970 (after Kozloff).
B. Amphiinoiias globosa Kent, x480 (after Lenimermann). C. Amphiynonas
cyclopum (Kent) Blochmann, xl500 (after K.). D. Diploniita socialis Kent,
with lorica; xlOOO (after Lenimermann). E. Bodo caudatus HoUande, x2250
(after H.). F, G. Streptomonas cordata (Perty) Klebs; different views show-
ing keel; xl334 (after Lemniermann). H. Pleuromonas jaculans Perty, x767
(after Lemniermann). L Spongomonas iivella Stein; gelatinous matrix con-
tains many granules; x347 (after Lemmerniann). J. Proteromonas lacerti
(Grassi), showing "parabasal body" and a ring which encircles the flagellar
axoneme and parabasal rhizoplast; x2550 approx. (after Grasse). K. Colpo-
nema loxodes Stein; trailing flagelhnn extends along prominent ventral
groove; endoplasm is "granular"; xl200 (after Klebs). L. Pseudobodo m'mima
Hollande; compact "parabasal body" anterior to the nucleus; x3600 (after
H.). M. Dinomonas tuberculata Kent; xl7I0 (after K.). N. Phyllomitus amy-
lophagus Klebs; ventral view showing pharyngeal groove and elongated
"parabasal body"; x3375 (after Hollande).

180 The Mastigophora

plasma. Species of Cryptobia occur in the seminal vesicles and digestive
tract of molluscs and certain other invertebrates, and in the digestive
tract of marine fish. Species of Trypanoplasma occur in the blood of
marine and fresh-water fishes.

Family 5. Amphimonadidae. These are naked or loricate types with
two equal flagella (181). Naked types may be either free-swimming or
sessile. Colonial forms are assigned to several genera. The group as a
whole is much in need of investigation.

Solitary types include Amphimonas Dujardin (245; Fig. 4. 42, B, D), Diplomita Kent
(Fig. 4. 42, D), Spiromonas Perty (181), and Streptomonas Klebs (Fig. 4. 42, F, G).
Colonial types are assigned to Cladotnonas Stein (Fig. 1. 3, F), Rhipidodendron Stein
(181) and Spongomonas Stein (Fig. 4. 42, I).

Family 6. Bodonidae. These are solitary naked flagellates reported from
fresh and salt water, and from the digestive tract of certain reptiles and
Amphibia. One of the two flagella is usually trailed in swimming. A para-
basal apparatus is known in several genera. The Feulgen-positive para-
basal body of Bodo divides in fission (110) and is thus similar to the
kinetoplast of Trypanosomidae.

The following genera are included: Bodo (Ehrbg.) Stein {Proiuazekia Hartmann and
Chagas) (110; Fig. 4. 42, E); Cercobodo Krassiltschick (109, 110; Fig. 1. 10, P); Cerco-
monas Dujardin (113, 282); Colponema Stein (151; Fig. 4. 42, K); Dinomonas Kent
(130; Fig. 4. 42, M); Phyllomitus Stein (110; Fig. 4. 42, N); Pleuromonas Perty (181;
Fig. 4. 42, H); Proteromonas Kunstler {Prowazekella Alexeieff) (98; Fig. 4. 42, J), from
the intestine of lizards and salamanders; Pseudobodo Hollande (110; Fig. 4. 42, L).

Order 3. Polymastigida

Erection of the Order Trichomonadida by Kirby (147) has re-
moved from the old Order Polymastigida several families of closely
related uninucleate and multinucleate flagellates. As retained here, the
Polymastigida include families which are excluded from the Trichomo-
nadida but form an otherwise heterogeneous group. This arrangement
will serve a practical purpose until accumulated data permit a more
satisfactory classification. In this restricted sense, the Polymastigida
usually have 3-8 flagella and one, two, or occasionally a number {Micro-
rhopalodina) of nuclei. A parabasal apparatus is known in the Hexa-
mitidae but its homology with that of the Trichomonadida is not yet
certain. Seven families are retained in the order: Trimastigidae, Tetra-
mitidae, Streblomastigidae, Retortomonadidae, Callimastigidae, Poly-
mastigidae, and Pyrsonymphidae.

Family 1. Trimastigidae. There are three flagella, one anterior and
two trailing (181). Almost nothing is known about the cytology of the
group. One genus has been reported from salt water and two others
from fresh water. The family includes Dallingeria Kent and Trimastix

The Mastigophora 181

Fig. 4. 43. A. Retortomunas gryllotalpae (Grassi) Stiles, ventral view
showing peristomial fibril, two flagella, nucleus (in outline); x2000 (after
Wenrich). B. Chilonwstix intestiiialis Kuczynski, ventral view showing peri-
stomial fibril, four flagella, food vacuole, nucleus (in outline): x2000 (after
Wenrich). C. Macrumastix lapsa Stokes, x2250 (after Lemmermann). D, E.
Costia necatrix (Henneguy) Leclerq; ventral view showing groove and bases
of flagella; lateral view; x3750 (after Tavolga and Nigrelli). F. Coprnmastix
prowazeki Aragao, showing groove (at left), nucleus and rhi/ostyle; xI444
(after A.). G. Retortomonas gryllotalpae, lateral view (after Wenrich). H.
Relortomonas agilis Mackinnon, x2880 (after Ludwig). I, J. Chilomastix
magna Becker, showing nucleus, peristomial fibril, and intracytoplasmic
band but not the cytostomal flagellum (I), x2160; protargol technique, show-
ing peristomial fibril and flagella (J), x2850 (after Kirby and Honigberg).
K. Tetrarnitus rostratus Perty, showing groove, rhizostyle, nucleus; x2250
(after Hollande). L. Tetrarnitus salinus (Entz) Kirby, showing groove, ante-
rior nucleus, and food vacuole developing at base of gullet; x2320 (after K.).
M. Streblomastix strix Kofoid and Swezy, showing flagella and long slender
nucleus; xHOO (after K. & S.).

182 The Mastigophora

Kent, both with a long anterior flagellum, and Macromastix Stokes (Fig.
4. 43, C) with a short anterior flagelkim. A lateral membrane (or keel?),
which is not an undulating membrane, extends the length of the body
in Trimastix. The flagellar equipment of Macromastix resembles that of
the chrysomonad genus Prymneshim Massart (Fig. 4. 8, A). Similarly,
Chrysochromiilina (Fig. 4. 8, D) is similar to DaUingeria and Trimastix.
Perhaps the Trimastigidae should be investigated for possible affinities
with the Chrysomonadida.

Family 2. Tetramitidae. There are four unequal or equal flagella, one
or two of which may be trailed. No parabasal body or axostyle has been
reported, although a rhizostyle is present in Tetramitus (Fig. 4. 43, K)
and Copromastix (Fig. 4. 43, F). A dimorphic cycle involving flagellate
and amoeboid stages is known in Tetramitus (29, 110).

The following genera have been included in the family: Costia Leclerq
(7, 59, 269; Fig. 4. 43, D, E), from the skin of fish; Tetramitus Perty (29,
110, 153, 245; Figs. 4. 43, K, L, 2. 14, C-E), in which the life-cycle includes
amoeboid and flagellate stages; and Tricercomonas Wenyon and O'Con-
nor (22, 65, 285; Fig. 11. 2, A-E), from the intestine of man. Enteromonas
Fonseca may be an additional valid genus, although Dobell (65) has con-
cluded that Tricercomo7ias is merely a synonym of Enteroynonas. How-
ever, da Cunha and Muniz (53), as well as Fonseca, have described
Entero?nonas intestinalis with one long and two short flagella, and in
contrast to Tricercomonas, without any trace of a fourth flagellum or
caudal extension. The status of Copromastix Aragao is uncertain. C.
prowazeki Aragao (Fig. 4. 43, F) is so similar to Tetramitus rostratus (29,
110) that the two flagellates probably should be referred to the same
genus.

Family 3. Streblomastigidae. These parasites of termites {Termopsis),
have an unusually slender body with a few spirally wound ridges and an
anterior group of four flagella (131, 158). The flagella arise from the
anterior tip of the body which can be extended as a slender holdfast
organ. The only known genus is Streblomastix Kofoid and Swezy (Fig.
4. 43, M).

Family 4. Retortomonadidae. This family (278) includes Retortomonas
Grassi {Embadomonas Mackinnon) (Fig. 4. 43, A, G, H) and Chilomastix
Alexeieff (Fig. 4. 43, B, I, J). Both Retortomonas (18, 150, 187, 278) and
Chilomastix (149, 198, 278) possess a cytostomal groove, in the margin of
which a cytoplasmic fibril extends across the anterior end and posteriorly
along each side. A true parabasal body is lacking. The significance of a
differentiated intracytoplasmic "band/' sometimes apparent just beneath
the right limb of the peristomial fibril (149), is uncertain. In both genera,
a single trailing flagellum emerges from the cytosomal groove. Retorto-
monas is distinguished from Chilomastix by the presence of one instead
of three anterior flagella. The cytostomal flagellum in Chilomastix has
been interpreted as part of an undulating membrane by Nie (198) and

The Mastigophora 183

Fig. 4. 44, A, B. Monocercomonoides pilleata Kirby and Honigberg; pro-
targol technique (A), showing pelta, "costa," axostyle, trailing flagellum,
bases of anterior flagella; specimen showing flagellar connections, nucleus,
and axostyle (B); x2880 (after K. & H.). C. Callimastix equi Hsiung, show-
ing heavy tuft of flagella; xll66 (after H.). D. Dinenympha ftmbriata Kirby;
nucleus, heavy axostyle, four adherent flagella which become free posteri-
orly, and bacteria attached to the body; xIOOO (after K.). E. Pyrsonympha
minor Powell; nucleus, axostyle (split posteriorly); the adherent flagella
arise from the apical "centroblepharoplast" and extend posteriorly as eight
spiral cords; x900 (after P.). F. Oxymonas dimorpha Connell, non-flagel-
lated attached form with extended rostellum; axostyle and subpellicular
supporting fibrils extend posteriorly from rostellum; nucelus and ingested
wood chips indicated; x425 (after C). G. O. dimorpha, motile form, rostel-
lum not extended; xI750 (after C). H. Polymastix phyUophagae Travis and
Becker; nucleus, axostyle, adherent bacilli; x2400 (after T. & B.). I. Micro-
rhopalodina {Proboscidiella) multinucleata (Kofoid and Swezy), showing
rostellum (which may be extended to several times body length), multiple
karyomastigonts (each with a heavy axostyle); bacteria are usually attached
to the body; xllSO (after K. & S.). J. Saccinobacubis doroaxostylus Cleve-
land; broad axostyle, nucleus, flagella; x600 (after C),

184 The Mastigophora

several earlier workers. Such a relationship remains doubtful in certain
species of Chilomastix (149).

Both Retortomonas and Chilomastix are represented by species in in-
sects and vertebrates. Chilomastix mesnili and Retortomonas intestinalis
of man are discussed in Chapter XI.

Family 5. Callimastigidae. This family includes Callimastix Weissen-
berg (Fig. 4. 44, C), represented by species from the stomachs of cattle,
goats and sheep, from the cecum and colon of horses, and from the body
cavity of Cyclops. The most striking feature is a compact antero-lateral
group of flagella which beats as a unit.

Family 6. Polymastigidae. Four flagella arise as two pairs from the
anterior end of the body. There is an axostyle but apparently no para-
basal body. A pelta is present in Moriocercomonoides pilleata (149), and
a possibly homologous structure ("parabasal body") occurs in Polymastix
phyllophagae (272). The family includes Polymastix Biitschli (98; Fig.
4. 44, H) from insects and Monocercomonoides Travis (149, 271; Fig. 4.
44, A, B) from rodents and insects.

Family 7. Pyrsonymphidae. These are uninucleate or multinucleate
flagellates. Each karyomastigont usually contains four, but sometimes
eight or twelve flagella, and one axostyle. An intranuclear spindle ap-
pears in mitosis (39). Some members of the family (e.g., Kirbyella,
Oxymonas) are attached, by means of an extensible rostellum, to the gut
wall of termites.

The family (139) includes several uninucleate genera — Dinenympha Leidy (133, 160;
Fig. 4. 44, D), Pyrsonympha Leidy (160, 233; Fig. 4. 44, E), Saccinobaculus Cleveland
(39; Fig. 4. 44, J) from the wood roach, Metasaccinohacuhis de Freitas (87), and
Oxymonas Janicki {Opisthomitus Duboscq and Grasse) (41, 52; Fig. 4. 44, F, G) — and
the multinucleate Microrhopalodina Grassi and Foa (Proboscidiella Kofoid and Swezy)
(159a; Fig. 4. 44, I) and Kirbyella Zeliff (286).

Oxymonas, Microrhopalodina, and Kirbyella seem to be restricted to the termite
genus Kalotermes; Saccinobaculus, to the wood roach; the rest of the group, to
Reticulotermes.

Family 8. Hexamitidae. These are binucleate organisms with six or
eight flagella and, in at least certain genera, parabasal bodies and axo-
styles. Bilateral symmetry is typical of the family. The group includes
free-living and parasitic types. Species of Giardia are widely distributed
intestinal parasites of vertebrates. Giardia lamhlia of man is discussed in
Chapter XI. Hexamita meleagridis (105, 194) is associated with a catarrhal
enteritis in young turkeys. Other species of Hexamita have been reported
from monkeys (279), Amphibia (267), fishes (58), leeches (17), reptiles
and rodents, and also as free-living flagellates. The genus Trepomonas
also contains both free-living and parasitic species.

The family includes the following genera: Giardia Kunstler (Fig. 4. 45, H), Gyro-
nionas Seligo (245; Fig. 4. 45, E, F), Hexamita Dujardin (Octomitus Prowazek) (Fig. 4.

The Mastigophora 185

Fig. 4. 45. Hexamitidae. A. Urophagus rostratus (Stein) Klebs, xl200
(after K.). B. Hexamita pitheci (da Cunha and Muniz) Wenrich, from Ma-
cacus rhesus: paired nuclei, axostyies and flagella; x3465 (after W.). C. Hexa-
mita gigas Bishop, from a leech (Haemopsis sangiiisugae); elongated nuclei,
two axostyies, food vacuoles; x2640 (after B.). D. Trigonomonas compressa
Klebs, x833 (after K.). E, F. Gyromonas ambulans Seligo, narrow and broad
surfaces; x945 (after S.). G. Trepomonas agilis Dujardin; two comma-shaped
nuclei, paired flagella, ingested bacteria; x2500 (after Bishop). H. Giardia
muris (Grassi), showing axostyle, paired nuclei, parabasal bodies, and flag-
ella; concave ventral area indicated in outline; x2550, schematic (after
Kofoid and Christiansen).

45, B, C), Trepomonas Dujardin (19; Fig. 4. 45, G), Trigonomonas Klebs (153; Fig. 4.
45, D), and Urophagus Klebs (Fig. 4. 45, A). It is possible that Urophagus should be
considered a synonym of Hexamita.

Order 4. Trichomonadida

These flagellates have an axostyle, a parabasal body (not a kineto-
plast), and a mastigont of 3-6 flagella (147). One flagellum is a trailing
flagellum which may or may not form part of an undulating membrane.
Each mastigont is typically associated with one nucleus, although a
partial or complete dissociation has occurred in certain multinucleate
species. A paradesmose appears in mitosis. Members of the order, as now
known, are uninucleate or multinucleate, not binucleate.

186 The Mastigophora

Fig. 4. 46. A. Devescovina vestita Kirby, showing adherent baciUi, trail-
ing flagellum, projecting axostyle, basal portions of anterior flagella; xll65
(after K.). B. Hexamastix termopsidis Kirby; nucleus, axostyle, jDarabasal
body, ingested bacteria; x2100 (after K.). C, D. Tricercomitus termopsidis
Kirby; rounded form showing nucleus and axostyle (C), xl650; slender form
(D) from recently molted nymph, xl600 (after K.). E. Pseudotrichomonas
keiliui Bishop, with short undulating membrane; x2970 (after B.). F. Deves-
covina arta Kirby; ribbon-like trailing flagelhmi, small cresta, parabasal
body curled aroimd axostyle; xI190 (after K.). G. Parajoenia grassii Janicki;
stout axostyle with anterior expansion, branched parabasal body, pennant-
like costa, four flagella, adherent spirochetes; subcuticular inclusions shown
posteriorly; x875 (after Kirby). H. Monocercomonas verreus Honigberg,
with projecting axostyle; x3420 (after H.). I. Monocercomonas phyllophagae
(Travis and Becker); heavy axostyle, long trailing flagellum; x2700 (after
T. & B.).

The Mastigophora 187

Family 1. Monocercomonadidae. There is either a free or an adherent
trailing flagelhini but no cresta and no undulating membrane with its
underlying costa. The group includes parasites of the digestive tract in
termites, certain other insects, and all classes of vertebrates. However, the
distribution of particular genera ranges from that of Tricercomitus, in
termites only, to that of Monocercoconas, reported from various groups
of vertebrates and insects, including termites.

The family contains the following genera: Hexamastix AlexeiefF (136; Fig. 4. 46,
B). Monocercomonas Grassi (Eiitrirlininastix Kofoid and Swezy, Trichomastix Bloch-
mann) (137; Fig. 4. 46, H, I), Protrichnmonas Alexeieff (2), Pseudotrichomonas Bishop
(21; Fig. 4. 46. E), Tetratrichomastix Mackinnon (190), and Tricercomitus Kirby (136;
Fig. 4.^46, C, D).

Family 2. Devescovinidae. A group of three anterior flagella is char-
acteristic and there is also a trailing flagellum which becomes a rather
broad ribbon in some species. The trailing flagellum is often adherent to
the body through part of its length but there is no undulating membrane.
Bacteria are commonly attached to the surface of the body. The charac-
teristic cresta varies from a small narrow structure to a wide band ex-
tending almost the length of the body. The parabasal body ranges from
a short rod to a long structure coiled around the axostyle. The axostyle
may curve forward along one side of the nucleus. More commonly, the an-
terior part of the axostyle is flattened into a capitulum. The Devesco-
dinidae are known from termites, almost entirely from the Kalotermitidae.
The occurrence of encystment is doubtful and flagellates probably are
transferred by proctodeal feeding.

The following genera are included: BuUanympha Kirby (148; Fig. 1. 8, E), Caduceia
Franca (142; Fig. 4. 47, B), Devescoviua Foa (141; Fig. 4. 46, A, F), Foaina Janicki (143;
Fig. 4. 47, C), Gigantomonas Dogicl (146; Fig. 2. 14, H-J), Hyperdevescovina Kirby
(148; Fig. 4. 47, E). Macrotriclwinonas Grassi (142; Fig. 4. 47, D), Metadevescovina
Light (145; Fig. 4. 47, A), Parajoenia Janicki (143; Fig. 4. 46, G), and Pseudodevescovina
Sutherland (145; Fig. 4. 47, F). Gigantomojjas differs from the others in that the cycle
includes an amoeboid stage, sometimes multinucleate, in which elements of the mas-
tigont may be much reduced.

Family 3. Calonymphidae. These are multinucleate flagellates with
eight (Coronympha) to hundreds of mastigonts (Snyderella), each usu-
ally containing four flagella. One of the four is typically a trailing flag-
ellum. The cresta is well developed in some species but is small or else
lacking in others. The axostyles range from fairly heavy separate struc-
tures to slender filaments which form a compact axial bundle. Coronym-
pha, Metacoronympha, and Stephanonympha contain karyomastigonts
exclusively. In Calonympha there are both karyomastigonts and masti-
gonts, while the mastigonts and nuclei are completely dissociated in
Snyderella. The Calonym25hidae have been reported mostly from the

188 The Mastigophora

Fig, 4. 47. A. Metadevescovina modica Kirby, x750 (after K.). B. Cadu-
ceia bugnioni Kirby; adherent spirochetes indicated, bacilli not shown; axo-
style expanded anteriorh; long parabasal body coiled around axostyle; x700
(after K.). C. Foaina taeniola Kirby, xl310 (after K.). D. Marrotrichomonas
lighti (Connell) Kirby; large cresta (stippled), long coiled parabasal body:
x700 (after K.). E. Hy perdenescovina mitrata Kirby, x750 (after K.). F.
Pseudodevescovina iiniflagellnta Sutherland; axostyle expanded anteriorly,
complex parabasal apparatus; x750 (after Kirby).

termite genus Kalotermes; Snyderella seems to be limited to a single
species of that genus.

The family includes Calonympha Tok (122), Coronympha Kirby (135a, 140; Fig. 4.
48, F), Metacoronympha Kirby (140), Snyderella Kirby (135a; Figs. 1. 8, C; 1, 10, C),
and Stephanonympha Janicki (134; Fig. 1. 10, D, E),

Family 4, Trichomonadidae. These are uninucleate types with an un-
dulating membrane and an underlying costa. In addition, a pelta occuzs

The Mastigophora 189

Fig. 4. 48. A. Trichomonas limacis Dujardin, showing pclta, beaded
and bifurcated parabasal body, axostyle, and nucleus; x2415 (after Koz-
loff). B. Tritrichomouas augusta Alexeieff; axostyle, nucleus, parabasal
body, heavy costa; xl680 approx. (after Samuels). C. Tritrichomouas foetus,
parabasal body not shown; x2795 (after Wenrich and Emmerson). D. Tri-
chomonas gallinae (Rivolta) Stabler, x3400 (after S.). E. Pseudotrypano-
soma gigantea Grassi; heavy costa, long parabasal body parallel to axostyle,
long undulating membrane; x575 (after Kirby). F. Coronympha clevelajidi
Kirby, showing anterior circle of karyomastigonts, axostyles extending pos-
teriorly; xl400 (after K.).

in some species. The group is widely distributed in vertebrates and cer-
tain invertebrates. Several parasites of man are discussed in Chapter XI.
TricJwmonas gallinae is a pathogen in the anterior digestive tract of
pigeons (261, 262); Tritrichonionas foetus is a parasite of the genital
tract in cattle (196, 281); Trichomonas gallinarum occurs in the ceca of
chickens and turkeys and the liver of turkeys. Like Histomonas rnele-

190 The Mastigophora

agridis, T. gaUinarum is associated with "blackhead" in poultry (4, 5).

The family includes the following genera: Pentatrichomonas Mesnil (Fig. 11. 3, AC);
Pentatrichomonoides Kirby (137); Pseudotrypanosoma Grassi (137; Fig. 4. 48, E);
Trichomonas Donne (Fig. 4. 48, A, D), for which Morgan (195) has published a host-
parasite catalog; and Tritrichomonas Kofoid (Fig. 4. 48, B, C).

Order 5. Hypermastigida

These are uninucleate organisms with many flagella. Multiple axo-
styles and parabasal bodies also are characteristic. All known species are
intestinal parasites of termites, wood roaches or cockroaches. Feeding
methods may be saprozoic or holozoic, and some species ingest wood chips
swallowed by the host (77). Two suborders, Lophomonadina and Tricho-
nymphina, have been recognized.

Suborder 1. Lophomonadina. In this group, the flagella and associated
structures are arranged in one anterior group which is resorbed in fission.
The suborder includes three families which differ in arrangement of the
flagella.

Family 1. Lophomonadidae. The blepharoplasts form an anterior ring
so that the flagella, if numerous (Fig. 4. 49, C), form a distinct tuft. The
axostyle, at least in Lophomonas and Torquenympha (Fig. 4. 49, B), is
a bundle of fibrils enclosing the nucleus anteriorly. The fibrillar bundle
may be split posteriorly into several fibrils in Torquenympha (27). Mem-
bers of the group are known from the digestive tract of cockroaches
[Lophomonas), the wood roach (Prolophomonas), and certain termites
(Torquenympha).

The family includes Prolophomonas Cleveland (39), Lophomonas Stein (168, 169),
and Torquenympha Brown (27). The flagella number 24 or less in Prolophomonas
(Fig. 4. 49, A) and Torquenympha, but are more numerous in Lophomonas.

Family 2. Joeniidae. Although limited to an anterior area, the blepharo-
plasts are arranged in longitudinal rows instead of a compact ring. As a
result, there may be an anterior tuft of flagella, as in Joenia and Joenopsis,
while the rest of the flagella are trailed. The flagellar rows may extend
past the middle of the body in Joenopsis (55), but are shorter in Micro-
joenia (27, 55; Fig. 4. 49, D). A paired parabasal apparatus is quite
simple in Microjoenia. In Joenopsis and Joenia, however, there are two
filaments to which are attached numerous rod-like parabasal bodies (55).

The following genera have been reported from termites: Joenia Grassi, Joenopsis
Cutler, Joenina Grassi (98a), Mesojoenia Grassi and Foa, and Microjoenia Grassi.

Family 3. Kofoidiidae. The flagella are arranged in a spiral series of
permanent bundles. The nucleus lies within a "suspensorium" from
which filaments radiate into the cytoplasm. These filaments may be

The Mastigophora 191

0^''

^S^' > \ " "^1^ i;//i 'ijf'

^1 M K .^ ri#

/;/ V

Fig. 4. 49. A. Prolopliomouas tocopola Cleveland, showing axostyles,
nucleus, food vacuoles; xl200 (after C). B. Torqitetiyinpha octoplus
Brown, showing parabasal bodies and fibrillar axostyle which surrounds
the nucleus anteriorly; xl645 (after B.). C. Lophomonas striata Biitschli,
showing axostvlar filaments which form a "calyx" enclosing the nucleus;
adherent bacteria (Fusiformis lophomonadis Grassc) indicated on body;
xl475 approx. (after Kudo). D. Microjoenia ratcliffei Brown, showing two
parabasal bodies, axost^le, nucleus, and anterior rows of blepharoplasts;
x2380 (after B.). E. Koifoidia loriculata Light, showing bundles (loriculae)
of flagella; xl75. F. A', loriculata, anterior end of body showing nucleus
suspended in membranous "suspensorium," bases of several loriculae, and
body fibrils extending into cytoplasm; x750 (after L.).

analogous to the axostylar bundle in Torque?iympha and Lophomonas.
The general organization, although more complex, is similar to that in
Lophomonas. The type genus is Kofoidia Light (183; Fig. 4. 49, E, F),
reported from one species of Kalotermes.

Suborder 2. Trichonymphina. The retention of flagella and associated
structures in fission is characteristic. Organization is basically bilateral,
and there are either two or four sets of organelles which are separated
equally in fission. Encystment is known for species of Macrospironympha
and Trichonympha in the wood roach (39), but not for the Trichonym-
phina of termites.

192 The Mastigophora

Fig. 4. 50. A. Staurojoenina assimilis Kirby. showing four flagellar groups,
rhizoplast bands extending anteriorly from the nucleus, and the major body
filaments extending posteriorly; cuticular striations indicated at lateral mar-
gins; X330 (after K.). B. Optical section, anterior end of S. assimilis, showing
four flagellar groups; X330 (after Kirby). C. Barbitlanympha ufalula Cleve-
land; two anterior flagellar gioups. nucleus surrounded by parabasal bodies;
axostylar filaments extend posteriorly; x20o (after C). D. Uiinyinpha talca
Cleveland; two flagellar groups, nucleus suspended by nuclear sleeve; axo-
stylar filaments extending posteriorly; X3r,o (after C). E, F. Hoplonympha
natator Light; surface view showing two flagellar tufts and spiral pellicular
grooves (E); optical section showing nucleus suspended by rhizoplast bands
(enclosed in granular column); a delicate endoplasmic thread (primitive
axostyle?) extends posteriorly; X855 (after L.).

Family 1. Hoplonymphidne. The flagella arise in tAvo anterior groups.
One group passes to each daughter organism in fission. Hoplonympha is
represented in termites; three other genera, in the wood roach (Crypto-
cercus).

The Mastigophora 193

^S'

■'■/■iv.:' :i'-

i''\'iV''

Fie. 4. 51. A. Holomastigotoides hemigymnum Grassi; nucleus, axostyle
(expanded anteriorly), flagellar bands (flagella indicated only at sides of
body); x320 approx. (after Mackinnon). B. Spirotrichonympha elegans
(Mackinnon); rostellar tube, nucleus, axostUe (expanded anteriorly), flag-
ellar bands (only the marginal flagella are shown); xl820 (after M.). C.
Spirnnympha porteri Koidzumi; axostvle, nucleus, flagellar l)ands with at-
tached parabasal bodies; marginal flagella indicated; adherent spirochetes
posterior to the flagellar bands have sometimes been mistaken for flagella;
xl600 approx. (after Brown).

The family includes Barbulanymplta Cleveland (39; Fig. 4. 50, C), Hoplonympha
Light (182; Fig. 4. 50, E, F), RhyncJwnympha Cleveland (39), and Urinympha
Cleveland (39; Fig. 4. 50, D).

Family 2. Staurojueuinidae. The flagella are arranged in four anterior
groups. A number of slender fibrillar axostyles are attached to each
flagellar group, and in Idionympha four groups of slender parabasal
"cords" are associated with the flagellar groups.

The family includes Staurojoenina Grassi (133a; Fig. 4. 50, A, B) from
termites and Idionympha Cleveland (39) from the wood roach.

Family 3. Holomastigotidae. The flagella arise from bands of basal
granides which extend spirally around the body. Two, four, or more
bands have been reported in different species. Apparent variations within
a species possibly involve duplication in fission.

The family includes Holomastigotes Grassi (72, 160), Holomastigotoides Grassi and
Foa (14, 160, 192; Fig. 4. 51, A), Leptospironympha Cleveland (39), Macrospironympha
Cleveland (39), Spironympha Koidzumi (28; Fig. 4. 51, C), Spirotrichonymphella
Grassi, Spirotrichonympha Grassi (54, 72, 160, 193; Fig. 4. 51, B) and Spirotrichosoma
.Sutherland (266). Leptospironympha and Macrospironympha have been reported from
the wood roach; the other genera, from termites.

194 The Mastigophora

i;Vr->v'-',AOi.

^Mi.

■■:^y

IP^P-

Fig. 4. 52. A. Teratonympha sp. from Reticulotermes speratus; anterior
end of body showing rostral tube, rostra! flagella, nuclear "sleeve" extending
from nucleus into rostral tube, and supporting fibrils surround nuclear
sleeve and nucleus; the fibrils end posteriorly in the first flagellar band;
x840 (after Cleveland). B. Surface view of Teratonympha showing circular
flagellar bands; flagella indicated diagrammatically; x280 (after Cleveland).
C. Eucomonympha inula Cleveland, showing rostrum with anterior cap
(operculum), nucleus, and fibrillar axostyles extending posteriorly; x350
(after C). D. Triclwnympha corbula Kirby, showing three flagellar zones
and the parabasal bodies surrounding the nucleus; x475 (after K.).

Family 4. Trichonymphidae. Except for the tip of the rostrum, the sur-
face of the body is flagellated in certain genera {Deltotrichonyynpha ,
Eucomonympha, Mixotricha, Pseudotrichonympha). In others, a small or
a large posterior portion is bare. The flagella are arranged in longitu-
dinal rows, and may form two or three transverse zones diffiering in

The Mastigophora 195

length of the flagella. The parabasal apparatus consists of a number of
parabasal cords, usually encircling the nucleus and attached by filaments
to the parabasal lamella at the base of the rostrum (71, 144). Differences
in form, size, and number of the cords are useful taxonomic features. In
the rostrum, the conical anterior end of the body (Fig. 4. 52, C, D), the
blepharoplasts and the parabasal lamella, internal to them, form a rostral
"tube." This tube is sometimes widened posteriorly into a cone, as in
Eucomonympha (Fig. 4. 52, C).

The family contains Deltotrichonympha Sutherland (72, 266), Eucomonympha Cleve-
land (39; Fig. 4. 52, C), Mixotricha Sutherland (266), Pseudotrichonympha Grassi
(39, 160), and Trichonympha Leidy (39, 138. 144; Fig. 4. 52, D). Trichonympha is
represented in termites (three families) and in the wood roach (39). Eucomonympha
has been reported from the wood roach; the other genera, from single families of
termites.

Family 5. Teratonymphldoe. This family was erected for Teratonytnplm
Koidzumi (Cyclonympha Dogiel) from termites. The rostrum is similar
to that of Trichonymphidae, but the post-rostral flagella arise from cir-
cular bands underlying grooves which give the body a segmented appear-
ance (40, 160; Fig. 4. 52, A, B).

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196 The Mastigophora

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125. and T. L. Jahn 1942. Physiol. Zool. 15: 89.

126. Kamptner, E. 1928. Arch. f. Protistenk. 61: 38.

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132. Killian, C. 1924. Arch. f. Protistenk. 50: 50.

133. Kirby, H. 1924. Univ. Calif. Publ. Zool. 26: 199.
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134. 1926. Univ. Calif. Publ. Zool. 29: 103.

135. 1928. Quart. J. Micr. Sci. 72: 355.

135a. 1929. Univ. Calif. Publ. Zool. 31: 417.

136. 1930. Univ. Calif. Publ. Zool. 33: 393.

137. 1931. U7iiv. Calif. Publ. Zool. 36: 171.

138. 1932. Univ. Calif. Publ. Zool. 37: 349.

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198 The Mastigophora

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142. 1942. Univ. Calif. Publ. Zool. 45: 93.

143. 1942. Univ. Calif. Publ. Zool. 45: 167.

144. 1944. Univ. Calif. Publ. Zool. 49: 185.

145. 1945. Univ. Calif. Publ. Zool. 45: 247.

146. 1946. Uriiv. Calif. Publ. Zool. 53: 163.

147. 1947. /. Parasit. 33: 214.

148. 1949. Univ. Calif. Publ. Zool. 45: 319.

149. and B. Honigberg 1949. Univ. Calif. Publ. Zool. 53: 315.

150. and 1950. Univ. Calif. Publ. Zool. 55: 35.

151. Klebs, G. 1892. Ztschr. f. wiss. Zool. 55: 322.

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153. Klug. G. 1936. Arch. f. Protistenk. 87: 97.

154. Kofoid, C. A. 1899. Bull. III. St. Lab. Nat. Hist. 5: 273.

155. 1909. Arch. f. Protistenk. 16: 25.

156. 1911. Univ. Calif. Publ. Zool. 8: 187.

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159. and 1921. "The Free-living Unarmored Dinoflagellates." Univ. Calif.

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161. Korshikov, A. A. 1926. Arch. f. Protistenk. 55: 439.

162. 1927. Arch. f. Protistenk. 58: 441.

163. 1927. Arch. f. Protisteiik. 58: 450.

164. 1928. Arch. f. Protistenk. 61: 223.

165. 1929. Arch. f. Protistenk. 67: 253.

166. Kozloff, E. 1948. /. Morph. 83: 253.

167. Krichenbauer, H. 1937. Arch. f. Protistenk. 90: 88.

168. Kudo, R. 1926. Arch. f. Protistenk. 53: 191.

169. 1926. Arch. f. Protistenk. 55: 504.

170. Kuschakewitsch, S. 1931. Arch. f. Protistenk. 73: 323.

171. Lackey, J. B. 1929. Arch. f. Protistenk. 66: 175.

172. 1934. Biol. Bull. 67: 145.

173. 1936. Biol. Bull. 71: 492.

174. 1939. Lloydia 2: 128.

175. 1940. Amer. Midi. Nat. 23: 463.

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179. 1923. /. Mar. Biol. Assoc. 13: 271.

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183. 1927. Univ. Calif. Publ. Zool. 29: 467.

184. Loefer, J. B. 1931. Arch. f. Protistenk. 74: 449.

185. 1937. Physiol. Zool. 12: 161.

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193. 1927. Quart. J. Micr. Sci. 71: 47.

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The Mastigophora 199

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203. 1916. Arch. f. Protistenk. 37: 31.

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208. 1928. Arch. f. Protistenk. 63: 241.

209. 1929. Arch. f. Protistenk. 68: 637.

210. 1929. Atin. Protistol. 2: 157.

211. 1930. Beih. Bot. Centralhl. 47: 271.

212. 1930. Arch. f. Protistenk. 69: 401.

213. 1930. Arch. f. Protistenk. 72: 311.

214. 1931. Beih. Bot. Centralbl. 48: 317.

215. 1931. Arch. f. Protistenk. 73: 315.

216. 1932. Beih. Bot. Centralbl. 49: 293.

217. 1932. Arch. f. Protistenk. 76: 1.

218. 1932. Arch. f. Protistenk. 77: 305.

219. 1932. Beih. Bot. Centralbl. 49: 549.

220. 1940. Arch. f. Protistenk. 93: 331.

221. 1940. Arch. f. Protistenk. 94: 295.

222. 1942. Arch. f. Protistenk. 96: 75.

223. 1942. Beih. bol. Centralbl. 61: 462.

224. 1943. Intern. Rev. ges. Hydrobiol. Hydrogr. 43: 110.

225. 1943. Arch. f. Protistenk. 96: 288.

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250. 1925. Arch. f. Protistenk. 51: 1.

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200 The Mastigophora

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V
The Sarcodina

Class 1. Actinopodea
Order 1. Helioflagellida
Order 2. Heliozoida

Suborder 1. Actinophrydina
Suborder 2. Acanthocystidina
Suborder 3. Desniothoracina
Order 3. Radiolarida
Life-cycles
Taxonomy

Suborder 1. Actipylina
Suborder 2. Peripylina
Suborder 3. Monopylina
Suborder 4. Tripylina

Class 2. Rhizopodea

Order 1. Proteomyxida
Family I. Labyrinthulidae
Family 2. Pseudosporidae
Family 3. Vampyrellidae

Order 2. Mycetozoida
Suborder 1. Acrasina
Suborder 2. Plasmodiophorina
Suborder 3. Eumycetozoina

Order 3. Araoebida

Family 1. Dimastigamoebidae

Family 2. Amoebidae

Family 3. Endamoebidae
Order 4. Testacida

Pseudopodia

Contents of the test

Life-histories

Ecological relationships

Taxonomy

Family 1. Arcellidae
Family 2. Difflugiidae
Family 3. Euglyphidae
Order 5. Foraminiferida

Pseudopodia and their activities

Tests

The endoplasm

Life-cycles

Reproduction of the agamont
Gametogenesis and syngamy
Duration of the life-cycle

Taxonomy

Family Allogromiidae
Literature cited

.HE Sarcodina are mostly floating or creeping organisms, al-
though a number are sessile. The thin periplast permits the formation
of pseudopodia and the amoeboid movement of naked species. Locomo-
tion may or may not involve the formation of definite pseudopodia.
Certain amoebae, for instance, move by a protoplasmic flow which in-
volves the body as a whole and does not depend upon pseudopodia. Some
Sarcodina also develop flagella at certain stages in the life-cycle. Flagel-
late stages occur as gametes in various Foraminiferida; in certain other
Sarcodina, a similar status of the flagellate stage is suspected but not
proven. In addition, there are cases in which the flagellate stage seems
to be merely a second active phase in a dimorphic life-cycle. The ability

201

202 The Sarcodina

to develop a test is widely distributed. Such structures are found in
Testacida and Foraminiferida and in the majority of Heliozoida. The
lattice-work skeletons of many Radiolarida are analogous developments.

The Sarcodina as a group are widely distributed in fresh and salt
water and in the soil. However, the Radiolarida have remained marine
and the Foraminiferida which have invaded fresh water are primitive
types sometimes considered Testacida. A number of the Sarcodina are
parasitic. Various sessile forms may be epiphytic or epizooic, but endo-
parasitism is limited to the more primitive species or to possibly degen-
erate representatives of certain groups.

On the basis of pseudopodial equipment, the Sarcodina are often di-
vided into two classes, Actinopodea and Rhizopodea. By definition, the
Actinopodea possess axopodia. The Rhizopodea may have any other kind
of pseudopodia but do not develop axopodia.

CLASS 1. ACTINOPODEA

These are mostly floating or sessile organisms, although flagellate
stages are known in a few genera. Accessory lobopodia are developed oc-
casionally, at least in certain species. The class may be divided into three
orders: (1) Helioflagellida, with one or more flagella as either a perma-
nent feature or a characteristic of the dominant phase in a dimorphic
cycle; (2) Heliozoida, in which a flagellate stage apparently is rare and
the inner cytoplasm is not separated from the outer zones by a central
capsule; (3) Radiolarida, in which a central capsule is characteristic and
skeletal structures are more highly developed than in Heliozoida.

Order 1. Helioflagellida

The relationships of this group are uncertain, and members of the
order have been classified as Rhizomastigida (Mastigophora) and Pro-
teomyxida, as well as Helioflagellida. The presence of axopodia, and also
a "central granule" in certain genera, suggests closer affinities with the
Heliozoida than with the Rhizomastigida or Proteomyxida. The Helio-
flagellida are of interest as possible sources of data bearing on phylogeny
of the Actinopodea.

The following genera may be assigned to the order: Acinetactis Stokes (141, 143;
Fig. 5. 1, A); Actinomonas Kent (45; Fig. 5. 1, K); Ciliophrys Gruber (45; Fig. 5. 1, D,
E); Dimorpha Gruber (Fig. 5. 1, I-J); Dimorphella Valkanov (143; Fig. 5. 1, B, C);
and Tetradimorpha Hsiung (61; Fig. 5. 1, F-H). A "central granule," from which the
axoncmes of the axopodia radiate, has been demonstrated in Dimorpha, Dimorphella.
and Tetradimorpha. This central granule behaves as a centrosome during mitosis in
Dimorphella elegans (Fig. 5. 1, C). With the possible exception of Tetradimorpha, the
pseudopodia show the granules characteristic of axopodia; streaming of the granules
has been described in Acinetactis and Dimorphella. More or less complete retraction
of the pseudopodia occurs in swimming stages of Acinetactis, Ciliophrys, Dimorpha,
and Tetradimorpha. Both marine and fresh-water species of Ciliophrys have been
described; the other genera have been reported from fresh water.

The Sarcodina 203

/m\

.-''' -' / '• < 'i '•

i-*;v^*i

Fig. 5. 1. Helioflagellida. A. Acinetactis arnaudoffi Valkanov; two fiagella.
granular axopodia; x800 (after V.). B, C. Dimorphella elegans Valkanov; fiag-
ella and axopodia arising from a central gianule (B); stage in division (C);
x2400 (after V.). D, E. Ciliophrys marina Caullery;" axopodia retracted in
flagellate stage (D); granular axopodia extended (E); x960 (after Griessmann)'.'
F-H Tetradimorpha radiata Hsiung; axopodia extended, nucleus central, x325
(F); typical swimming stage, x480 (G); stained preparation showing nucleus,
blepharoplast, axonemes of retracted axopodia (H), x490 (after H.). I-J. Di-
morpha mutans Gruber; axopodia and flagella arising from a central granule
(I); axopodia retracted (J); xl060 approx. (after Blochmann). K. Actitiomonas
mirabiUs Kent, one flagellum, axopodia extended; xl360 (after Griessmann).

Order 2. Heliozoida

The Heliozoida possess radially arranged axopodia which rarely
anastomose, and typically contain globules or granules. A flow of granules
along the axopodia is characteristic. The finer structure of the pseudo-

204 The Sarcodina

podia has been discussed by Roskin (126). The inner and peripheral zones
of cytoplasm are not separated by a central capsule. Most Heliozoida are
approximately spherical floating types, and except for a few species of
Acanthocystis, Camptonema, and certain other genera, occur in fresh
water. The recognition of typical Heliozoida is easy enough. However,
it is difficult to detect axonemes in the delicate pseudopodia of certain

/ 7/ / I \ \ '•

Fig. 5. 2. Basic morphological types in Heliozoida; diagrammatic. A. Acan
thocystis-type: test composed of separate plates, spines sometimes present
nucleus not central; axopodia radiate from a central granule. B. Clathrulina
type, as in Desmothoracina: perforated test not composed of separate scales;
stalk often present. C. Acti7iophrys-type: no test; nucleus approximately cen
tral in uninucleate forms. D. Nuclear division in Acanthocystis aculeata, show
ing supposed central granules at the poles of the spindle; xlOlO (after Belar)

forms and there are some species in which axonemes have not yet been
reported.

With respect to the peripheral cytoplasm and its derivatives, Heliozoida
may be divided into naked types and those which secrete some sort of a
test. The test may contain discrete scales or spines (Fig. 5. 2, A), or it
may be a continuous capsule containing many pores (Fig. 5. 2, B). In
such naked types as Actinophrys (Fig. 5. 2, C), the outer cytoplasm con-
tains many vacuoles, one or more of which may be contractile. The vac-
uolated layer encloses a thick granular zone of cytoplasm within which,
in uninucleate species, a large nucleus is more or less centrally located.

The Sarcodina 205

Around the nucleus, there is a hyaline layer in which the axonemes end.
In the Acanthocystis-type (Fig. 5. 2, A), the vacuolated zone is lacking
and the body is covered with a test composed of skeletal elements em-
bedded in a capsule. Some such covering is found in the majority of
Heliozoa. Beneath the relatively thin ectoplasm there is a thick granular
zone containing one or more contractile vacuoles, food vacuoles, and
other inclusions. Within the granular layer, a zone of clear cytoplasm
contains the "central granule" and a nucleus. The central granule, in
which the axonemes converge, resembles a centrosome in its behavior
dining mitosis (Fig. 5. 2, D). However, Stern (139), on the basis of multi-
nucleate and other abnormal stages seen in cultures, has argued that the
central granule does not really function as a centrosome.

Fig. 5. 3. A-D. Ingestion of a fiagellate by Acanthocystis aculeata, succes-
sive stages; xl215 (after Stern). E. Formation of a food vacuole outside the
test in Hedriocystis pellucida; xl050 (after Hoogenraad). F. A large lobo-
podium, in addition to axopodia, in Raphidocystis infestans; x8I5 (after
VVctzcl). G. Cytostome-like structure, with food vacuole at the base of the
"gullet," in Actinosphaerium eichorni; x34 (after Okada). H. A ciliate (Para-
mecium) attacked by a group of Raphidocystis infestans; xl28 (after Wetzel).
I. A ciliate completely surrounded by such a group; stained preparation; x238
(after Wetzel).

206 The Sarcodina

Feeding is predominantly holozoic, and food includes other Protozoa,
algae, and occasionally rotifers or other small invertebrates. After cap-
ture of such organisms, axial filaments may disappear in the immediate
region and a layer of cytoplasm surrounds the prey (Fig. 5. 3, E). Occa-
sionally, captured microorganisms pass immediately into the deeper cy-
toplasm where digestion is completed (Fig. 5. 3, A-D). In addition to
axopodia, lobopodia are sometimes formed (158) and the ingestion of
food by means of gullet-like "food cups" also may occur (Fig. 5. 3, G).
A protozoan version of the hunting pack has been described in Raphi-
docystis infestans (158). A ciliate, for example, may be attacked by a
number of these Heliozoida, which adhere to the prey and may fuse to
form a continuous layer of protoplasm enclosing the captured food (Fig.
5. 3, H, I). A simple life-cycle — including an active stage and a cyst — has
been reported in a number of Heliozoida. Cysts with a siliceous ectocyst
have been described in certain species (108). An alternation of genera-
tions, in one of which flagellate gametes are produced, has been reported
in Wagnerella borealis (163), although this account has not been con-
firmed. The formation of a flagellate daughter organism (Fig. 5. 7, D,
L), which leaves the parental test, has been described in Monomastigo-
cystis (129) and Hedriocystis (54).

The work of Belar and his predecessors has established the occurrence
of pedogamy in certain Heliozoida, or at least the occurrence of syngamy
following a gametic meiosis (Chapter II). The zygote so produced nor-
mally undergoes encystment.

Subdivision of the Heliozoida has been based largely upon the presence
or absence of skeletal elements and their structure. On such a basis, the
group may be divided into three suborders: (1) Actinophrydina, the
naked types; (2) Acanthocystidina, with a gelatinous capsule in which
separate skeletal elements are usually embedded; and (3) Desmothora-
cina, with a continuous test containing a number of pores.

Suborder 1. Actinophrydina. There is no capsule or test enclosing the
outer zone of vacuolated cytoplasm. Since there is no central granule, the
axopodia may end near the nuclear membrane (Fig. 5. 4, E) in uninu-
cleate species, or near a nucleus or inner margin of the vacuolated layer
in inultinucleate types. Just beneath the vacuolated zone of Actinosphae-
rium eichorni, there is a finely granular layer (Fig. 5. 4, B) which may
serve as a support for the bases of the axopodia (83, 108). Inside the
granular layer lies the finely vacuolated endoplasm. The boundary be-
tween endoplasm and ectoplasm is not so sharply defined in the smaller
Actinophrydina.

The suborder includes the following genera: Actinophrys Ehrenberg (5, 6, 83. 108;
Fig. 5. 4, C-F), uninucleate fresh-water types; Actinosphaerium Stein (83, 108; Fig. 5.
4, A, B), multinucleate fresh-water types; Cawptonema Schaudinn (133), multinucleate
marine forms. Actinosphaerium eichornii, which often measures more than 300^^ and

The Sarcodina 207

rM.

T--.;»-: ■ y.V.*^-:---

^J'-^'^'^-^x

I F

Fig. 5. 4. Actinophrydina: A. Actinosphaerium eichorni Ehrenberg (diam-
eter may reach or exceed SOOfi); axopodia, peripheral zone of vacuoles; in-
gested food (after Penard). B. Portion of peripheral cytoplasm, A. eichorni,
showing an axoneme ending in a granular layer just beneath the vacuolated
zone; diagrammatic (after Penard). C, D. Actinophrys pontica Valkanov;
stained specimen (C), xl200; fused aggregate of three organisms (D), x800
(after V.). E, F. Actinophrys sol Ehrenberg; stamed section of small specimen
showing axonemes extending to nucleus, x975; living specimen from culture,
x325 (after Belar).

may exceed 1000/^ in diameter, is the largest of the Actinophrydina. Other species fall
within the range, 25-150/1.

Suborder 2. Acanthocystidina. There is typically a secreted capsule,
sometimes "gelatinous" (Fig. 5. 5, A, G), in which skeletal elements are
embedded. The ectoplasm is not extensively vacuolated. In some genera at
least, the axonemes are known to end in a central granule. Data are
lacking in other cases, In Astrodiscuhis (Fig. 5. 5, A), the capsule is thick

208 The Sarcodina

but contains no skeletal elements. In other genera, the capsule varies in
thickness and may be reduced to a thin membrane which binds the
skeletal structures together. With the apparent exception of Heterophrys,
the skeletal elements are siliceous. Aside from a few species such as
Lithocolla glohosa (108), in which foreign particles are cemented to a
thin capsule, the skeletal scales and spicules are products of the organism.

^ ! /

--t'

k
9

■^■:^<H' ...

^^^

^ s -*/•■; © - A * . lib ^>j3

f D

Fig. 5. 5. Acanthocystidina: A. Astrodisculus radians Greet, "gelatinous"
covering without scales; x575 approx. (after Penard). B. Pinaciophora fliivia-
tills Greef (diameter, 45-50fi), test composed of scales (after Penard). C.
AcantJiocystis rubella Penard (diameter, 23-27/i); portion of body showing
tangential scales; radially arranged spines are enclosed within the axopodia
(after P.). D. Cienkowskya mereschkowskyi (diameter about 60(a), a sessile
form; scales embedded in gelatinous mantle; distal portions of axopodia not
shown (after Villeneuve). E-G. Raphidocystis infestans Wetzel; cyst being
released from ruptured test (E) and freed cyst (F), x570; dividing form,
skeletal elements dissolved with HFl to show gelatinous envelope (G), x820
(after W.). H. Raphidiophrys pallida Schulze, x2.50 (after Penard).

The Sarcodina 209

The differentiation of genera is based to an important extent upon
thickness of the capsule and the form and arrangement of the skeletal
elements (Fig. 5. 6, A-H).

The following genera arc included in the suborder: Acarithoryslis Carter (83, 108
154; Fig. 5. 5, C); Actinolophus Schulze (149; Fig. 5. 6, I); AstrocUsculus Greef (108
Fig. 5. 5, A); Cienkowskya Schaudinn (149; Fig. 5. 5, D); Elaeorhanis Greef (108)
Heterophrys Archer (108; Fig. 5. 6, J); Lithocolla Schulze (108); Oxnerella Dobell (34)

t 1

'f-i

^1 G

E F

^\{ml :/'/

. . ^\\^^\.

^/' I

Fig. 5. 6. Acanthocystidina: A-H. Skeletal elements: A. Raphidocystis
ambigua; B. Acanthocystis mimetica, spine and scales; C. A. aculeata, spine
and scales; D. Raphidiophrys elegans, surface and edge views of scales; E.
Raphidocystis glutinosa; F. Raphidiophrys intermedia, surface and edge
views of scales; G. Spine of Heterophrys myriopoda; H. Raphidocystis
leniani; schematic (after Penard). I. Actinolophus pedunculatus Schulze
(body, 35 x 30;n), sessile on Bryozoa; radially arranged bodies within test
believed to be ingested food (after Villeneuve). J. Heterophrys myriopoda
Archer, x330 (after Penard). K. Pompholyxophrys punicea Archer, x400
approx. (after Penard).

210 The Sarcodina

Pinaciocystis Roskin (128); Pinaciophora Greef (108; Fig. 5. 5, B); Pompholyxophrys
Archer (108; Fig. 5. 6, K); Raphidiophrys Archer (108, 158; Fig. 5. 5, H); Raphidocystis
Penard (108, 158; Figs. 5. 3, F, H, I, 5. 5, E-G); and Wagnerella Mereschkowski (163).
The status of Myriophrys Penard (108) is uncertain. The secreted envelope with
adherent scales, the slender granular pseudopodia, and the large eccentric nucleus
would seem to qualify the genus for the Acanthocystidina. A coat of undulating "cilia
or flagella" complicates matters. Perhaps these "flagella" should be investigated as
possible bacteria adherent to the body. The genus Chondropus Greef (108) must re-
main unassigned until more is known about the organisms.

^k.-

C£ i^-Q

/- ,.c^

■•-• :•■.■:■■ J

g G

Fig. 5. 7. Desmothoracina: A-F. Monomastigocystis brachypoiis De Saede-
leer (width, 9-15//): specimen with short stalk (A); optical cross-section (B);
in fission (C, D) one daughter organism develops into a flagellate (E); cyst
(F) with double membrane (after De S.). G-L. Hedriocystis pellucida:
young specimen without test (G), schematic (after Valkanov); mature form
(H), x700; fission (I, J), x315; one daughter organism becomes a biflagellate
stage which leaves the test (K, L), x525 (after Hoogenraad). M. Clathrulina
ehgans Cienkowski; diameter of test, 60-90;i (after Penard).

The Sarcodina 211

Suborder 3. Desmothoracina. In this group, there is a non-siliceous
(108) one-piece test (Fig. 5. 2, B) containing pores through which pseu-
dopodia are extended. Certain genera contain sessile types with stalks.
The stalk in Hedriocystis (Fig. 5. 7, G, H) is said to be merely an exten-
sion of the body resembling a slender pseudopodium (144). In Clathru-
lina (Fig. 5. 8, E-H), the young organism first develops a protoplasmic
stalk by outgrowth from the body. An outer covering is then secreted

B i

/ ,

...'"v\«i!iV'- i .-ly

.^....:::fj||^M|^::.::::.....

.^^^■■

' C

'"%^'

•art

• : '3' \ ■-

Hill

Fig. 5. 8. Desmothoracina: A. Hedriocystis reticulata Penard, x500 ap-
prox. (after P.). B. Choanocystis lepidula Penard, x730 approx. (after P.).
C. Clathrella foreli Penard; diameter of test, 40-55/i (after P.). D. Elastcr
greefi Grimm, x700 (after Penard). E-H. Clathrulina e/ega?J5 ^ienkowski: '
cytoplasm grows down over the original stalk (F) and produces a hollow
stalk (G), which becomes continuous with the test in older forms (H);
schematic (after Valkanov).

212 The Sarcodina

and the protoplasmic core disappears, leaving a tubular mature stalk
attached only to the test (144).

Although little is known about the life-cycles, fission within the test,
the development of a flagellate stage from one of the daughter organisms,
and encystment have been described (Fig. 5. 7) in Monomastigocystis
(129) and Hedriocystis (54).

The taxonomic relationships of the Desmothoracina are still debatable.
Superficially, they show striking resemblances to typical Heliozoida. Al-
though the granular pseudopodia seem to be axopodia, they are some-
times so slender that the presence of axonemes is uncertain. The nucleus
is central in some species and eccentric in others, but no central granule
has been demonstrated. In view of the apparent absence of axonemes
and a central granule, Valkanov (148) suggested transfer of the Desmo-
thoracina to the Foraminiferida as another monothalamous group.

The following genera have been assigned to the suborder: Choaiiocystis Penard (108;
Fig. 5. 8, B); ClathruUna Cienkowski (83, 108, 144; Fig. 5. 7, M); Blaster Grimm (108;
Fig. 5. 8, D); Hedriocystis Hertwig and Lesser (54, 108, 144; Fig. 5. 7, G-L); Monomasti-
gocystis de Saedeleer (129; Fig. 5. 7, A-F).

Order 3. Radiolarida

These marine organisms, with a geological history dating at least
from Lower Silurian and probably from Cambrian time, are apparently
the oldest known group of animals. Their most striking feature is their
skeleton, which has undergone specialization to a remarkable degree. The
general organization of the body and the possession of axopodia relate
them to the Heliozoida, but the central capsule, separating inner and
outer zones of protoplasm, is a differential feature.

The central capsule is nearly always a distinct layer, usually single
but sometimes double (Fig. 5. 13, A), and can be detected without diffi-
culty except in a few Actipylina (Acantharina). The capsule may be
spherical, ovoid, or sometimes lobate or branched (Fig. 5. 11, C), and is
composed of organic material designated variously as chitin, pseudochitin,
or tectin. The capsule may be resorbed more or less completely in fission
of the simpler species, it may increase in diameter with growth of the
organism, and it may be somewhat changeable in form even in the mature
organism. Perforations, either distributed uniformly or concentrated in
one or more groups, permit cytoplasmic continuity and also serve as
taxonomic features.

The skeleton of the Actipylina may be composed largely of strontium
sulphate, usually with a radial arrangement of the skeletal elements. The
basic components are spines which extend radially from the center of the
body, passing through the central capsule (Fig. 5. 9, A). At the surface of
the body there may also be a lattice-work test, or shell, which is fused with
the radial spines (Fig. 5. 9, D). For the other groups of Radiolarida, silice-

The Sarcodina 213

ous skeletal elements are the rule. Rods and spines, if present, always
lie outside the capsule. In addition to rod-like elements, or in their
absence, one or more lattice-work layers may be deposited, peripheral
to, and concentric with, the central capsule. The lattice framework may
be spherical or non-spherical (bell-shaped, helmet-shaped, etc.), and in

/';'

ti^^^-

/'/ hwyr

Fig. 5. 9. A. Acanthometra pellucida, showing central capsule, axial rods
and "myonemes" (myophrisks) joining the superficial cytoplasm and the
sheaths of the axial rods; x200 (after Moroff and Stiasny). B, C. Axial rods
and myophrisks in Actipylina; ectoplasmic layer expanded and myophrisks
contracted (B); ectoplasmic layer contracted and myonemes extended (C);
schematic (after Schewiakoff). D. Dorotaspis lieteropora Bernstein, showing
lattice-work shell and axial rods; schematic (after B.).

the latter case may approach bilateral symmetry. Complicated skeletons
already had been developed early in the known history of the Radiolarida
(Fig. 5. 11, A, B).

The intracapsular cytoplasm contains the nucleus or nuclei, stored re-
serves, pigment granules in some species, and the so-called "yellow cells"
in the Actipylina. The number of nuclei varies. The Actipylina are typ-

214 The Sarcodina

ically multinucleate, while the Monopylina and Tripylina are usually
uninucleate. The extracapsular cytoplasm is concerned primarily with
flotation, capture of food, and digestion. Several layers may be recog-
nizable (Fig. 5. 11, D): the sarcomntrix, a so-called digestive layer next to
the central capsule; the vacuolated calymma, which is a thick zone in some
species; a thin layer outside the calymma; and the zone of axopodia whose
axonemes often arise in the sarcoma trix. Food is captured much as in the
Heliozoida. Since the size of solitary species ranges from about 50[x to
several millimeters, the larger Radiolarida are able to feed on copepods
and other small Crustacea, as well as on algae and Protozoa which come
in contact with the pseudopodia.

The "yellow-cells" (zooxanthellae), present in many Radiolarida al-
though not in the Tripylina, are more numerous in species with a well-
developed calymma. They are typically intracapsular in the Actipylina,
extracapsular in other groups. In the living host, these parasites are com-
monly spherical to ovoid. After death of the host, they may develop into
palmella stages which give rise to flagellates. Certain of these flagellates
have been referred to the Dinoflagellida (22, 58). Their reputed status as
symbiotes remains somewhat uncertain.

Some of the Radiolarida, such as Collozoum and Sphaerozoum, are
colonial forms (16, 140) in which a number of central capsules are em-
bedded in an elongated or more or less spherical mass of extracapsular
cytoplasm. In certain species at least, each central capsule contains a
number of nuclei. Skeletal elements are often reduced to scattered spic-
ules, although lattice-work shells occur in some species.

Life-cycles. As a result of the difficulties in obtaining adequate material
for study, little is known about the life-cycles of Radiolarida. Various
accounts in the older literature suggest that the life-cycles may be fairly
complex, but more extensive observations are needed. Since some of the
shallow-water species will survive in the laboratory for reasonable periods,
perhaps the application of techniques which have already been so pro-
ductive for Foraminiferida would yield valuable information on Radio-
larida.

Although reproduction has been traced in relatively few species, fission
occurs in species with simple skeletal elements. The central capsule is
divided, and any skeletal elements are passed on to the two daughter
organisms. Fission also has been reported within the helmet-shaped skele-
ton of certain Tripylina. One daughter organism retains the old shell;
the other leaves and develops a new one. According to Brandt (14), cer-
tain Thallophysidae may undergo a complicated plasmotomy which fol-
lows dedifferentiation of the adult, and results in a number of small
organisms, each with several nuclei. Budding possibly occurs in a few
species (15), but the process needs further investigation.

Evidence for sexual phenomena in Radiolarida is still inconclusive, al-

The Sarcodina 215

though the literature contains repeated descriptions of flagellate stages
(flagellispores) — supposedly gametes. However, syngamy has not been ob-
served, and Chatton (23) concluded that some of these supposed flagellate
stages are probably parasites. This conclusion certainly seems justified for
"flagellispores" which are similar to dinoflagellates. However, some of
these flagellates (80) obviously are not dinoflagellates (Fig. 5. 10) and
they show a marked resemblance to flagellate gametes of Foraminiferida
(Fig. 5. 42).

Although the Radiolarida are not swimmers, at least some of them
apparently can rise or sink in response to changing environmental con-
ditions. A collapse of vacuoles in the calymma increases the specific grav-
ity of the organism and thus induces sinking; regeneration of the vacuoles
reverses this effect. Such a mechanism enables species living near the

Fig. 5. 10. "Flagellispores" (gametes?) of Radiolarida: A, B. Acantho-
metra pellucida, dividing gametocyte showing paradesmose (A) and biflag-
ellate gamete (B); x4000 (after Le Calvez). C. Xiphicantha alata, x4000
(after Le Calvez). D, E. Coelodendrum ramosissimum, living (D) and
stained (E); x2550 (after Le Calvez).

surface to sink when disturbed by rough wave action or when the tem-
perature becomes unfavorable.

The majority of species probably live within the upper 1,500 feet, al-
though a few forms have been dredged from depths of 2-3 miles. Within
this vertical range, the fauna varies to a considerable extent with depth.
The majority of the Peripylina are found within the upper 200 feet, while
the Actipylina are most abundant below 150-200 feet. The Tripylina are
to be found mainly within a range of 1,200 to 3,500 feet. The group as a
whole is widely distributed over the oceans, although specific distribution
varies considerably. Some species show essentially universal distribution
while others may be limited to tropical or to polar waters. The greatest
variety of species occurs within the equatorial zone. Radiolarian skeletons,
sinking to the bottom, make up deposits of radiolarian ooze, and many
fossil types are known.

216 The Sarcodina

Taxonomy.^ The Radiolarida are subdivided, on the basis of skeletal
structure and the distribution of pores in the capsule, into four suborders:
(1) Actipylina ("Acantharia"), with a skeleton composed basically of
radial spines which penetrate the central capsule to converge in the mid-
dle of the body; (2) Peripylina ("Spumellaria"), often with no skeleton
or one limited to disconnected extracapsular rods and less commonly with
a perforated shell; the spherical central capsule shows uniformly dis-
tributed pores; (3) Monopylina ("Nasselaria"), with a thick central cap-
sule in which the pores are limited to one zone, or "porous plate" (Fig.

•..■"•-•!-^^»_-'^ ■ '^ >' ' ' ■ '■-' ' ■'/.•••■'; .-•'' .•-

■■■■.::t — T' ir ' ^•— \ , ' -■[, ■ ^-^i .•"■;.■■■".'■

..."::-.>-^,'i:'-;-- :_ /■ : • ',- ■• ." - ■'• , ./;

•^\::^>'"*^\: : ';. ,-^- ■.;; ^.j '- -r,::"--*,^,_^.Zj;vz'.'.

•'A

••^;fe^^'%?

Fig. 5. 11. A. Ceiiosphaera tnacroponi Riist, from Ordovician (Lower
Silurian) deposits; x]20 approx. (after R.). B. Staurolonche micropora Riist
(Ordovician), xl20 approx. (after R.). C. Branched central capsule of Cyto-
r'ndus spinosus, x5 (after Schroder). D. ThalassicoUa nucleata, from living;
central capsule (surroimded by zone of small vacuoles), layer of hyaline
cytoplasm, calymma, and axopodia (after Huth).

5. 12, F); and (4) Tripylina ("Phaeodaria"), in which the central capsule
has one major and two accessory openings (Fig. 5. 13, A).

Suborder 1. Actipylina. The central capsule, sometimes irregular in
shape, is rather uniformly perforated, although arrangement of the pores
in rows or fields is often recognizable. The skeleton consists mainly of
rods which converge inside the central capsule (Fig. 5. 9, A-C) and usually
show an arrangement described by Miiller's "law." There are often twenty

^ More detailed information will be found in such special monographs as the follow-
ing: general: Haeckel, E. 1887. Challenger Rep., Zool. 18; Hertwig, R. 1879. Der
Organismus der Radiolarien (Jena); Actipylina: Popofsky, A. 1904. Ergebn. Plankton-
exped. 3, 1907. Nordisches Plankton 16; Schewiakoff, W. 1926. Fauna Flora G. Neapel
37; Peripyliyia: Schroder, O. 1914. Nord. Plankt. 17; Monopylina: Popofsky, A. 1913.
Ergebn. Deutsch. Siidpol.-exp. Bd. 14, Zool. 6; Tripylina: Borgert, A. 1903-1911. Ergebn.
Planktonexp. 3.

The Sarcodina 217

(sometimes multiples of twenty) rods which form a characteristic pattern.
An equatorial group emerges from the body in a plane essentially 90°
from either pole, and two other groups emerge in planes about 45° above
and below the equatorial plane. The basic skeleton is sometimes modified
by lateral outgrowths from the rods which form a perforated shell, com-
posed typically of twenty plates. Two such shells, concentric with the
central capsule, are present in certain species. The outer layer of extra-
capsular cytoplasm is joined to the skeletal rods, apparently by contrac-
tile fibrils ("myophrisks") which are said to bring about minor changes
in form and volume of the body (Fig. 5. 9, B, C) and thus to aid in
controlling flotation.

The suborder includes such genera as the following: Acantlwcliiasma Krohn, Acan-
thometm Midler (Acaiithoinetron Haeckel) (94; Fig. 5. 9, A), AcantJionia Haeckel,
Actmelius Haeckel, Amphilonche Haeckel, Diplocolpus Haeckel, Diploconus Haeckel,

Fig. 5. 12. A-C. Skeletal features of Monopylina: tripod and central cap-
sule (A); tripod and ring enclosing central capsule (B); helmet-like skeleton
(cephalis, capitulum) derived from the more primitive tripod and ring (C);
schematic (after Haeckel). D. Helmet-like skeleton of Eucyrlidium cranioidcs
Haeckel, xllO approx. (after H.). E. Skeleton of Dictyophimus gracilipes
Bailey, schematic (after Bernstein). F. Lithocircus annularis Hertwig, skel-
eton, central capsule with perforated plate, nucleus (in outline); schematic
(after H.).

218 The Sarcodina

Dorotaspis Haeckel (Fig. 5. 9, D), Hexaconus Haeckel, Litholopus Haeckel, Lithoptera
Miiller, Phractaspis Haeckel, Podactinelius Schroder, Sphaerocapsa Haeckel, and Tlioro-
capsis Haeckel.

Suborder 2. Peripylina. There is a fairly thick spherical central capsule
in which numerous pores are uniformly distributed. There is no skeleton
at all in some species. In others, a relatively simple skeleton consists of
scattered extracapsular spicules, a perforated shell, or both. The lattice-
work shells may be single, or in certain families, often multiple in a con-
centric series. In CoUosphaera, Collozoum, and Sphaerozoiim the central
capsules, instead of separating after fission, remain embedded in a com-
mon extracapsular mass to form colonies which may measure several
centimeters.

The following genera have been included in the Peripylina: Acanthosphaera Ehren-
berg, Arcliidiscus Haeckel, Cenolarcus Haeckel, CenospJiaera Ehrenberg (Fig. 5. 11, A),
Chitoanastrum Haeckel, CoUosphaera Miiller, Collozoum Haeckel (140), Cromyodrymus
Haeckel, Cytocladus Schroder, Druppula Haeckel, Euchitonia Haeckel, Lampoxanthiurn
Haeckel, Orosphaera Haeckel, Physeiuaticum Haeckel, Pipetla Haeckel, Sphaerozoum
Meyen, Staurocyclia Haeckel, Staurosphaera Haeckel, Thalassicolla Huxley (62; Fig. 5.
II, D), Thalassolampe Haeckel, Thallasophysa (14), Thalassothamnus Hacker.

Suborder 3. Monopylina. The thick-walled central capsule (Fig. 5. 12,
F), which may be radially or bilaterally symmetrical, shows a single porous
plate or, more often, a single field of small pores with thickened walls.
The pseudopodia usually arise opposite this field. The siliceous skeleton,
composed of solid elements, may show three distinct parts (tripod, capit-
ulum, and ring). The basic form of the tripod (Fig. 5. 12, A) suggests
the name applied to the structure. The ring, if present, is attached to
the tripod (Fig. 5. 12, B). Outgrowths from the ring and tripod may result
in a hemlet-shaped shell, the capitulum (Fig. 5. 12, C-E). Modification of
these three basic elements, by suppressions or by the addition of append-
ages and decorations, gives rise to a variety of skeletons.

The suborder includes the following genera: Cortiniscus Haeckel, Cystidium Hert-
wig, Dictyophimus Ehrenberg (Fig. 5. 12, E), Eucyrtidium Haeckel (Fig. 5. 12, D),
Lithocircus Miiller (Fig. 5. 12, F), Protympanium Haeckel, Stichoformis Haeckel,
Theopera Haeckel, Theophormis Haeckel, Triplagia Haeckel, Zygostephanus Haeckel.

Suborder 4. Tripylina. The central capsule has one major (the astro-
pyle) and two accessory openings (parapyles), the latter usually lying
opposite the first (Fig. 5. 13, A). The astropyle typically is covered with
a striated plate, in which the central openings are often drawn out into
tubes. A characteristic feature is an accumulation of greenish-brown mate-
rial (perhaps the reinnants of diatoms and other food) just outside the
astropyle. This colored material ("phaeodium") is responsible for the
name, "Phaeodaria," often applied to this suborder.

The siliceous skeletons show a wide range in complexity. The skeletons

The Sarcodina 219

Fig. 5. 13. A Central capsule of Trip)lina, showing inner and outer
layers, astropyle, two parapyles, and large nucleus; diagrammatic (after
Gamble). B. Bivalve shell and its appendages, galea with nasal process, or
rhizocanna; astropyle drawn out into a tube; diagrammatic (after
Gamble). C. Costanidiuin sol Hacker, portion of skeleton showing lattice-
work shell and radial elements; diameter of shell, 400-500^ (after H.). D.
Skeleton of Challengeron armatum Borgert, xl70 (after B.).

of Aulacantha and related genera consist of separate elements, hollow
radially arranged rods and sinaller tangentially distributed spicules. The
latter are often replaced by a lattice-work shell (Fig. 5. 13, C, D); or two
shells may be present, one just outside the central capsule. In some genera,
only the inner shell is developed. Several families show a bivalve inner
shell (Fig. 5. 13, B), and each valve sometimes bears a hollow apj^endage,
the galea.

The group includes the following genera: Aulacantha Haeckel (13), Aulosphaera
Haeckel, Cannosphaera Haeckel, Castanidium Haeckel (Fig. 5. 13, C), Challengeron
Haeckel (Fig. 5. 13, D), Circoporus Haeckel, Coelacantha Hertwig, Coeloden-
drum Haeckel, Coementella Borgert, Conchoceras Haeckel, Euphysetta Haeckel,
Medusetta Haeckel, Tuscarilla Haeckel, Tuscarora Murray.

CLASS 2. RHIZOPODEA

These Sarcodina may have lobopodia, filopodia, or myxopodia but
do not develop axopodia and do not show a foamy peripheral cytoplasm.
Tests, well developed in certain groups, may be composed mainly of
organic material, with or without added foreign particles, or largely of

220 The Sarcodina

inorganic materials such as calcium salts. Binuclearity and multinuclear-
ity are not uncommon.

The group is usually divided into five orders: (1) Proteomyxida, which
often develop slender filopodia, sometimes delicate ones which super-
ficially resemble axopodia; (2) Mycetozoida, plasmodial organisms, which
move primarily by protoplasmic flow, and certain other types which de-
velop a pseudoplasmodium; (3) Amoebida, naked forms which usually
show lobopodia; (4) Testacida, which have a simple test and may form
filopodia or lobopodia in different genera; (5) Foraminiferida, which have
either a simple or a multi-chambered test and typically develop myxo-
podia.

Order 1. Proteomyxida

This order is not clearly defined and the interrelationships of the
families usually assigned to it need investigation. The mature stage in
certain genera is a large plasmodium; in others, an amoeboid uninucleate
organism. Both flagellate and amoeboid stages occur in certain genera;
in other cases, a flagellate stage is unknown. Three families are often
included in this order: (1) Labyrinthulidae, uninucleate organisms which
grow in "nets" and may form an aggregate (pseudoplasmodium) before
encystment; (2) Pseudosporidae, uninucleate forms with amoeboid and
flagellate stages; (3) Vampyrellidae, in which the mature stage is a plas-
modium.

Family 1. Labyrinthulidae. These are little known Proteomyxida which
parasitize eel grass and various algae. The organisms usually form a
peculiar network (Fig. 5. 14, A, D), the organization of which has been
disputed in Lobyrinthula. According to one interpretation, the individual
organisms are joined by cytoplasmic processes; according to another view
(145), they are held together by a tubular membrane in L. zopfi (Fig. 5.
14, A, B). Neither interpretation is supported by the observations of
Young (162) on Labyrhitluila mocrocystis, in which the "connections"
are interpreted as filamentous "tracks" secreted by the individual organ-
isms. At the advancing end of a net the organisms first form a clump (Fig.
5. 14, D). Then, hyaline filaments, one from each individual, "dart" for-
ward to a length several times that of tlie organism. The filaments wave
about until they meet and fuse to form a track. The organisms, by a
method still undetermined, glide along such a track "like a drop of
glycerin rolling down a taut silk thread" (162). One organism may over-
take and pass another without either one leaving the track. Since the
organisms may leave the track independently, they apparently do not lie
within a tube. Growth of the net involves fission of the organisms.

The life-cycles need more investigation. A slowly moving pseudoplas-
modium, composed of a mass of organisms embedded in a thin matrix,
has been observed in Labyrinthula macrocystis (162). Encystment of 1-8

The Sarcodina 221

Fig, 5. 14. A-C. Labyrinthula zopfi Valkanov (individual organisms reach
8/i in length): portion of Hving network (A); two organisms stained (B);
encysted stage, from hving (C); schematic (after V.). D, E. Labyrinthula
macrocystis Cienkowski: vegetative network (D), x380 approx.; single organism,
stained, showing nucleus and vacuole (E), x2700 approx. (after Young).

organisms within one membrane has been described in Labyrinthula
zopfi (Fig. 5. 14, C), and in L. macrocystis, a membrane may be formed
around a pseudoplasmodium composed of 5-100 organisms (162).

The family includes Labyrinthula Cienkowski (145, 162), reported from eel grass and
certain marine algae (Cladophora, Chaetomorpha); and Labyrinthomyxa Duboscq (35),
reported from Laniinaria. Labyrinthula macrocystis has been found associated with a
fungal disease of eel grass (120, 162), and it is possible that the organisms, by attack-
ing the plant cells, contribute to the spread of infection.

Family 2. Pseudosporidae. These organisms invade filamentous algae
and Volvocidae. The parasitic stages are amoeboid. Either flagellate or
amoeboid "swarm-cells" may be produced, depending apparently upon
the species. The best known genus is Pseiidospora Cienkowski (127, 134,
135; Fig. 5. 15). Several other genera- — Protomonas Cienkowski, Apheli-
dium Scherffel, Amoeboaphelidiujn Scherffel, Aphelidiopsis Scherffel,
Pseudosporopsis Scherffel, Barbetia Dangeard — appear to be related to
Pseudospora and presumably belong to the same family (134).

A fairly complex life-cycle has been described for Pseudospora parasi-
tica (135). Growth of the young amoeboid stage into a mature form may
be followed by formation of a "zoocyst," or reproductive cyst (Fig. 5. 15,
A-D). According to Schussnig (135), gametes eventually are produced

222 The Sarcodina

Fig. 5. 15. A-J. Pseudospora parasitica Cienkowski: A, B. Young and older
amoeboid stages. C, D. Formation of "zoocyst." E, F. Production of uni-
nucleate amoebae and their escape from the cyst. G. An amoeboid "gamete."
H, Stage supposedly produced by fusion of two gametes. I. Nuclear fusion is
said to have occurred in the "zygote." J. A "sporocyst" has developed from
the encysted zygote; schematic (after Schussnig). K, L. Pseudospora rovig-
nensis Schussnig, amoeboid and flagellate stages; schematic (after S.). M-O.
Pseudospora volvocis Cienkowski, flagellate, amoeboid, and encysted stages;
xlHO approx. (after Roskin).

and syngamy occurs (Fig. 5. 15, G-I). The supposed zygote promptly en-
cysts. Within the "zoocyst," a second membrane ("sporocyst") is secreted to
produce a resting cyst (Fig. 5. 15, J). Flagellate "swarm-cells" and small
amoebae also have been reported in P. rovignensis (135), P. eudorini, and
P. volvocis (127).

The taxonomic position of the family is still uncertain and it has been

The Sarcodina 223

V'.

■'mi:

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B \ '^

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I7,^^r!i^

"^^^^^

'^■;.^■■

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Fig. 5. 16. A-D. Vampyrella lateritia Leidy, schematic (after Hoogen-
raad): specimens showing different forms of pseudopodia (AC); organism
ingesting contents of a Spirogyra cell. E-G. Vampyrella closterii I'oisson and
Mangenot (after P. and M.): E. Specimen attached to Closterhim and ingest-
ing contents of the alga; xl58. F. Cyst attached to empty cell wall of
Closterium; xl58. G. Section of cyst showing central mass of ingested food,
nuclei, mitochondria, and a peripheral zone of neutral-red-stainable vacu-
oles; x563. H, I. Arachnula impatiens Cienkowski (after Dobell): small speci-
men with a number of nuclei and several contractile vacuoles, x200; cyst
with several nuclei and ingested diatoms, x500.

suggested that Pseudospora may belong in the Dimastigamoebidae (Order
Amoebida) rather than in the Proteoinyxida (127).

Family 3. Vampyrellidae. The mature stage, in the type genus, is a
fairly large plasmodium. Reproduction involves plasmotomy, and multi-
nucleate cysts are formed by plasmodia. These general characteristics are

224 The Sarcodina

clearly represented in Arachnula Cienkowski (Fig. 5. 16, H, I), Leptomyxa
Goodey (Fig. 5. 17), and Vampyrella Cienkowski (Fig. 5. 16, A-G). Arach-
nula may be a synonym of Vampyrella (33). Chlamydomyxa Archer,
as represented by C. montana Lankester, closely resembles Leptomyxa
Goodey and it is not certain that the two should be placed in separate
genera. The mature stage of C. montata is a large plasmodium, the pseu-
dopodia are similar to those of Leptomyxa, and several endocysts are
produced within an ectocyst.

The life-cycles appear to be fairly simple. Excystment of a young plas-
modium is followed normally by growth and nuclear division. In addi-

Fig. 5. 17. A-G. Leptomyxa reticulata Goodey: A. Multinucleate plas-
modium, which may reach lengths of 2-3 mm.; x550. B. Ectocyst with six
endocysts; x880. C. Single endocyst; x880. D. Small plasmodium after emer-
gence from cyst. E. Plasmodium penetrating a root; x3I2. F. Plasmodium ex-
tending through several cells; x312. G. Stamed cyst with many nuclei; xl83.
A-D, after Singh; E-G, after MacLennan. H. Biornyxa merdaria Hollande,
x960 approx. (after H.). I. Biornyxa vagans Leidy, xl25 (after L.).

The Sarcodina 225

tion, fusion of several plasmodia into a single large one measuring as
much as I500[jl has been described in J'ampyrella closterii (112). Plas-
motomy within the cyst has been reported in Arachnula (33) and Vajn-
pyrella (112). The details of encystment may vary slightly. In a strain of
Leptomyxa reticulata recovered from hops (88), the cysts (Fig. 5. 17, G)
were large (425-900[j.) and contained only one endocyst. In other strains
(43, 138) several multinucleate endocysts have been found within an
ectocyst (Fig. 5. 17, B).

Leptomyxa reticulata occurs in the soil (138) and as a secondary in-
vader of diseased hops (88). Arachmila itnpatie7is has been described from
fresh and brackish water (33), while species of Vampyrella attack Spiro-
gyra (85) and Closteriinn (112) by digesting a portion of the cell wall
and sucking out the contents.

Both large plasmodial forms (Fig. 5. 17, I) and smaller uninucleate
organisms (52; Fig. 5. 17, H) have been assigned to Biomyxa Leidy but

Fig. 5. 18. A-G. Hyalodiscus rubicundus Hertwig and Lesser: oval forms
(35-70 X 20-50yn) seen from above (A) and from the side (B); invading cells
of Oedogoniutn (C, D); resting form with radiating pseudopodia (E); speci-
mens in locomotion, seen from above (F) and from the side (G); A-D, after
Hoogenraad; E-G, after Penard. H-K. Vampyrellidium vagana Zopf: various
amoeboid forms (H-J); resting cyst (K); schematic (after Ivanic).

226 The Sarcodina

the life-cycles are still unknown. Hyalodiscus Hertwig and Lesser (Fig.
5. 18, A-G) includes small organisms which may attack filamentous algae.
Although several morphological varieties occur, the production of a
large plasmodial stage has not been demonstrated for this genus. Schaeffer
(132) concluded that Hyalodiscus belongs in the Amoebidae. Vampy-
rellidmm Zopf (Fig. 5. 18, H-K) is similar to Hyalodiscus. The "axopodia"
of V. vagajis (63) resemble the ectoplasmic ridges of Thecamoeba (132).

/•

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n C

Fig. 5. 19. A. Actinocoma ramosa Penard (14-26/i); pseudopodia may
show small granules in movement (after P.). B-D. Nnclearia caulescens
Penard (16-20^); free stage (B); form temporarily attached by pseiido-
podiinn (C); specimen with a gelatinous sheath (D); after P. E. Gephyra-
jnoeba delicatula Goodey, specimen clinging to cyst from which it has just
emerged; x375 (after G.).

The Status of Gephyramoeba Goodey (Fig. 5. 19, E) is somewhat un-
certain. Although Gephyramoeba delicatula occasionally reaches lengths
of 250jjL, the organisms remain uninucleate and their cysts apparently
have a single membrane (43). Nuclearia Cienkowski (Fig. 5. 19, B-D)
includes uninucleate and multinucleate forms, either naked or with a
capsule through which the pseudopodia extend. Actinocoma Penard, as
represented by A. ramosa (Fig. 5. 19, A) is similar to noncapsulated uni-
nucleate forms of Nuclearia. These organisms apparently have little in
common with the plasmodia of Vampyrella and Leptomyxa.

The Sarcodina 227

Order 2. Mycetozoida

The mature stage of the Mycetozoida^ is either a large plasmodium
or a pseudoplasmodium. On the basis of differences in morphology and
life-history, three suborders may be recognized: (1) Acrasina ("Acra-
siales"), in which the structural unit is the uninucleate stage, although
pseudoplasmodia may be formed by aggregation of myxamoebae with-
out cytoplasmic fusion; (2) Plasrnodiophorina ("Plasmodiophorales"),
parasites which are plasmodia at maturity but do not produce sporangia;
(3) Eumycetozoina (Euplasmodida, "Myxogastres"), the typical free-

^ '•■

V>-''^A

PJL

Fig. 5. 20. A, B. Dirtyostcliinn inucoroides Brefeld (after Schuckmann):
active amoeboid stage with ingested bacteria, x440 (A); portion of pseudo-
plasmodium showing spindle-shaped organisms, x440. C-M. Dictyosteliiim
discoideuni Raper (after Bonner): C-I. Successive stages in development of
a pseudosporangium from a pseudoplasmodium; diagrammatic. J. Pseudo-
sporangium, almost mature, showing basal disc, stalk, and spores; schematic,
xl40 approx. K-M. Diagrams illustrating changes in position of "cells"
during development of a pseudosporangium (M) from a pseudoplasmodium
(K). KEY: b, basal disc "cells"'; s, spore "cells"; 1, 2, 3, stalk "cells" of three
different regions.

^ Detailed discussions of the Mycetozoida will be found in several monographs (46,
84, 86) and modern data have been reviewed by Martin (89).

228 The Sarcodina

living Mycetozoida in which the mature stage is a migiatory plasmodium;
more or less complex sporangia are produced in many genera.

Suborder 1. Acrasina. In this group, a small uninucleate "myxamoeba"
is released from the cyst ("spore"). Sexual phenomena have not been
demonstrated. These myxamoebae (Fig. 5. 20, A) lead an active life,
feeding typically on bacteria and undergoing fission. Under certain con-
ditions, which include a favorable humidity (Hi/) and perhaps partial
exhaustion of food (117), a pseudoplasmodium is developed by the ad-
hesion of myxamoebae to one another (Fig. 5. 20, B). In spite of its
organization, the pseudoplasmodium of Dictyostelium discoideiim moves
as a polarized vmit (116) and may grow by fission of the component
myxamoebae. The myxamoebae are said to cease feeding after formation
of the pseudoplasmodium in Dictyostelium (137) and the aggregate ap-
parently is a preliminary step toward sporulation.

Sporulation in some of the simpler Acrasina, such as Guttulina, in-
volves merely a heaping up of the m)T{amoebae into a compact mass and
then secretion of a cyst membrane (117). In such specialized types as
Dictyostelium discoideum (11), a pseudoplasmodium, under favorable
conditions, may first vmdergo a certain amount of migration. At sporula-
tion, the pseudoplasmodium gradually assumes an upright position and
becomes reorganized into a pseudosporangium (Fig. 5. 20, C-I). During
the late migratory phase, the posterior components of the pseudoplas-
modium are differentiated into intensely staining pre-spore cells; the
anterior units become stalk-cells; those at the base of the pseudoplasmo-
dium, basal-disc cells. Later on, the pre-spore cells are transformed into
spores. Morphogenesis also involves changes in position of the units. The
stalk-cells most anterior in the migratory stage are pushed up to and over
the top of the stalk-sheath and do^vn toward the basal disc during de-
velopment of the pseudosporangium. As a result, the relative positions
of various groups of cells are reversed (Fig. 5. 20, K-M). The pseudo-
sporangia are quite specialized also in Polysphojidylium. One interesting
feature of this commimal process is that sporulation follows a specific
pattern. Even after being crushed and mixed together, pseudoplasmodia
of two different species may reorganize and then produce their typical
spore-bearing structures (118).

The best known genus is Dictyostelium Brefeld, species of which have been investi-
gated in detail by several workers (II, 114, 116, 137). Certain species of Dictyostelium
have been maintained in cultures (12, 25, 115, 116, 137). Other genera (102) include
Acrasis Van Tieghem, Coenonia Van Tieghem, Guttulina Cienkowski, GuttuUnopsis
Olive, and Polyspliondylium Brefeld. The status of Sappi7jia Dangeard, sometimes
included in this group, is uncertain (117).

The Acrasina are free-living forms found commonly in soil and on decaying wood,
leaves, and straw, and all of them apparently feed on bacteria.

Suborder 2. Plasmodiophorina. These organisms invade cells in the
roots and underground stems of higher plants. Infections are often ac-

The Sarcodina 229

companied by the hypertrophy of tissues and formation of galls. A host
index has been published by Karling (70). The mature stage is a plas-
modium which may divide into small plasmodia or may give rise to
uninucleate cysts ("spores"). Although chitin has been reported, cellulose
apparently is not produced by the Plasmodiophorina.

Fig. 5. 21. A-I. Typical life-cycle of Plasmodiophorina, diagrammatic
(after Cook): A. Uninucleate cyst ("spore"). B. Excystment. C. Flagellate
stage. D. Amoeboid stage, after loss of flagellum. E. Amoeboid stage sup-
posedly formed by fusion of two flagellates. F. Binucleate amoeboid stage.
G. Plasmodium in host cell. H. Products of plasmotomy. I. Developing
spores. J-M. Sporomyxa tenebrionis Rietschel (from Tenebrio moUtor), xl890
(after R.): uninucleate stage (J); amoeboid form with four nuclei (K); de-
veloping "spores" in sporocyst (L); uninucleate spore (M).

In a typical life-cycle (Fig, 5. 21, A-I) excystment releases a myxoflag-
ellate in the soil. This flagellate ("swarm-cell") penetrates a cell in a
root-hair of the plant host and becomes a myxamoeba. Or, according to
some accounts (27), two myxoflagellates or two amoebae may fuse to
produce a diploid myxamoeba. At any rate, the myxamoeba develops
into a Plasmodium which, at maturity, mav undergo plasmotomy or
produce uninucleate cysts (Fig. 5. 21, H, I).

230 The Sarcodina

Relationships to the Eumycetozoina are not yet clear and further in-
vestigation of the life-cycle is needed. In certain species, meiosis is sup-
posed to precede formation of spores (28, 57, 155). For the group as a
whole, however, data on gametogenesis and syngamy are inadequate from
a cytological standpoint.

About a dozen genera have been erected, largely on the basis of the
arrangement of spores in the spore-masses and the shape of the masses.
However, Palm and Burk (105), in preparations of Sorosphaera from one
host species, found so much variation in the spore-masses that they
questioned the validity of the conventional generic criteria. On this basis,
they suggested that six generic names (Clathrosoriis Ferdinandsen and
Winge, Ligniera Maire and Tison, Mernbranosonis Ostenfeld and Peter-
sen, Ostenjeldiella Ferdinandsen, Sorodisciis Lagerheim and Winge,
Spongospora Brunchorst) might be considered synonyms of Sorosphaera
Schroeter. Furthermore, the authors suggested the advisability of placing
all described Plasmodiophorina in only two genera, Plasmodiophora
Woronin and Cystospora Elliott (37).

Cook (27), on the other hand, recognized the following genera: Plas-
modiophora Woronin (29, 91), Spongospora Brunchorst (77, 104), Lig-
niera Maire and Tison (26), Sorodisciis Lagerheim and \Vinge (157),
Sorosphaera Schroeter (10, 155) and Tetramyxa Goebel. These genera are
differentiated partly by the arrangement and form of the spores (27).
Spherical spores occur in groups of four without a common membrane
in Tetramyxa; ellipsoidal or pyriform spores are grouped in irregular
"spore-balls" within a common membrane in Sorosphaera; and in hollow
spore-balls without a common membrane in Spongospora. A flat "spore-
cake," composed of urn-shaped spores, is surrounded by a membrane in
Sorodiscus; and in Ligyiiera and Plasmodiophora the spores are neither
aggregated nor enclosed in a common inembrane. The taxonomic status
of Sporomyxa Leger (125; Fig. 5. 21, J-M), Peltomyxa Leger, Cystospora
Elliott, and Trematophlyctis Patouillard has been disputed. According to
Cook (27), these genera do not belong in the Plasmodiophorina.

Suborder 3. Eumycetozoina. The Eumycetozoina (Euplasmodia) in-
clude several hundred species of "slime-molds." The mature stage is a
migratory plasmodium which reaches a length of several inches to a foot
or more. Examined microscopically, the plasmodium in such types as
Physarum shows many channels of various sizes. Through the channels
flows a liquid containing many granules, the direction of flow being
reversed at intervals (92). As the plasmodium moves, vessels may be re-
sorbed in some areas and formed anew in others. The cytoplasm may be
hyaline, or with inclusions and pigments, may be white or various shades
of violet, blue, green, yellow, orange, red, and brown. Unfortiuiately,
these colors vary so much, under both natural and experimental condi-
tions, that they are not thoroughly reliable as taxonomic characteristics

The Sarcodina 231

(69). The diet may influence color of the plasmodium, since some species
become pink in association with Serratia marcescens (69). The pigment
of Physarum polycephalum is a pH-indicator, changing from yellow-
green at pH 8.2 to a deep red-orange at pH 1.0 (136). Certain species
with yellow pigment apparently require light for completion of the life-
cycle, while several non-pigmented species develop sporangia equally well
in light and in darkness (44).

The Plasmodium is holozoic, feeding largely on bacteria and other
microorganisms. A number of species have been grown in cultures with
a variety of microorganisms as food (19, 44, 60, 100). In addition, Fiiligo
septica, BadJiamia foliicola, and several others have been grown in pure
cultures on autoclaved yeast (25), but the specific food requirements of
these organisms are yet to be determined.

The Eumycetozoina occur on rotting leaves and logs, and the plasmo-
dium usually grows in or beneath such decaying materials. The plasmo-
dium penetrates decaying wood by extending slender processes through
the interstices and, under experimental conditions, may pass through
filters with pores measuring about 1.0[a (92). Shortly before sporulation,
the Plasmodium creeps to an exposed position, sometimes on trunks or
stems of nearby plants, where conditions will facilitate desiccation and
dispersal of spores. Subsequent behavior varies in different species. In
the simpler cases a plasmodium merely gives rise to a compact flattened
mass, or aetJiaUinn (Fig. 5. 23, A), or to an irregularly lobate body
(plasmodiocarp) which retains to some extent the outline of the plasmo-
dium (Fig. 5. 23, B). In either case, the entire mass becomes enclosed in
a membrane and may be considered a single large spore-case (sporocarp).
More often, the plasmodium produces individual sporangia (Fig. 5. 23,
C-I), stalked in many species but not in others.

The sporangia usually begin development as dense areas which become
segmented into knob-like masses. In many cases, the young sporangium
undergoes vertical growth, followed by differentiation of a stalk and a
spore case; in others, the sporangia remain sessile. The surface of the
sporangium typically becomes enclosed in a resistant wall (peridium),
which is commonly wrinkled at maturity. In stalked types, the peridium
is usually continuous with the covering of the stalk, and the stalk extends
to the substratum to end in a basal network, the hypothallus. Inside the
peridium, a capillitium (a network of threads or bands) is often devel-
oped, although lacking in Cribraria, Licea, and related genera. The first
indication of the capillitium in Physarum polycephalum (60) is the ap-
pearance of lacunae within the sporangium. These channels develop into
hollow threads whose junctions (nodes) become filled with calcium salts
as the sporangium approaches maturity. In other species, calcium may be
deposited throughout the capillitium, may be limited to the peridium or
its inner surface, or may not be deposited at all. The capillitial net, per-

232 The Sarcodina

haps by contractions induced by desiccation, probably helps to distribute
the spores after rupture of the peridium. During development of the
capillitium, nuclear division may continue in the sporangial protoplasm
for a time, but uninucleate pre-spores eventually are produced. These
become enclosed in membranes to form the characteristic spores.

In addition to sporulation, another method of producing resistant
stages is known in the Eumycetozoina. An entire plasmodium may be-

Fig. 5. 22. A-I. Physariim polycephalum, xl360 (after Howard): A. Spore.
B. Completion of mitosis; spore membrane ruptured. C. Completion of fission
at excystment. D. Amoeboid flagellate. E. Swimming flagellate. F. Flagellate
zygote shortly after fusion of gametes. G. Amoeboid zygote after loss of
flagella. H. Encysted zygote; gametic nuclei not yet fused. I. Zygote after first
nuclear division in formation of young plasmodium. J-L. Arcyria cinerea
(after Kranzlin): J-K. Stages in development of sporangiimi, x23. L. Portion
of cross-section through a sporangium, showing spores, peridituii, and part
of a capillitial thread ("elater"), x375.

come sclerotized (17) upon subjection to desiccation. The plasmodium
becomes partly dehydrated and is enclosed in a membrane, the sclerotiuni,
said to consist inainly of cellulose. Once sclerotized, the organism can re-
main viable for several months and then become active again in the
presence of adequate moisture and oxygen.

Development of the spores after liberation seems to be a complicated
process. Prior to germination, each spore in Ceratiomyxa (41) develops
four nuclei, so that a quadrinucleate amoeboid stage is released. The
amoeboid stage is said to produce eight uninucleate myxoflagellates, sup-

The Sarcodina 233

posedly gametes which fuse in pairs to produce amoeboid zygotes. In a
number of other Eumycetozoina (40), one or two amoeboid "swarm-cells"
are liberated and each amoeba then develops a flagellum. In Physarum
polycephalum, for instance, the spore nucleus divides once at the begin-
ning of germination and fission produces two amoeboid stages. One
amoeba emerges, develops a flagellum, and then swims away. The second
amoeba then repeats the process (59). Syngamy of the myxoflagellates
produces amoeboid zygotes (60). Except perhaps for slight differences in

C ,M

Fig. 5. 23. Sporangia in various Eumycetozoina (after MacBride): A. An
aethallium of Fuligo septica, xO.75. B. A plasmodiocarp of Hcmitrichia
serpula, x2.2. C. Sessile sporangia of Trichia inconspiciia, xll. D. Physarum
leiicopus, xll. E. Didymiiirn annulatum, xl3.5. F. Trichia decipiens, x6 ap-
prox. G. Didymiiim melanosperrnum, x7.5. H. Section showing capillitium
in sporangium of Physarella oblonga, x24. I. Badhamia magna, \.l.b.

vital staining, there is no evidence for two distinct types of "swarm-cells"
(69), and this question remains open for the suborder. Although meiosis
has been reported just before formation of the uninucleate "protospores"
in Ceratiomyxa (41), there is much uncertainty as to the exact stage in
which this process occurs in Eumycetozoina generally.

The following genera have been included in the suborder: Arcyria Wiggers (74),
Amaurochaete Rostafinski, Badhairiia Berkeley, Ceratiomyxa Schroter (41), Cribraria
Persoon, Didymium Schriider, Fuligo Haller, Licea Schrader, Lycogala Adanson (153),
Margarita Lister, Orcadella Wingate, Physarum Persoon (59, 60), Reticularia Bulliard,
Stemonitis Gleditsch (7), Trichia Haller, Tubulina Persoon.

Order 3. Amoebida

The Amoebida normally form lobopodia in locomotion, or else
move by a wave-like protoplasmic flow. Some species form slender acces-

234 The Sarcodina

sory pseudopodia which may have httle or no function in locomotion.
A hyahne ectoplasm and a granular endoplasm are usually distinguish-
able. A flagellate stage has been reported in several species usually
assigned to the order; in the rest, the cycle apparently is monomorphic.
Many species occur in the digestive tract of invertebrates and vertebrates;
others are free-living in fresh and salt water and in the soil.

The order is often divided into three families: Dimastigamoebidae,
in which the life-cycle includes both a flagellate and an amoeboid phase;
Amoebidae, free-living species without a flagellate stage; and Endamoe-
bidae, the endoparasitic amoebae.

Family 1. Dimastigamoebidae. The dimorphic cycle includes a domi-
nant amoeboid phase and a flagellate phase of relatively short duration.
Members of the family have been reported from fresh water and from
cultures inoculated with feces of certain insects and of various verte-
brates (including man).

Naegleria griiberi is the best known representative (113, 156, 161). The
small amoeboid stage (Fig. 5. 24, A, B, I) commonly forms one large
lobopodium. The nucleus contains a large Feulgen-negative endosome
which divides in mitosis. The flagellate stage (Fig. 5. 24, C, D, M), which
has two equal flagella, is a temporary one under the conditions reported;
ingestion of food has been described in only one instance (113). The
transformation from amoeba to flagellate is induced by diluting the cul-
ture medium with water (113, 161). Cysts (Fig. 5. 24, E-H) are usually
but not always uninucleate. The cyst membrane shows two well-defined
layers and also several opercula, through one of which the organism
emerges during excystment.

The generic composition of the family has been disputed. The type
genus, Dimastigamoeba Blochmann (9), is based on Dimastigamoeba
(Dimorpha) radiata (Klebs). The amoeboid phase (73) develops slender
radially arranged pseudopodia; the flagellate stage has two unequal flag-
ella, one of which is usually trailed. Dimastigamoeba simplex Moroff
(Fig. 5. 24, Q) is similar to D. radiata (93). The genus Naegleria Alexeieff
em. Calkins (2, 18) includes species with a flagellate stage showing two
equal flagella, and an amoeboid stage which moves by means of a blunt
lobopodium. There seems to be no sound reason for assuming that
Naegleria is a synonym of Dimastigamoeba. The status of Trimastiga-
7noeba Whitmore is uncertain, since stages with two, three, and four
approximately equal flagella were figured for T. philippinensis (159).
Such material might suggest a biflagellate organism in various stages of
flagellar duplication prior to fission. Hollande (53) has suggested that
Naegleria Alexeieff is a synonym of Vahlkampfia Chatton and Lalung-
Bonnaire (Fig. 5. 26, A-F). However, a flagellate stage was not reported
in V. punctata (24), and the structure of the dividing nucleus, although
similar to it, is not identical with that described for N. griiberi (113).

The Sarcodina 235

Fig. 5. 24. Dimastigamoebidae. AM. Naegleria gruberi: A. Unusually
elongated amoeba. B. Amoeba with four nuclei. C. Flagellate stage, from
living. D. Flagellate with three nuclei. E, F. Cysts with one and three nuclei.
G. Amoeba leaving cyst. H. Cyst showing several pores and unusual separa-
tion of inner and outer membranes. I-M. Stages in development of flagellate
(M) from amoeboid stage (I). A-G, xl600 (after Wilson); H, x2400 (after
Wenyon); I-M, xl215 (after Rafalko). N-P. Naegleria (Vahlkampfia) tachy-
podia (Glaser): rounded amoeba, two blepharoplasts on nuclear membrane
(N), x2010; amoeba, from living (O), xlI20; flagellate (P), x2010 (after
Pietschmann). Q. Dimastigamoeba simplex MorofE (20-40 X 10-12^), flagellate
stage showing long trailing fiagelluxn (after M.).

Until it is shown that the type species o£ Vahlkampfia has a flagellate
phase, there is no justification for placing this genus in the family
Dimastigamoebidae as now constituted. "Vahlkampfia" tachy podia Glaser
does show a flagellate stage (HI) closely resembling that of N. gruberi

236 The Sarcodina

^z^^ Xz^

yo^ ^

Fig. 5. 25. Various types of amoeboid activity in Amoebidae: A. Locomo-
tion without formation of distinct pseudopodia, cctoplasmic ridges distinct,
as in Tliecamoeba verrucosa. B. Formation of conical pseudopodia along
anterior margin and on free siuface during locomotion, as in Mayorella
bigemma. C. Formation of large pseudopodia which direct locomotion, as in
Amoeba proteus. D. Formation of a ninnber of large pseudopodia, including
several which direct locomotion, as in Amoeba dubia. E. Floating form with
slender and sometimes spiral pseudopodia, as in Astramoeba flagellipodia.
F. Slug-like forms moving by protoplasmic flow, as in Trichamoeba clava;
uroid (slender cytoplasmic projections at posterior end) present. G-L. Loco-
motion of "walking" type, as seen in thriving cultures of Chaos (Pelomyxa)
carolinensis. M-O. Acanthamoeba castcUanii (Douglas) \'olkonsky (12-30/i),
showing different forms of pseudopodia in one species. A-F, schematic (after
Schaeffer); G-L, schematic (after Wilber); M-O, after Volkonsky.

The Sarcodina 237

(Fig. 5. 24, N-P) and therefore should be transferred to the genus
Naegleria.

Family 2. Amoebidae. These are the free-living amoebae which lack a
flagellate phase. Although complex cycles involving polymorphism and
syngamy have been described, such interpretations apparently were based
on cultures contaminated with other species of Amoebidae, Mycetozoa,
and water-molds (67). At present, it appears that the life-cycle is limited
to the amoeboid stage and a cyst.

Classification of the Amoebidae is not yet on a satisfactory basis and
there remains a certain amount of disagreement concerning the genera
which should be recognized. Furthermore, the concept of a single family
for all the free-living amoebae is subject to the objection that habitat is
not necessarily an accurate gauge of zoological relationships. Conse-
quently, there is at least a reasonable basis for various suggestions that
the group should be split into less heterogeneous families. In a sense,
problems of taxonomy are complicated by the very simplicity of amoebae.
Lack of the more obvious fixed characteristics typical of many other
groups necessarily limits the taxonomist to consideration of range in size,
form of the body, type of pseudopodia, methods of locomotion, structure
of the nucleus, and the form and nature of cytoplasmic inclusions. Aside
from the nuclear picture, which should show reasonable constancy, these
characteristics vary within greater or lesser limits and presumably are
subject to significant environmental influences. The effective utilization
of such dynamic traits in taxonomy obviously demands extensive knowl-
edge of amoebae, particularly as living organisms. Consequently, there is
much need for the detailed study of many species which are not yet
thoroughly characterized. In some cases, adequate characterization may
depend upon pure-line cultures for determining the range in form and
behavior to be expected of jiarticular species. The systematic investiga-
tion of nuclear structure and division, on the order of some recent work
with Naegleria (113), also should yield information of taxonomic value.
For instance, a nucleus with a large endosome is characteristic of both
Vnhlkampfia (24) and Acanthamoeha (150), but the mitotic pictures are
strikingly different, the endosome being resorbed in the latter.

It has been pointed out very clearly (132) that amoebae differ char-
acteristically (Fig. 5. 25) with respect to types of pseudopodia, methods
of locomotion, form of the body and the nature of its changes in form,
presence or absence of a "uroid" (a gioup of thin cytoplasmic projections
at the posterior end), form of the nucleus, and even the types of cyto-
plasmic crystals in certain large fresh-water species. Some amoebae, for
example, form determinate pseudopodia which grow to a more or less
definite size and are then withdrawn, never becoming large enough to
include the entire amoeba and thus not directing locomotion. Others
develop indeterminate pseudopodia which are not restricted in size and

238 The Sarcodina

Fig. 5. 26. A-F. Vahlkampfia punctata (Dangeard) Chatton and Lalung-
Bonnaire: amoeba stained to show nucleus (A), xl710; stages in mitosis,
showing division of the endosome and other features (B-F), x3420 (after C.
& L-B.). G. Astramoeba Stella Schaeffer in active locomotion, x875 approx.
(after S.); compare with floating stage of A. flagellipodia (Fig. 5. 25, E). H.
Mayorella conipes Schaeffer, showing conical pseudopodia which do not
direct locomotion; xll55 approx. (after S.). I. Trichanweba pallida Schaef-
fer, a marine type showing typical uroid; xllOO approx. (after S.). J-M.
Amoeba proteus (Pallas) Leidy average length about 600 fi): amoeba in loco-
motion, showing typical pseudopodia and ectoplasmic ridges (J); broad (K)
and narrow (L, M) aspects of typical discoid nuclei (after Schaeffer). N-P.
Amoeba dubia Schaeffer (average length usually about 400^^): typical amoeba
(N), polar and lateral views (O, P) of the elongated nucleus (after S.).

The Sarcodina 239

may, as "main pseudopodia," become large enough to include the whole
organism and thus direct locomotion. And there are also certain amoebae
which develop no typical pseudopodia at all during locomotion. Such is
the case in Trichamoeba and Thecamoeba, in which locomotion is best
characterized as protoplasmic flow. The eventual correlation of such
characteristics with adequate cytological data should furnish a much
clearer picture of generic boundaries and relationships than is now
available for the free-living amoebae.

Some of the genera which have been proposed for various types of Amoebidae are
listed below; certain others have been characterized by Schaeffer (132).

Acanthamoeba \'olkonsky (50, 150; Fig. 5. 25, M-O); Amoeba Ehrenberg (Fig. 5. 26,

Fig. 5. 27. A. Flabelhda mira Schaeffer (marine), in locomotion; xl740
approx. (after S.). B. Dinamoeba mirabilis Leidy, characteristic spine-like
pseudopodia; some specimens show adherent rods, possibly bacteria; xl25
(after L.). C. Thecamoeba orbis Schaeffer, in locomotion, showing typical
ectoplasmic ridges; xl600 (after S.). D. Hartmanella kUtzkei Arndt, many
ingested bacteria, stained preparation; xl250 approx. (after A.). E. Pelo-
myxa ("gray type," P. palustris), longitudinal section of slug-like body
showing many nuclei, several food vacuoles, central axis, and tail-piece
("telspn") of hyaline cytoplasm; x53 approx. (after Okada).

240 The Sarcodina

J-P), represented by Amoeba proteus (Pallas) Leidy em. Schaeffer and A. dubia Schaeffer
(131); Astramoeba Vejdowsky (132; Figs. .5. 25, E, 5. 26, G), erected for A. radiosa
(Ehrenberg); Chaos Linnaeus, represented by Chaos (Pelomyxa) caroUnensis (71, 75,
76, 160; Fig. 5. 25, G-L), multinucleate types which sometimes measure 4-5 mm. in
length; Dinamoeba Leidy (83, 107, 132; Fig. 5. 27, B), erected for D. mirabilis; Flabel-
luhi Schaeffer (132; Fig. 5. 27, A); Hartmanella Alexeieff (150; Fig. 5. 27, D); MuyoreUa
Schaeffer (132; Figs. 5. 25, B, 5. 26, H); Pelomyxa Greef (Fig. 5. 27, E), represented
by P. palustris (83, 101, 107, 152), multinucleate types which move by protoplasmic
flow and may reach a length of more than 2 mm; Thecamoeba Fromentel (132; Fig. 5.
27, C), established for T. {Amoeba) verrucosa (Ehrenberg); Trichamoeba Fromentel
(132; Figs. 5. 25, F, 5. 26, I); Vahlkampfia Chatton and Lalung-Bonnaire (24; Fig. 5. 26,
A-F). It is possible that Hyalodiscus Hertwig and Lesser (Fig. 5. 18, A-G) also should be
included in this group.

Family 3. Endamoebidae. These are parasitic amoebae, found typically
in the digestive tract of invertebrates and vertebrates. The range of hosts

^-^^C-/

^jsd0i^'

■f: ■ -' -^ J'j \ ' • " ■ ■' \

Fig. 5. 28. A-C. Entamoeba invadens Rodhain: amoeba in liver smear
from Coluber constrictor (A); binucleate cyst with many chromatoid bodies
(B); cyst with four nuclei (C); xl260 (after Geiman and Ratcliffe). D.
Endolimax terjnitis Kirby, xI600 (after K.). E. Endamoeba granosa Hender-
son, from termites; x500 (after H.). F, G. Endamoeba simulans Kirby, from
termites; amoeba with much ingested material (F); cyst with fom^ nuclei
(G); x530 (after K.). H. Hydramoeba hydroxena (Entz) Reynolds and
Looper, section through the outer surface of Hydra showing destruction of
the epithelium; x560 (after R. & L.).

The Sarcodina 241

may be wide even within a single genus, since different species of Endo-
limax have been reported from termites and from primates. Most Enda-
moebidae are probably endocommensals, or else approach such a status.
However, there are notable exceptions, such as Entamoeba histolytica of
man (Chapter XI), and E. invadens which may produce fatal infections
in various reptiles (39, 119). As in the case of the Amoebidae, the assign-
ment of genera to this family is based upon their sharing a common
habitat rather than upon a consideration of more valid taxonomic cri-
teria. It is not impossible that some of the Endamoebidae are more closely
related to certain free-living amoebae than they are to other members of
their own "ecological" family. The following genera have been included
in the Endamoebidae:

Endamoeba Leidy (Fig. 5. 28, E-G), erected for Biitschli's Amoeba blattae (90, 95),
contains parasites of cockroaches and termites (49).

Entamoeba Casagrandi and Barbagallo (Fig. 5. 28, A-C) includes species from the
major groups of vertebrates. Although the validity of this generic name, as distinct
from Endamoeba Leidy, has been disputed extensively, reasons for retaining Entatnoeba
as a generic name for E. coli and related amoebae are ably presented by Kirby (72).
This usage emphasizes the fact that E. blattae and E. coli cannot logically be placed
in the same genus. The three species parasitic in man are discussed in Clhapter XI.

EndoUmax Kuenen and Swellengrebel (Fig. 5. 28, D) is represented in termites and
cockroaches as well as various \ertebrates. E. nana of man is described in Chapter XI.

Dientamoeba Jepps and Dobell includes a parasite of the human colon, while
lodamoeba Dobell is represented in pigs and in man (Chapter XI).

Hydramoeba Reynolds and Looper (121, 122; Fig. 5. 28, H) includes a rather large
amoeba which attacks the epithelial layers of Hydra, often with fatal results.

Order 4. Testacida

These are typically creeping organisms which develop lobopodia
or filopodia and possess one-chambered tests. The primitive test is com-
posed of an apparently single secreted layer. The material is said to be
"pseudochitin" (1). The flexibility of the test in Pamphagus and Cochlio-
podimn, for instance, indicates there is no significant addition of inor-
ganic material. Mixtures of silica with the basic "chitinous" material are
found in relatively firm tests which maintain a characteristic shape, as in
Hyalospheiiia.

The test of most Testacida apparently contains two layers (Fig. 5. 29,
J). The inner layer is composed of "chitin," sometimes mixed with sili-
ceous material. The structure of the outer layer varies in different genera.
Although apparently bivalve in Clypeolina rnarginata (109), this layer
seems to be continuous in other Testacida. In Arcella (Fig. 5. 29, C-F),
more or less spherical elements are cemented together in a honeycomb
pattern. In Amphizonella (Fig. 5. 29, L), the test is sometimes covered
with a "gelatinous" layer. Difflugiidae ingest sand grains, and occasionally
diatom shells, which are used with little or no modification in construc-
tion of the test. Such particles are embedded in a "chitinous" cement.
The test of Centropyxis (Fig. 5. 29, K) apparently is constructed of a

242 The Sarcodina

"chitinoid-siliceous" material which is usually, although not always, en-
crusted with sand grains. In Lecquereusia (Fig. 5. 29, A), sand grains or
diatom shells are ingested and then modified in form before addition to
the test (107). In the Euglyphidae (Fig. 5. 29, G-I), foreign particles are
replaced by scales, which are formed and stored in the cytoplasm prior
to fission. These scales are insoluble in hot sulfuric acid in Nebela collaris
and seem to be completely siliceous (87). The Euglyphidae are thought
to produce such scales from absorbed minerals, rather than by the modi-
fication of ingested particles. In Euglypha (47), it is possible to observe
cytoplasmic inclusions showing similar optical properties and forming a
graded series from small globules to typical scales. Such a "series" implies
a gradual growth of the scale by addition of material from the cytoplasm.

The color of the test varies with the species and often to some extent
with the individual specimen. Various shades of yellow and brown are
the rule, and the color may become darker as the animal grows older.
The yellow-brown tests presumably contain iron, w^hile the occasionally
observed violet tints (Heleopera) are attributed to manganese.

Pseudopodia. The pseudopodia of Testacida are of two general types,
slender lobopodia (Fig. 5. 29, B, C) and typical filopodia (Fig. 5. 29, G).
The former have rounded tips while the latter type tapers to a point.
Extended filopodia may show some degree of rigidity, although they are
flexible and may be swung about like sluggish flagella, as in Trinema
lineare (36). In addition to these clearly defined types, pseudopodia
somewhat intermediate in form have been described in Cryptodifflugia
and Cochliopodium. The form of the pseudopodia seems to be a reliable
taxonomic feature, and their relative number also may be fairly char-
acteristic. Such species as Hyalosphenia punctata (107) normally move
by means of one large pseudopodium. Other species typically extend
several pseudopodia at once. In addition to the usual functions, filopodia
in particular serve in attachment of Testacida to the substratum.

Contents of the test. Within the cytoplasm are found the nucleus or
nuclei, ingested food, one or more contractile vacuoles, stored food, and
often reserve shell-plates (Euglyphidae) or ingested sand giains to be used
for construction of a new test. The majority of species have only one
nucleus, which usually lies near the aboral pole of the test. However,
Arcella (Fig. 5. 29, C) is binucleate, Avhile such large species as Diffiugia
urceolata (107) are multinucleate. The perinuclear cytoplasm ("chromid-
ium," "chromidial zone") of the Euglyphidae usually contains stored food
which, in Nebela collaris (87), consists mainly of a glycogen-like carbo-
hydrate. This chromidium has been implicated in various accounts of
the chromidial origin of nuclei. Since the chromidium is sometimes
stained so intensely that the nuclei are obscured, it was believed at one
time that the nuclei periodically disintegrate into chromidia to form the
chromidial zone. Since the nuclei could be seen in specimens without a

The Sarcodina 243

Fig. 5. 29. A, B. Lecquereiisia spiralis (Ehrbg.) Penard (test, 125-140(14
long); oral view, showing surface pattern (A); optical section (B), schematic
(after P.). C-F. Arcella vulgaris Ehrenberg (test, 80-140;u in diameter);
horizontal section (C), schematic (after Penard); optical section through
wall of test (D), schematic (after Awerinzew); vertical section (E) through
test (after Penard); surface pattern (F), schematic (after Awerinzew). G.
Euglypha aspera Penard, plates shown at margin and around mouth; x206
(after P.). H, I. Plates from mouth region and from other parts of test
(after P.). J. Stained section of Heleopera rosea (test 90-105/i, long), show-
ing test membrane with overlying plates; reserve plates in cytoplasm
(after MacKiniay). K. Test of Centropyxis aculeata Stein, spineless variety
(C. ecornis); xlOO (after Leidy). L. Amphizonella violacea Greef (test 125-
250^ long); chitinous test covered with a gelatinous layer which is often
lacking (after Penard).

well-developed chromidium, it was assumed that the chromidia had been
utilized for reconstitution of the normal nuclei.

In addition to the usual inclusions, non-contractile vacuoles supposedly
filled with gas are frequently seen in species of Arcella. It has been sug-
gested that these vacuoles function in flotation by increasing the buoyancy
of the organism (8).

244 The Sarcodina

Life-histories. Fission in Testacida typically involves retention of the
old test by one daughter organism. As traced in living Nebela collaris
(87), stored sand grains are passed into the lower part of the cytoplasm
which is protruded from the mouth of the test at the beginning of fission.
This naked portion gradually assumes the form of an adult and then de-
velops a new test. Nuclear .division occurs next and is followed by fission
and separation of the two organisms. In the Euglyphidae, reserve shell

Fig 5 30 A. Cyst of Heleopera picta Leidy, test closed by operculum,
organism within cyst membrane; x250 (after L.). B. Cyst of Triuema
eruhelys. cyst membrane, "cyst-shell," and original test (mouth plugged
with debris); xll90 (after Volz). C. Encapsulated, or "drought-stage." m
Eughpha laevis, as found on dry moss; shell closed by a secreted membrane
(mouth of test sometimes plugged with debris); x800 (after Volz). D-H.
"Association" in Nebela collaris, from living (after MacKinlay).

plates appear in the cytoplasm, are stored in the perinuclear region, and
are used later for construction of the new test in fission.

Binary fission may not be the only method of reproduction. Occasional
production of a number of small amoebulae has been reported in Dij-
flugia (42), Centropyxis (20), and Arcella (21, 64). Perhaps this phenom-
enon is to be correlated with the reported occurrence of multinucleate
stages in Arcella (107). These small amoebae may undergo fission, but
they increase in size sooner or later and secrete a normal test. The ob-
servations of Cavallini (20, 21) were based on clone cultures. Although

The Sarcodina 245

such reports have encountered scepticism, they suggest the desirabihty of
further investigation under conditions which would eliminate possible
contamination of cultures with other forms of Protozoa.

Phenomena suggesting syngamy also have been reported in Testacida.
In the usual account, two mature organisms fuse with the mouths of
their tests in contact and the binucleate mass is drawn into one test (36,
42, 106, 142). Unfortunately there is no real evidence that meiosis and
the fusion of haploid nuclei occur. Until such data are available, inter-
pretations must remain tentative. However, the actual occurrence of such
cytoplasmic fusions (Fig. 5. 30, D-H) is attested not only by descriptions
of stained material but also by continuous observations on living speci-
mens (87). These findings, in conjunction with the occurrence of syngamy
in Heliozoida and Foraminiferida, stress the need for more intensive
study of life-cycles in Testacida.

Although Testacida are generally capable of surviving drought — re-
maining viable for some time on dried moss, for example — they often do
not develop typical cysts. Instead, the pseudopodia are withdrawn, usually
bringing into the mouth of the test a mass of debris which forms a plug.
Inside the test, a chitinous membrane is secreted (Fig. 5. 30, C). The
result is an effectively sealed "capsule-stage" (151), seemingly quite re-
sistant to desiccation. Perhaps less commonly, true cysts (Fig. 5. 30, A, B)
are produced. In such cases, the reserve shell-plates of Euglyphidae may
be used for a "cyst-shell" within the test. A cyst membrane is then
secreted inside the cyst-shell.

Ecological relationships. The Testacida as a group, and many of the
individual species, are cosmopolitan inhabitants of fresh water. Eco-
logically, however, their distribution is more restricted. Some of the
Testacida are commonly found in wooded areas or along streams on moss
which is not constantly submerged. Others are typical of fauna reported
for peat bogs, and a few species are commonly found in deep lake ^vaters
(depths of 60 feet or more). In general, the Testacida thrive best in acid
waters and may be either rare or absent in neutral or alkaline waters.
In surveys of various European bogs, species representing 18 genera and
all three families have been found within the range, pH 5.0-6.4. Within
these limits, differences in pH seemed to show little correlation with
specific composition of the fauna, but relatively few organisms were found
in an environment at pH 4.6 (103). The Testacida may prove interesting
material for studying the relations of pH to the utilization of minerals.

Taxonomy. Subdivision of the order is usually based upon the structure
of the test. Genera with a secreted test, either apparently homogeneous
or containing minute structural elements, are assigned to the family
Arcellidae. The family Difflugiidae is characterized by arenaceous tests,
composed usually of sand grains although sometimes of other materials.
The test of the Euglyphidae shows an outer layer of scales, or plates.

246 The Sarcodina

Fig. 5, 31. Arcellidae: A. Corycia flava (Greef) Penard (80-100/x), elastic
membranous test, pseudopodia retracted (after P.). B, C. Hyalosphenia
cuneata Stein (60-70^), broad surface of specimen in locomotion, narrow
surface of test (after Penard). D, E. Pyxidicula operculata Ehrenberg, view
from above, vertical section of test (after Penard). F, G. Pseudochlamys
patella Claparede and Lachmann (diameter about -iOfi), specimens seen from
above and from side (after Penard). H, I. Cryptodiffliigia compressa Penard
(I6-18;tt lo'ig)' broad and narrow surfaces (after P.). J, K. Plagiophrys parvi-
punctata Penard (test averages 50yn long), broad and narrow surfaces (after
P.). L, M. Cocliliopodium gratiiilatum Penard (test 70-90/i long), lateral and
polar views (after P.). N, O. Pamphagus mutabilis Bailey (test usually 70-
90^ long), test transparent, elastic, often twisted; in locomotion (N), and view
from above (after Penard).

This system is convenient in that it is based upon fairly obvious char-
acteristics, but it ignores such features as structure of the pseudopodia.
The family Arcellidae, for instance, includes Arcella and Pseudochlamys
with slender lobopodia and also Pamphagus and DifflngieUa with typical

The Sarcodina 247

lit*

..,-.„ f /■?

\^l,^ <i.

-V

Fig. 5. 32, Difflugiidae. A. Diffiugia pyriformis Perty; test 65 to (rarely)
400-500^ (after Penard). B, C. Pontigulasia incisa Rhumbler (test 85-150;x
long), lateral view; oral view showing "bridge" inside test (after Penard).
D, E. Cucurbitella mespiliformis Penard (test \25-\A0ii long), lateral view
and oral view of test showing collar (after P.). F. Frenzelina reniformis
Penard (test 26-30/i in diameter), hemispherical test, filopodia (after P.).
G-I. ClypeoUna marginata Penard (test 80-140/i long), view of broad surface,
optical cross-sections of two tests (after P.). J. Nadinella tenella Penard,
filopodia, collar, surface details shown at margin of test; schematic (after
P.). K. ParmuUna cyathus Penard (test usually 40-45/i long), surface detail
shown at upper margin; schematic (after P.). L, M. Heleopera picta Leidy
(x250), broad surface showing surface details, narrow surface in outline (after
L.). N. Centropyxis aculeata Stein, x300 (after Hoogenraad). O, P. Pseudo-
difPugia fulva (Archer) Penard (test 15-23/i long), lateral and oral views
(after P.).

248 The Sarcodina

WmM¥m

Fig. 5. 33. AC. Difflugiidae; D-L. Euglyphidae. A, B. Cannjyasciis trique-
ter Penard (test 90-120yii long), lateral view (collar, surface detail at margin
of test); optical cross-section of test (after P.). C. Diaphorodon mobile Archer
(test 40-111^ lorig); t^cst is somewhat plastic, with added foreign particles
(after Penard). D. Assidina semilunum Leidy, x375 (after Hoogenraad). E,
F. Paraeuglypha reticulata Penard (test 55-'10/jl long), organism showing shape
of test; surface pattern (after P.). G, H. Paulinella chromatophora Lauter-
born (test usually 20-30;:i lo"g); surface view; optical cross-section of test,
position of mouth indicated (after Penard). I, J. Cyphoderia trochus Penard
(test usually 110-120;^ long); schematic longitudinal section; siuface pattern
of the hyaline scales (after P.). K, L. Qiiadrula discoides Penard (test 30-40/i
long), lateral and polar views (after P.).

filopodia. Objections to such disregard of pseudopodial structure have
been raised (130), and it is possible that division of the order into a
larger number of appropriate families would illustrate natural relation-
ships somewhat more clearly than the present arrangement.

The Sarcodina 249

Genera included in the Arcellidae, Difflugiidae, and Euglphidae are
listed below.

Family 1. Arcellidae. Amphizouella Greef (Zounmyxa Niisslin) (107; Fig. 5. 29, L);
Arcella Ehrenberg (31, 83, 107; Fig. 29. C-F); Coch'liopodium Hertwig & Lesser (Ss'.
107; Fig. 5. 31, L, M); Corycia Dujardin (107; Fig. 5. 31, A); Cryptodiffliigia Penard
(107; Fig. 5. 31, H, 1); Hyalosphenio Stein (107; Fig. 5. 31, B, C); Pamphagus Bailey
(Chlamydophrys Cienkowski) (4, 56, 83, 107; Fig. 5. 31, N. O); Plagiophrys ClaparMe &
Lachmann (107; Fig. 5. 31, J, K); Pseudochlamys ClaparMe & Lachmann (83, 107;
Fig. 5. 31, F, G); PyxidicuJa Ehrenberg (107; Fig. 5. 31, D, E).

Fig, 5. 34. Euglyphidae. A, B. Trinema enchelys (Ehrbg.) Leidy (test 40-
lOOfi long); oral view showing surface pattern; schematic longitudinal section
(after Penard). C-E. Sphenoderia lenta Schlumberger (test averages 35;^ long);
lateral view, lest in optical section; oral view (outline); surface pattern (after
Penard). F-H. Nebela vitraea Penard (test 170-200^ long), broad and narrow
aspects (in outline); surface pattern (after P.). I, J. Placocista lens Penard
(test 65-67yLi long); broad aspect, hyaline plates shown at margin; narrow
aspect in outline (after P.).

Family 2. Difflugiidae. Campascus Leidy (83, 107; Fig. 5. 33, A, B); Centropyxis Stein
(32, 68, 83, 107; Fig. 5. 32, N); Clypeolina Penard (107, 109; Fig. 5. 32, G-I); Cucurbitella
Penard (107; Fig. 5. 32. D, E); Cystidina Volz (151); Diaphorodon Archer (107; Fig. 5.
33, C); Difflugia Leclerc (68, 83, 107; Fig. 5. 32, A); Frenzelina Penard (107); Heleopera
Leidy (83, 107; Fig. 5. 32, L, M); Lecquereusm Schlumberger (107; Fig. 5. 29. A, B);
Nadinella Penard (107; Fig. 5. 32. J); Oopyxis Jung (68); Parmulina Penard (107;' Fig.
5. 32, K); Phryngalella Penard (107); Pontigulasia Rhumbler (107; Fig. 5. 32, B, D);
Pseiidodifflugia Schlumberger (83, 107; Fig. 5. 32, O, P).

250 The Sarcodina

Family 3. Euglyphidae. Assulina Ehrenberg (55, 83. 107; Fig. 5. 33 D; Corythton
Taranek (107); Cyhhoderia Schlumberger (83, 107; Fig. 5. 33, I, J); Euglypha Dujardm
(83 107; Fig. 5. 29, G-I); Nebela Leidy (68, 83. 107; Fig. 5. 34, F-H); Pareuglypha
Penard'(107' Fig- 5. 33, E, F); Paulinella Lauterborn (78, 107; Fig. 5. 33, G, H);
Placocista Leidy (53, 107; Fig. 5. 34, I. J); Quadrula Schulze (83, 107; Fig. 5. 33 K, L);
Sphenoderia Schlumberger (83, 107; Fig. 5. 34, C-E); Trinema Dujardin (36, 83, 107,
151; Fig. 5. 34, A, B).

Order 5. Foraminiferida

Two features are characteristic— myxopodia and a test surrounded
by cytoplasm. The majority of living species measure less than 10 milli-
meters and are thus relatively small as compared with some of the extinct
species. However, there are exceptions, such as Bathysiphon filiformis in
which the test reaches a length of 50 mm (82). Most species are found in
salt and brackish water, and the few reported from fresh water are rela-
tively simple types. A small group contains specialized pelagic forms.
More typically, however, the Foraminiferida are slowly creeping organ-
isms, or else are migratory when young but sessile as adults. Various
sessile species have been found attached to eel grass and seaweed. Attach-
ment to seaweed or other floating objects presumably would be a signifi-
cant factor in the distribution of such species.

The Foraminiferida as a group are distributed throughout the oceans,
but there are characteristic local faunas restricted to particular areas.
Vertical distribution is influenced by the type of shell, since calcareous
tests go into solution at deeper levels. Geologically, Foraminiferida are
represented from Silurian to Recent time, although they vary in abun-
dance in different strata. The association of specific types with particular
deposits has been applied to the determination of geological correlation,
especially in drilling for oil. The most common type of modern deposit
is Globigerina-ooze, formed from tests of pelagic Globigerinidae and
Globorotaliidae at depths of 500-2500 fathoms.

Pseudopodia and their activities. Myxopodia are typical of Foramini-
ferida These sticky pseudopodia form a meshwork when extended and
show streaming of protoplasm, as indicated by the movement of granules
which may reach a rate of 400-500ij. per minute in Iridia lucida (81). As
described in Elphidium {Polystornella) crispum (66), this circulation may
be noted even in small branches of the network, granules moving up and
down the pseudopodia and occasionally reversing directions. Granules
flowing in opposite directions are often seen on opposite sides of a single
pseudopodium. Length of the myxopodia may equal or may greatly ex-
ceed the diameter of the test. In some instances, an organism with a test
measuring about 1.0 mm may form a myxopodia! net covering an area
20-40 mm in diameter. Myxopodia may show considerable activity. In
Elphidium crispum they are sometimes withdrawn at "lightning speed";
or they may be shot out "like little rockets" and then wave about in the

The Sarcodina 251

water, "bending, undulating, quivering, and putting out side branches
which meet and fuse and so establish the reticulum" (66). Myxopodia
often appear to be covered with mucus which leaves a trail as the pseudo-
podia are retracted. The pseudopodia, in species with imperforate tests,

Fig. 5. 35. AC. Addition of a new chamber to the test in Discorbina
bertheloti, x45 (after Le Calvez): A. Pseudopodia have been retracted and
an arenaceous cyst formed over the area of the future chamber; test shown
schematically, pores indicated in one chamber. B. Pseudopodia have been
retracted further and the form of the new chamber is evident. C. The first
layer, a thin chitinous membrane, has been secreted; pores are formed. D-F.
Changes in form of the pseudopodia during early development of Iridia
diaphana (after Le Calvez): D. Creeping "embryo" as it emerges from the
parental test, xlOO. E. Later pelagic stage with bristle-like pseudopodia, xl50.
F. At the beginning of fixation; typical pseudopodia are developing and the
"bristles" of the pelagic stage are disappearing, xll5. Compare with later
stage of development (Fig. 5. 40, E) in which the myxopodia have increased
in size.

252 The Sarcodina

arise mainly from an ectoplasmic stalk (raphe, peduncle) which extends
through the aperture (Fig. 5. 43, G). In types with perforate tests, most
of the pseudopodia may arise from the ectoplasm enclosing the test.

The pseudopodial pattern varies to some extent with environmental
conditions, and in at least certain species (Fig. 5. 35, D-F), may undergo
marked changes during development of the young organism. Although
myxopodia are often characterized as pseudopodia showing a more fluid
outer layer and a less fluid core, it is sometimes impossible to distinguish
the two zones. However, even the more delicate myxopodia may show a
certain degree of stiffness, in that they tend to follow a straight line and
often extend unsupported for considerable distances through the water.
The pseudopodia of certain species (Fig. 5. 44) sometimes assigned to
the order apparently are filopodia.

The Foraminiferida are markedly holozoic. The pseudopodial net
traps other Protozoa, algae (especially diatoms), and sometimes larval
Crustacea and other small invertebrates. The captured food is proinptly
surrounded by cytoplasm. Food is usually drawn toward the test by a
shortening of appropriate pseudopodia, and such particles may move at
the rate of several millimeters an hour. Unless size is prohibitive, the
prey may be drawn inside the test; diatoms, in particular, are often
found in the endoplasm. However, the myxopodial net itself may have
marked digestive abilities, although variation is noted from species to
species. In general, the shorter myxopodia show greater digestive activity
than the long delicate pseudopodia (82). Digestion often begins soon after
the food is surrounded by cytoplasm and may be completed before the
material reaches the test. In such types as Elphidium crispwn this seems
to be the normal method, to the exclusion of digestion within the endo-
plasm (66).

Other pseudopodial activities include the construction of tests and
cyst walls. Such activities are especially noticeable in species which build
arenaceous tests and cysts. In multilocular types, the addition of a new
chamber to the test is often carried out within an arenaceous cyst wall
laid down by the pseudopodia outside the area of the new chamber (Fig.
5. 35, A-C). Within the cyst, the pseudopodia form a reticulum outlining
the cavity of the new chamber and the new wall is developed at the
surface of the mass. If materials for a cover are excluded from cultures,
formation of a new chamber proceeds without encystment in Elphidium
crispum (66).

Many Foraminiferida are motile. Their characteristic creeping depends
upon the contraction of distally attached pseudopodia, the body being
dragged along the substratum as a result. Although creeping species may
seem restless under laboratory conditions and can travel several milli-
meters in an hour, such locomotion is relatively sIoav in terms of size of
the organism.

The Sarcodina 253

Fig. 5. 36. A, B. Iridia serialis Le Calvez, arenaceous test, upper and lower
surfaces; xl9 (after Le C). C, D. Webbinella crassa Rhumbler, lower and
upper surfaces of hemispherical test; pseudopodia are extended between
base of test and substratum; x23 (after Le Calvez). E. Allogromia laticollare
Arnold, half of approximately spherical test (diameter, \OOA50fi), showing
aperture (after A.). F, G. Test of Camerina elegans, dissection showing septa
and foramina (F), external view showing sutures (G); x5 (after Jones). H, I.
Central portions of test in Planorbulina mediterranensis d'Orbigny, megalo-
spheric type (H) and microspheric type (I); xlOO (after Le Calvez).

Colored granules or globules (xanthosomes) — brown, reddish, or yel-
low— are commonly seen in the myxopodia. Such inclusions often occur
in the endoplasm, from which they apparently pass outside to be dis-
carded in the trails left by retracted pseudopodia. The chemical nature
of the xanthosomes is unknown, although they are often referred to as

254 The Sarcodina

"excretory granules." It has been suggested that these inclusions are
accumulated pigments derived from diatoms or other food.

Tests. On the basis of their construction, two varieties of tests may be
recognized — tests composed entirely of secreted materials; and arenaceous
tests consisting mainly of foreign materials held together by a secreted
cement. Throughout the order there appears a primitive "chitinous" test
which may become the definitive test of the adult, as in Allogromiidae
(Fig. 5. 43, 44) and such types as Iridia lucida (81). It is uncertain
whether this test is secreted by the myxopodia or is produced by actual
transformation of an outer pseudopodial reticulum. In most species, the
initial chitinous test is strengthened by the addition of inorganic salts or
of foreign particles during development of the organism. In any case, the
test is enclosed in a layer of cytoplasm continuous with the pseudopodia.

The basic structure of the arenaceous test is a thin "chitinous" layer.
Onto this layer are cemented sand grains, sponge spicules, ambulacral
plates of echinoderms, fragments of other tests, and the like (Fig. 5. 36,
A-D). Such particles are picked up by the pseudopodia and pulled to the
initial chitinous layer, where they are cemented into a wall. The more
primitive species show no discrimination. Others tend to use a particular
type of structural element, so that different species collected from the
same area may show characteristic differences in construction of their
tests. The nature of the cement varies with the species. In some of the
primitive forms, the cement is chitinous, like the initial layer of the test.
The most common is an orange to brownish material, supposedly con-
taining iron and often known as "ferruginous" cement. A number of
species produce a calcareous cement, and there are also a few in which
the cement is siliceous. Arenaceous tests are found in about a third of the
established families.

Calcareous tests, the predominant modern type, and the relatively rare
siliceous tests differ from arenaceous tests in the absence of foreign par-
ticles. There are, however, interesting cases in which the first few cham-
bers produced by the young organism are arenaceous while the later ones
are strictly calcareous. Such ontogenetic evidence, and the existence of
species forming graded series from typically arenaceous to completely
calcareous tests, support the assumption (30) that the cement of primitive
tests gradually became the predominant building material during evolu-
tion of the group.

Growth of the individual, in primitive types, may involve desertion of
the old test and the construction of a new and larger one (30). The more
specialized types merely add new chambers to the preceding ones as
growth continues (Fig. 5. 35, A-C). In different species, the new chambers
are added in characteristic series, and within reasonable limits, in rela-
tively characteristic numbers to produce the test of the adult. The result
is a wide variety of patterns (Fig. 5. 37).

The Sarcodina 255

Fig. 5. 37, Various forms of tests: A. Rectilinear, uniserial, Hyperam-
7ninoides elegans (Pennsylvanian deposits), x27 (after Cushman and Waters).
B. Rectilinear, biserial, Cribostomum bradyi (Carboniferous), xl9 (after
Moller). C. Rectilinear, triserial, Verneuilina schizea (Lower Cretaceous), x38
(after Cushman and Alexander). D. Spiral, elongate, Turrulina andreaei
(Oligocene), x49 (after Andreae). E. Spiral, conical, Turrispirillina conoidea
(Jurassic), after Paalzow. F, G. Spiral, planispiral, Cyclammina cancellata,
apertural and lateral views, x8 (after Brady). H, I. Stellate, Pseiidastrorhiza
silurica (Silurian), x45 (after Eisenack); Astrorhiza arenaria, x2.1 (after
Le Calvez). J. Flabelliform (fan-shaped), Pavonma fiabelUformis, x32 (after
Parr). K, L. Spherical, Saccammina fragilis, apertural and lateral views, x27
(after Le Calvez). M. Unusual branching type, Rhizonubecula adherens, x3.5
(after Le Calvez). N, O. Simple tubular type, Bathysiphon humilis, surface
view and section through apertural end, x23 (after Le Calvez). P. Ar-
borescent, Dendrophrya erecta, xl2 (after Brady).

256 The Sarcodina

The usual multilocular test opens to the outside through the aperture
in the last chamber. This opening, which may be single or multiple (Fig.
5. 38), commonly lies at the base of the chamber wall but tends to be-
come terminal in linear tests, shifting to the wall of the last chamber.
The position, size, and shape of the aperture, and the number of open-
ings are features of taxonomic importance. In addition to the aperture,
the walls of perforate tests contain many small pores. In imperforate
tests with a single aperture (Figs. 5. 42, 43), the ectoplasm extends to the
outside as a condensed oral plug (buccal ectoplasm, peduncle, raphe).

Fig. 5. 38. Various types of apertures: A. Simple terminal, Psammonyx
inilcanicus, megalospheric type (after Rhumbler). B. Aperture at base of wall
in last chamber, Eudothyra mtdia (Pennsylvanian), x32 (after Cushman and
Waters). C. Terminal, with tooth, Milliamina lata, x28 (after Heron-Allen
and Earland). D. Aperture with broad tooth, Biloculinella olobula, x32
(after Cushman). E. Simple terminal, at end of coiled tube, Cornuspira
planorbis, x52 (after Schultze). F. Aperture with bifid tooth, Dentostoniina
bermudiana, xl6 (after Cushman). G, H. Multiple, radiate, NevilUna coro-
nata, lateral and apertural views, xl5 (after Sidebottom). I. Simple terminal,
in coiled test, Fischerina helix, x52 (after Heron-Allen and Earland). J, K.
Multiple, cribate, in wall of last chamber, Polyphragma cribosum (Creta-
ceous) (after Reuss).

In perforate tests the ectoplasm emerges through the many pores as well
as through the aperture.

The initial chamber is known as the proloculum. In many multilocular
(polythalamous) species, there are two varieties of tests differing in rela-
tive size of the proloculum (Fig. 5. 36, H, I). As discussed below, the
microspheric type (with the smaller proloculum) is produced by an or-
ganism developing from a zygote. The megalospheric type (with the pro-
portionately larger proloculum) is produced by individuals resulting from
schizogony. As successive chambers are added, their limits are marked
externally by sutures, and internally by septa (Fig. 5. 36, F, G). The
sutures usually appear as grooves, but may be raised or else flush with
the surface in some species. As each new chamber is formed, the anterior

The Sarcodina 257

wall of the preceding chamber becomes a septum in the simpler cases and
the old aperture now becomes a foramen joining the two chambers. These
foramina were responsible for the name assigned to the group when the
Foraminiferida were still considered Mollusca. The foramina in such
multilocular species as Planorbulina mediterranensis (82) and Elphidium
crispiim (66) are gradually closed by "chitinous" deposits which first
appear as rings and then spread across the openings to form "plugs."
Periodically, the plugs break loose and are carried out of the test by cyto-
plasmic currents. The functional significance of such plugs is unknown.
The septa are double in many specialized Foraminiferida, a posterior
wall of each new chamber being deposited over the anterior wall of the
preceding chamber. The structure may be further complicated by a cana-
licular system composed of tubules running through the wall of the test
and within the double septa. The canals communicate with the chambers
and also open to the outside, independently of the usual pores.

The endoplasm. The endoplasm contains the nucleus, or the multiple
nuclei of mature agamonts, and various types of inclusions, including
xanthosomes in many species. In some of the multilocular types, freshly
ingested prey and undigested residues tend to be concentrated in the
last or the last few chambers. In others, which complete digestion outside
the test, the inner cytoplasm may be entirely free from such materials
(66).

Life-cycles. A dimorphic life-cycle involving an alternation of genera-
tions was attributed to Foraminiferida in the early work of Lister and
Schaudinn. Two adult forms were recognized, a megalospheric type (ga-
mont) and a microspheric type (agamont), on the basis of a difference in
size of the proloculum. Reproduction of the microspheric adult, by
schizogony, results in uninucleate organisms which develop a proloculum
larger than that of the parent. At maturity, each megalospheric organism
produces gametes. After syngamy each zygote secretes a small proloculum
and growth results in a microspheric adult.

Recent investigations have indicated that this concept is strictly ap-
plicable only to certain specialized Foraminiferida which produce flagel-
late gametes (82). In other cases, the two forms cannot be distinguished
by size of the initial chamber, and occasionally the significance of micro-
spheric and megalospheric forms in the life-cycle may appear to be re-
versed if only the absolute measurements of the initial chambers are
considered. In some of these apparent contradictions, however, the micro-
spheric chamber actually is smaller, in proportion to size of the test, than
the megalospheric chamber. Life-cycles are now known to vary consider-
ably in their details (Fig. 5. 39). Complications include the appearance,
in certain species, of two varieties of agamonts, one with a larger pro-
loculum than the other. In the terminology of Le Calvez (82), apogamic
life-cycles, as observed in Discorhis orbicularis, involve only the sequence

258 The Sarcodina

of stages 7-5-6-7 . . . (Fig. 5. 39). Dimorphic cycles, represented by
Patellina corrugnta (97), follow the sequence, 1-2-3-4-5-6-1. . . . Holo-
trimorphic cycles, represented by Rotalia beccari (51), involve both "mi-
crospheric" and "megalospheric" agamonts as well as a gamont and shows
the sequence, 1-2-3-4-5-6-7-5-6-1 . . . Paratrimorphic cycles, as noted in
Planorbulina mediterranensis (82), are complicated erratic modifications
which may produce such sequences as 1-2-3-4-5-6-1 . . . 6-7-5-6-7. ... In
the typical cycle the microspheric agamont is multinucleate; the megalo-
spheric gamont, uninucleate. However, both agamonts and gamonts of
various primitive species remain uninucleate until the beginning of
schizogony or gametogenesis (82), and the agamont of Spirillina vivipara
contains only a few nuclei until time for schizogony (98). In the tri-

gamonts

agamonts A
(m e go/o sphe ric)

schizogony

gametogenesis

syngamy

agamonts B
(microspheric)

Fig. 5. 39. The life-cycles of Foraminiferida (after Le Calvez).

morphic cycles, both the "microspheric" and the "megalospheric" aga-
monts are multinucleate.

Reproduction of the agamont. Reproduction is typically a schizogonic
process. Reproduction may occur within the parental test, as in Iridia
serialis (Fig. 5. 40, D); or the multinucleate agamont may leave the test
just before schizogony, as in Spirillina vivipara (Fig. 5. 40, A). The young
stages of multilocular species, when set free, usually have 1-5 chambers
formed by the "embryonic" ectoplasm at the expense of the parental
cytoplasm. Reproduction may be simpler in some of the primitive species.
Microgromia elegantula, for instance, undergoes fission and one of the
resulting organisms leaves the parental test as an amoeboid stage which
develops a new test after a few days (146). However, AUogromia lati-
collare undergoes schizogony to produce as many as forty young within
the parental test (3).

The Sarcodina 259

*^^Sf'

Fig. 5. 40. A-C. Spirillina iniiipara (Ehrbg.). in reproductive test, x330
(after Myers): A. Multinucleate againont leaving test in preparation for
schizogony. B. Young organisms shortly after schizogony. C. Yoimg gamonts
with tests. D. Young organisms, products of schizogony, in parental test of
Iridia serialis, xl5 (after Le Calvez). E. Stage in development of Iridia di-
aphana; halo of tangled pseudopodia is about to produce the definitive
chitinous test; compare with earlier stages in Fig. 5. 35, D-F; xll5 (after
Le Calvez).

Reproduction may be preceded by formation of a reproductive cyst,
composed of foreign particles as well as solids expelled by the organism
in preparation for reproduction. Reproductive cysts (Fig. 5. 40, A-C)
are characteristic of Patellina corrugata (96) and Spirillina vivipara (82,
98), for example.

Gametogenesis and syngamy. The details of gametogenesis (gamogony)
vary in different species. In some cases, represented by Patellina corrugata
(96, 97) and Spirillina vivipara, two gamonts become associated in a

260 The Sarcodina

process resembling syzygy in giegarines. In .S". vivipara, each pair produces
a common "fertilization-cyst" with an arenaceous wall (Fig. 5. 41, A-E).
Nuclear division occurs in each gamont and the multinucleate gamonts
then leave their tests and produce uninucleate gametocytes. Gametes are
formed by division of the gametocytes, nuclear division being meiotic.

Fig. 5. 41. A-E. Spirillina vivipara, x330 (after Myers): A. Two gamonts
in syzvgy within a fertilization cyst, nuclei dividing. B. Production of
amoeboid gametes. C. Gametes and zygotes in fertilization cyst. D. Zygotes
have developed into agamonts with several nuclei; still within fertilization
cyst. E. Immature agamont with three nuclei. F. Gamont of Iridia lucida
prior to formation of gametocytes; schematic (after Le Calvez).

In these species, the gametes are amoeboid, are produced in small num-
bers, and are rather large — gametes of S. vivipara measure 50-60[j.. The
gamont of Allogromia laticollare, in contrast to S. vivipara, may produce
as many as 400 small (4-6[j,) amoeboid gametes. Self-fertilization has been
reported, and the zygotes develop into multinucleate organisms before
leaving the old test (3).

The production of flagellate gametes, usually biflagellate but sometimes

The Sarcodina 261

uniflagellate (Fig. 5. 42), seems to be more common in Foraminiferida.
Gametogenesis in Iridia lucida (82) is representative. Final stages in the
process include a series of rapid nuclear divisions, resulting in many
small nuclei (Fig. 5. 41, F), and then segmentation of the cytoplasm to
produce uninucleate gametocytes (Fig. 5. 42). Each gametocyte develops

Fig. 5. 42. AG. Gametogenesis and syngamy in Iridia lucida, x4000
(after Le Calvez): A. Gametocyte. B. Uninucleate gametocyte with para-
desmose and two pairs of flagella. C. Stage with two nuclei, just before fission.
D. Biflagellate gamete. EG. Successive stages in syngamy. H. Gamete of
Iridia diapJiana, x484.5 (after Le Calvez). I. Gamete of Gromia oviformis,
x3400 (after Le Calvez). J. Gamete of Planorbulina mediterranensis, x4000
(after Le Calvez). K. Gamete of Iridia serialis, x4845 (after Le Calvez). L.
Gamete of Webbinella crassa, x2400 (after Le Calvez).

a pair of flagella and then undergoes flagellar duplication and nuclear
division. The paradesmose which appears in nuclear division is similar
to that of many Mastigophora. Cytoplasmic division results in active
flagellate gametes. The emergence of the mature gametes, which may
number many millions, has been compared to a cloud of smoke rolling
out of the test.

262 The Sarcodina

Flagellate gametes of Foraminiferida show such features as a densely
staining nucleus and a large retractile body, or perhaps a few smaller
inclusions, possibly representing stored food. It is interesting that similar
gametes (Fig. 5. 10) have been reported for Radiolarida. The foramini-
feran gamete usually has two flagella, but sometimes only one {Gromia
oviformis), or rarely three (Discorbis patellifortnis). In contrast to the
more common type, amoeboid gametes are produced in Patellhia cor-
rugata and SpirilUyia vivipara (97, 98), and also in Allogromia laticol-
lare (3).

The number of gametes produced by a gamont varies widely — millions
in Iridia lucida, which does not undergo syzygy (82); only 250-300 flagel-
late gametes in the syzygous Discorbis patellijormis (99).

Syngamy is rapid in Iridia lucida (82). Two gametes make contact at
their flagellar ends and fusion soon follows (Fig. 5. 42, E-G). From the
general appearance of the gametes, syngamy in /. lucida appears to be
isogamous, but there is no evidence for self-fertilization. In Allogrotnia
laticoUare, on the other hand, syngamy does involve fusion of gametes
produced within a single test (3), and thus resembles pedogamy in
Heliozoida (Chapter II).

Duration of the life cycle. Length of the cycle varies from species to
species — three weeks or less in Spirillina vivipara (98), and about six
weeks in Patellina corrugata (97), at laboratory temperatures; about a
year in various dimorphic species of Mediterranean waters (82); probably
about two years for a complete dimorphic cycle in Elphidium crispum
(66). For the large species of deeper waters, no accurate data are avail-
able. In the Mediterranean and North Seas, where there are well differ-
entiated summer and winter temperatures, correlation with seasons of the
year is evident in some species. Agamonts are dominant in winter and
early spring, while gamonts tend to replace the agamonts in early autumn.
In regions with mild winters, seasonal correlation becomes almost in-
significant and perhaps disappears completely in tropical seas (82).

The relatively slow pace of the cycles in large species is related to the
amount of growth the young gamont or agamont must undergo before
it reaches maturity. The gamont of Elpludium crispum, for example,
develops a test with about 45-50, or occasionally more, chambers. In
early development, at 55-60° F., the sixth chamber is completed after
about 11 days; the fifteenth chamber, in about one month; the usual 40
or so, after almost four months (66). As compared with growth, schizog-
ony and gamogony are comparatively rapid. Schizogony in Iridia dia-
phana, for instance, covers a period of about three days (82).

Taxonomy. Classification of the Foraminiferida is based upon form and
composition of the test. The available information on other morpho-
logical features, especially in the living organisms, is not yet extensive

The Sarcodina 263

-.^^-i.

"■•K i.

/ F

• / .

Fig. 5. 43. Allogromiidae: A-D. Mlcrogromia elegantula Penaid. upper
and lateral views (A, B), organism outside test (C), and cyst (D); x920
approx. (after Valkanov). E. Micrometes pahidosa Cienkowski, test 16-17;a
long (after Penard). F. Completion of fission within test of M. paluclosa (after
Valkanov). G. Rhyiichogroniia (Gromia) linearis (Penard), a number of
nuclei, ingested diatoms (after P.). H, I. Diplogromia (Gromia) brunneri
(Blanc), test 60-250/i long, uninucleate organism, polar view showing pseu-
dopodia (after Penard). J. LieberkUhnia wagneri Claparede and Lachmann,
specimen with test 96/j. long (after Penard).

enough to play a significant part in taxonomy. On the basis of structural
features of the test, 50 families have now been recognized.^

The possession of arenaceous tests more or less completely separates a
number of these families from others with a calcareous test. The absence

* For a detailed consideration of taxonomy, Cushman's (30) monograph may be
consulted.

264 The Sarcodina

of perforations distinguishes a few families with calcareous tests from the
majority which have perforate tests. Families with arenaceous tests are
differentiated by the number of chambers — one, two, or many — and by
the patterns in which the chambers are arranged in the multilocular
types. The form of the test (Fig. 5. 37) and the type of aperture (Fig. 5.

Fig. 5. 44. Allogromiidae: A. Amphitrema wrightianum Archer, schematic,
x375 (after Cash). B. Diplophrys archeri Barker (diameter of test, 8-20/i),
schematic optical section showing nucleus and large refractile inclusion; test
thin, hyaline (after Penard). C, D. Artodiscus saltans Penard (body, \8-2Sii);
entire organism showing pseudopodia unlike those of typical Foraminiferida
(C); single pseudopodium emerging through test (D); schematic (after P.).
E. Allogromia (Gromia) dujardini Schulze, x29 (after S.) F. Lecythium
granulatum (Schulze) Hopkinson, x360 (after S.). G. Amphitrema stenostoma
Nusslin, schematic optical section (after Penard).

38) also are bases for differentiating genera and species. In the most
primitive family, the Allogromiidae, a chitinous test is characteristic of
the adult organism.

Family Allogromiidae. The test is completely or mostly chitinous, usu-
ally with a single aperture, sometimes with an opening at each end. The
wall may be a single chitinous layer, thin in some forms and relatively

The Sarcodina 265

thick in others; or, foreign particles may be added to the outer surface.
In Diplogromia, the wall of the test is double, the outer layer being
arenaceous. The majority of species have been reported from fresh and
brackish waters.

It is unfortunate that so little is known about these organisms since
they appear to be favorable material for investigating basic characteristics
of the Foraminiferida. Some of the Allogromiidae, at least, can be main-
tained in the laboratory and extensive investigation of their life-cycles
should prove interesting. Reproduction by schizogony, much as in the
more specialized marine types, and the production of amoeboid gametes
have been observed in Allogromia under laboratory conditions (3). Per-
haps further studies of this nature will answer various unsettled questions
concerning the generic composition of the family.

The following genera have been referred to the Allogromiidae: Allogromia (3, 65,
124; Figs. 5. 36, E, 5. 44, E), test chitinous, ovoid to spherical; Amphitrema Archer (55,
107; Fig. 5. 44, A, G), foreign particles adherent to the chitinous test, one opening at
each end; Artodiscus Penard (107; Fig. 5. 44, C, D), affinities with the Allogromiidae
uncertain; Boderia Wright (124), peculiar marine types strikingly similar to a migra-
tory stage which occurs m early development of certain less primitive marine species
(Fig. 5. 35, D); Diaphorodnn Archer (107; Fig. 5. 33, C), with filopodia, sometimes
assigned to the Allogromiidae instead of the Testacida (Difflugiidae); Dactylosaccus
Rhumbler (123, 124), thin chitinous test tubular and twisted; Diplogromia Rhumbler
(124; Fig. 5. 43, H, I), double-walled test, outer layer of fine siliceous granules;
Diplophrys Barker (107; Fig. 5. 44, B), spheroid chitinous test, opening at each end;
Lecythiuin Hertwig and Lesser (Fig. 5. 44, F), filopodia, flexible chitinous test; L.
hyolinum H. and L. referred to genus Pamphagus by Penard (107); Lieberkiihnia
Clapar^de and Lachmann (107, 110, 124; Fig. 5. 43, J), chitinous test, ectoplasmic stalk
(peduncle) arises from the side of the body; Microgromia Hertwig and Lesser (146;
Fig. 5. 43, A-D), small, chitinous test; Micrometes Cienkowski (107, 147; Fig. 5. 43, E,
F), delicate chitinous test with several apertures; Myxotheca Schaudinn (38, 124),
marine, test thin, approximately spherical, usually with adherent foreign particles;
Plagiophrys ClaparMe and Lachmann (107; Fig. 5. 31, J, K), sometimes assigned to
the .Allogromiidae instead of the Testacida; Rhynchogromia Rhumbler (123; Fig. 5.
43, G), elongated chitinous test, terminal aperture; Rhynchosaccus Rhumbler (79, 123),
thin tubular chitinous test, opening at each end; Schulizella Rhumbler (124), delicate
spheroidal chitinous test, more than open aperture with variable positions.

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266 The Sarcodina

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The Sarcodina 267

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268 The Sarcodina

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Sporozoa

Class 1. Telosporidea
Subclass 1. Giegarinidia
Taxonomy

Order 1. Schizogregarinida
Family 1. Ophryocystidae
Family 2. Schizocystidae
Order 2. Eugregarinida
Suborder 1. Cephalina

Family I. Acanthosporidae
Family 2. Actinocephalidae
Family 3. Cephaloidophoridae
Family 4. Dactylophoridae
Family 5. Didymophyidae
Family 6. Gregarinidae
Family 7. Lecudmidae
Family 8. Menosporidae
Family 9. Monoductidae
Family 10. Porosporidae
Family 11. Stenophoridae
Family 12. Stylocephalidae
Suborder 2. Acephalina
Family 1. Aikinetocystidae
Family 2. Allantocystidae
Family 3. Diplocystidae
Family 4. Ganymedidae
Family 5. Monocystidae
Family 6. Rhynchocystidae
Family 7. Schaudinellidae
Family 8. Stomatophoridae
Family 9. Urosporidae
Family 10. Zygocystidae
Subclass 2. Coccidia
Life-cycles
Taxonomy

Order 1. Adeleida
Suborder 1. Adeleina
Family 1. Adeleidae
Family 2. Dobelliidae
Family 3. Klossiellidae
Family 4. Legerellidae
Suborder 2. Haemogregarinina
Family 1. Haemogregarinidae
Family 2. Hepatozoidae
Family 3. Karyolysidae

Order 2. Eimeriida

Family 1. Aggregatidae
Family 2. Caryotrophidae
Family 3. Cryptosporidiidae
Family 4. Eimeriidae
Famdy 5. Lankesterellidae
Family 6. Selenococcidiidae
Subclass 3. Haemosporidia
Order 1. Plasmodiida

Family 1. Haemoproteidae
Family 2. Plasmodiidae
Order 2. Babesiida
Genera of uncertain status
Dactylosoma Labbe
Toxoplasma Nicolle and Manceaux

Class 2. Cnidosporidea
Order 1. Myxosporida
Suborder 1. Eurysporina
Family 1. Ceratomyxidae
Family 2. Wardiidae
Suborder 2. Sphaerosporina
Family 1. Chloromyxidae
Family 2. Sphaerosporidae
Family 3. Unicapsulidae
Suborder 3. Platysporina
Family 1. Coccomyxidae
Family 2. Myxidiidae
Family 3. Myxobolidae
Family 4. Myxosomatidae
Order 2. Actinorayxida

Taxonomy
Order 3. Microsporida
Taxonomy

Famdy 1. Coccosporidae
Family 2. Mrazekiidae
Family 3. Nosematidae
Family 4. Telomyxidae
Order 4. Helicosporida

Class 3. Acn'idosporidea
Subclass 1. Sarcosporidia
Subclass 2. Haplosporidia

Literature cited

269

270 Sporozoa

/jLLL KNOWN Sporozoa are parasitic. The usual infective stage is
a sporozoite (Telosporidea), or an analogous sporoplasm (Cnidosporidea)
which may be ingested by a new host or inoculated by some vector.
Except in sjoecies transferred by inoculation, sporozoites are typically en-
closed within a spore membrane, the origin of which varies in different
groups. Sporozoa are not ciliated, and flagella are limited to the micro-
gametes of certain species. Nutrition is predominantly saprozoic, although
trophozoites of Nosema miitabilis apparently can ingest solid particles
(76).

The group as usually defined shows a lack of homogeneity which led
Wenyon (139) to restrict his Class Sporozoa to the Gregarinidia, Coccidia,
and Haemosporidia — the Telosporidea as listed beloAv — and to recognize
the Cnidosporidea as a group of equal taxonomic rank. This arrangement
expresses clearly the general belief that Sporozoa are not monophyletic in
origin. However, the more common usage will be followed here, dividing
the group into three classes, Telosporidea, Cnidosporidea, and Acnido-
sporidea.

CLASS 1. TELOSPORIDEA

The life-cycle typically shows asexual and sexual phases, both of
which, except in the Eugregarinida, are characterized by reproduction.
Reproduction in the sexual phase produces sporozoites, either directly
from the zygote or from intermediate sporoblasts arising by division of
the zygote. In such forms as malarial parasites sporozoites are clearly the
result of schizogony (or sporogony, in this phase), but the appropriateness
of this term is less obvious in certain TelosjDoridea which produce two
sporozoites from each sporoblast. Sporozoites may be naked, or they may
be produced within a spore membrane. The membrane, which often
consists of inore than one layer, may be secreted by th^ zygote, as in typical
gregarines; or an encysted zygote may divide into two or more sporoblasts,
each of which secretes a spore membrane. In the first case, the spore de-
velops from an oocyst (encysted zygote); in the latter, from sporocysts
(encysted sporoblasts). With a few possible exceptions, the membrane ap-
parently is not divided into valves. Furthermore, there are no polar cap-
sules in spores of the Telosporidea.

The asexual phase of the cycle is initiated by a sporozoite upon reach-
ing a host. Growth of the sporozoite into a mature trophozoite (schizont)
is followed by schizogony (or merogony, in this phase), except in the
Eugregarinida. The trophozoite remains uninucleate throughout much of
the growth period so that a plasmodium is usually developed shortly be-
fore merogony. Many Telosporidea are intracellular during this asexual
phase. However, some of the gregarines are intracellular only in the early

Sporozoa 271

stages of growth, while certain others (coelozoic parasites) are found only
in body cavities. The merozoites, produced in merogony, may repeat the
cycle of growth and merogony but they eventually become differentiated
into gamonts (gametocytes). The production of gametes generally in-
volves schizogony (gamogo7iy, in this stage), although in well defined
anisogamy, the process is often limited to the production of microgametes.
The Telosporidea may be divided into three subclasses: Gregarinidia,
Coccidia, and Haemosporidia. In their various hosts, development of
most Gregarinidia is largely or completely extracellular; that of the other
two groups, mainly intracellular. Sporocysts are usually developed within
the oocyst of Coccidia but not in that of Gregarinidia. In the Haemo-
sporidia, the sporozoites are not enclosed within spore membranes.

Subclass 1. Gregarinidia

The gregarines are typically parasites of the digestive tract and
body cavities of invertebrates, although a few occur in tunicates and
Enteropneusta. The early development of many species occurs within
tissue cells, but the trophozoites usually emerge to complete the cycle in
some body cavity of the host. In other gregarines young trophozoites may
be attached to an epithelium but there is no intracellular stage. With the
exception of such genera as M er ogre gar ina and Spirocystis, in which intra-
cellular merogony occurs, older trophozoites are typically free in the
lumen of the digestive tract or in some other body cavity. In cephaline
gregarines (Suborder Cephalina), the transition from the attached stage
to the mature free trophozoite {sporadin, or sporont) often involves loss
of the epimerite, an organelle of attachment. This is to be expected
especially if the epimerite is firmly attached to the host's tissue or em-
bedded in a tissue cell.

Gregarines vary widely in size, mature trophozoites ranging from about
10[jL to 3-4 mm in different species. Form of the body also varies con-
siderably (Fig. 6. 1). The fully grown gregarine is commonly an elongated,
often spindle-shaped organism, but there are a number of exceptions.
Among the elongated types, the body may be more or less cylindrical, or
it may be distinctly flattened. The typical individual gregarine also is
capable of undergoing contortions, which in some species, resemble
euglenoid movement of certain Euglenida.

There is usually a well differentiated cortex which is sometimes rather
thick — e.g., 5-6[jL in Rhynchocystis porrecta (133) — and is composed of
two layers, the sarcocyte and the myocyte (Fig. 6, 1, H). The outer sur-
face is covered with a cuticle (epicyte), oflen marked with ridges or other
decorations (Fig. 6. 1, G). In Rhyjichocystis pilosa (133), cuticular "hairs"
(Fig. 6. 11, G) are attached to the ridges. The sarcocyte, the layer under-
lying the cuticle, is usually homogeneous in appearance. The myocyte
contains the myonemes characteristic of many gregarines. In some species

272 Sporozoa

Fig. 6. 1. Variations in body form of Gregarinida. A. Taeniocystis mira,
resembling a cestode strobila; mature form (length reaches 400-500^) with-
out epimerite; the anterior "segment" is the protomerite (after L^ger). B.
Nematocystis anguillula, slender species reachin;^ a length of 500;^ (after
Berlin). C. Apolocystis minuta, approximately sp^^erical mature form, x930
(after Troisi). D. Schaudinella henleae, illustrating the spindle-shaped body
common among gregarines; x975 (after Nusbaum). E. Corycella armata, a
typical cephaline gregarine with barbed epimerite; mature forms reach 300jn
in length (after Leger). F. Aikinetocystis singularis, in which the anterior
part of the body is dichotomously branched, each tip ending in a "sucker";
tips of two branches shown; schematic, x400 approx. (after Gates). G.
Cross-section of Polyrhabdina spionis at level of nucleus; note heavily ridged
cuticle; x900 (after Mackinnon and Ray). H. Section through body wall of
Rhynchocystis porrecta showing cuticle, sarcocyte, myocyte, and portion of
the endoplasm with paraglycogen bodies; xl200 (after Troisi).

(Fig. 1. 13, D, E), both longitudinal and circular myonemes have been
described, but only longitudinal ones have been seen in R. pilosa (133).
The endoplasm, which contains the large nucleus, is rather homogeneous
except for the frequent appearance of many paraglycogen granules (Fig.
6. 1, C) measuring 2-7[jl in diameter in different species (34).

Sporozoa 273

Fig. 6. 2. Epimerites and mucrons. A, B. Lobate epimerite of Aclino-
cephalus parvus (after Weschenf elder): longitudinal section (A), x2560; polar
view (B), xl600. C. Anterior end of Rhynchocystis pilosa, an acephaline
gregarine with an epimerite-like organelle; the large nucleus and a portion
of the endoplasm are shown; x2800 (after Troisi). D. Epimerite of Polyrhab-
dina spionis, attached to epithelium; longitudinal section; xl750 (after
Mackinnon and Ray). E. Anterior end of Zygocystis wenrichi, showing
mucron; x331 (after Troisi). F. Attached trophozoite of Nina gracilis, ex-
panded protomerite with multiple filamentous epimerites extending into
intercellular spaces of an epitheliimi; tissue cells not shown; x600 (after
Goodrich). G. Anterior end of 7.ygosoma globosum, globular epimerite at-
tached to epithelium; x74 (after Noble). H. Trophozoite of Gregarina rigidn
with globular epimerite; xl3I5 (after Allegre). I. Epimerite of Lecytliion
thalassemae attached to epithelial cell; xl330 (after Mackinnon and Ray).

In one group (Cephalina) of the Eugregarinida, the body is differen-
tiated into two regions, an anterior protomerite and a posterior deuto-
merite. The two regions are separated by an optically distinct transverse
septum in most of the cephaline gregarines. The unusual protomerite of
Nina gracilis (37) may undergo marked changes in form (Fig. 6. 8, B-D),
and can be used as a sucker for attachment. The protomerite of the more

274 Sporozoa

typical cephaline gregarines is equipped with an epimerite, or hold-fast
organelle, which varies in structure in different species (Fig. 6. 2). The
multiple "epimerites" of Nina gracilis (Fig. 6. 2, F) are filaments se-
creted after the organism becomes attached by means of its expanded
protomerite (37). The epimerites of certain species remain embedded in
a tissue cell as the trophozoite emerges and the parasite may remain at-
tached through much of the growth period. In other cases, the epimerite
adheres to one or more epithelial cells. The epimerite is commonly, al-
though not always, lost when the gregarine becomes detached from its
anchorage. An analogous structure, the mucron (Fig. 6. 2, C, E), is present
in certain acephaline gregarines, and a sucker-like depression lies at the
anterior end of various others (Fig. 6. 12, A, B, F). The mucron serves
for attachment in such species as Rhynchocystis pilosa (133), but may be
rudimentary and apparently non-functional in other cases.

The trophozoite of the Schizogregarinida may either undergo merog-
ony, or give rise to one or a few gamonts (gametocytes). Since merogony
does not occur in Eugregarinida, surviving trophozoites develop into
gamonts. The gamonts of typical Gregarinidia become associated in pairs
or sometimes larger groups, a condition known as syzygy (Fig. 6. 3, A-C).
In many species, syzygy occurs early so that the trophozoites, immature
at first, are associated for some time before the differentiation of gamonts.
In other cases, association occurs much later and gamonts are differen-
tiated almost immediately afterward. In syzygy of cephaline gregarines
the anterior end of one sporadin (the satellite) often adheres to the
posterior end of another (the primite). Occasionally, two satellites may
be attached to one primite, and in exceptional cases several individuals
may form a chain. Such chains appear to be temporary associations.

Development of mature trophozoites into gamonts often involves no-
ticeable changes. Autotomy of a posterior portion of the body precedes
syzygy in Rhynchocystis pilosa (133), and elongated organisms tend to
round up. The epimerite of cephaline gregarines, if not already lost,
undergoes partial or complete resorption; likewise, cuticular decorations
disappear. The associated gamonts — usually two, occasionally three — se-
crete an enclosing membrane to produce a gametocyst. In some genera —
Hentschelia, Lecythia (85), and Nina (37, 79) — a gelatinous or mucous
ectocyst encloses the usual membrane. Within the cyst each gamont under-
goes gamogony (Fig. 6. 3, D). In such genera as Hentschelia, Lecythion,
and Nina, gametocysts are voided from the gut at an early stage so that
gamogony, and subsequent syngamy and sporogony, take place outside
the host. In Carcinoecetes and Cephaloidophora, the gamonts apparently
leave the intestine early in syzygy, often becoming attached to the exo-
skeleton of the crustacean host and developing a gametocyst as "ecto-
parasites" (5).

The original cuticle of each gamont persists within the gametocyst of

Sporozoa 275

Fig. 6. 3. Syzygy, gamogony, gametes, and syngamy. A. Chain in Nema-
topsis legeri, from living (after Hatt). B. Simple syzygy in Gregarlna rigida;
x665 approx. (after Allegre). C. Multiple, branching syzygy in Carcinoecetes
Hesperus, penultimate satellites partially fused; x275 (after Ball). D.
Gamogony within gametocyst (length, 185-223^), Monocystis ventrosa (after
Berlin). E, F. Microgamete (x4690) and macrogamete (xl875) of Nina gracilis
(after Goodrich). G, H. Microgamete and macrogamete of Urospora rliya-
codrili; xl710 (after Gabriel). I, J. Flagellated microgamete (showing axial
filament) and syngamy, Monocystis mrazeki; microgamete, about 9.5jti; macro-
gamete, about llyLc (after Hahn). K, L. A pair of gametes and syngamy,
Gregarina blattarum; x2830 (after Sprague). M. Syngamy in Hyalosporina
cambolopsisae, microgamete with pointed nucleus; x4725 (after Chakravarty).
N-P. Macrogamete, flagellated microgamete, and syngamy in Hentschelia
thalassemae; x2430 (after Mackinnon and Ray).

276 Sporozoa

Nina gracilis (37). The result is a partition separating the two groups of
gametes except for a short time in which the microgametes are passing
through to join the macrogametes. The cuticular sac of the microgameto-
cyte, which contains a milky residue after completion of gamogony and
migration of the microgametes into the other compartment, remains as
a so-called "pseudocyst." As the spores approach maturity, the pseudocyst
increases in volume, apparently through accumulation of gases, and
serves as a float if the gametocyst has been deposited in sufficient water.
The increasing internal pressure eventually ruptures the gametocyst.

In contrast to the Ophryocystidae, in which each gamont yields one
functional gamete (Fig. 6. 5, B, C), most gregarines produce many gam-
etes. In certain species, the gametes are obviously of two kinds (Fig.
6. 3, E-J, M-O), the microgamete being the smaller and sometimes bear-
ing a flagellum. Even in apparent isogamy, it is sometimes possible to
distinguish two types of gametes on the basis of cytoplasmic inclusions
(Chapter II). Comparable differences in inclusions have been noted in the
two gamonts within a gametocyst, as in Cephaloidophora communis (5).

As a rule, the gametes fuse completely in syngamy. Hyaolsporina
cambolopsisae (14) is an exception in which only the nucleus of the micro-
gamete enters the macrogamete (Fig. 6. 3, M). Soon after syngamy the
zygote of most gregarines encysts. Within the oocyst membrane, the zy-
gote divides into sporozoites and the oocyst thus becomes a spore. Poro-
spora is an unusual genus in which no oocyst membrane is secreted, and
sporozoites are thus not found in spores (42). The spores (Fig. 6. 4, A-Q)
may be spindle-shaped, ovoid, cylindrical, or spherical in different species
and are usually symmetrical, although asymmetrical or sometimes hetero-
polar types are produced by certain gregarines. The membrane is com-
monly smooth, although it may be equipped with long or short spines.

In most gregarines, the spores escape from the gametocyst by rupture
of the membrane. In certain genera, however, one or more tubular
sporoducts (Fig. 6. 4, R, S) are extruded from the wall of the mature
gametocyst. The sporoducts in Gregarina develop as tubular structures
extending inward from the gametocyst membrane and are everted shortly
before sporulation (2). Spores extruded through such sporoducts are
typically enclosed in mucous sheaths to form chains (Fig. 6. 4, P).

In parasites of the digestive tract, spores (or sometimes young game-
tocysts, or even gamonts in syzygy, depending upon the species) are
eliminated with the feces of the host. For species living in the coelom or
analogous body cavities, the distribution of spores may be more com-
plicated. The life-cycle of Gonospora is correlated with the breeding
habits of its host (46). Gamogony and the production of spores coincide
with spawning in the polychaete host, a circumstance which insures shed-
ding of spores from the coelom along with spermatozoa or ova. As for
Monocystidae in the seminal vesicles of earthworms, spores have been

Sporozoa 277

Fig. 6. 4. A-Q. Spores of different Gregarinida: A. Melamera reynoldsi,
x2880 (after Jones). B. Acanthospora repelini, x2170 (after Leger). C.
Machadoella triatomae, x2740 (after Reichenow). D. Stomatophora simplex,
xl200 (after Bhatia). E. Carcinoecetes hesperus, xl580 (after Ball). F.
Urospora rhyacodrili, about 23/tt long (after Gabriel). G, H. Gregarina
blattarum, with and without mucous sheath; x2770 (after Sprague). I.
Cystobia irregularis, x885 (after Minchin). J. Rhynchocystis porrecta, xlOOO
(after Troisi). K. Cephaloidophora communis, x29O0 (after Ball). L. Cera-
tospora mirabilis, schematic; 2770 approx. (after Leger). M. Diplocystis
schneideri, x2600 (after Kunstler). N. Cometoides capitatus, xl730 (after
Leger). O. Menospora polyacantha, 15x4^ (from Kamm, after Leger). P,
Q. Gregarinida rigida, spores in sheath as extruded from sporoduct (P),
and single spore (Q) about 8/i long (after Allegre). R. Everted sporoduct
in gametocyst of Gregarina blattarum, from living; optical section showing
expulsion of spores; x454 (after Sprague). S. Gametocyst (375/x) of Grc-
garina rigida, showing three everted sporoducts (after Allegre).

278 Sporozoa

found in the sperm ducts and in cocoons (9). Therefore, transfer of the
spores along with spermatozoa represents one method of dispersal. Death
of the host would also liberate spores in the soil, and the ingestion of
infected forms by birds and the elimination of spores in droppings has
been suggested as a mechanism for wider dispersal.

The life-cycles of the Porosporidae (42) are interesting exceptions to
the usual pattern in that both a crustacean and a molluscan host are

Fig. 6. 5. Schizogregarinida. A-E. Ophryocystis mesnili: syzygy (A);
gametocyles in gametocyst (B); one gamete formed from each gametocyte
(C); zygote after secreting oocyst membrane (D); sporozoites in oocyst (E);
xl600 (after Leger). F. Ophryocystis schneideri attached to epithelium;
xl600 (after Leger). G. Merogregarina amaroucii, anterior half of attached
sporont; xl600 (after Porter). H-N. Madiadoella triatomae: trophozoite (H);
schizont, four nuclei (I); merogony (J); merozoite (K); syzygy (L); gametes
formed within gametocyst (M); oocysts within gametocyst (N); L, xl012;
other figures, xl995 (after Reichenow).

Sporozoa 279

involved. Depending upon the species, trophozoites (Fig. 6. 10, F-H)
grow to maturity in the intestine of a crab or a lobster. At the end of
the growth period, each gregarine adheres to the lining of the rectum and
undergoes encystment as an individual, not as a member of a syzygous
pair. Encystment is followed by a series of rapid nuclear divisions and
then the production of a number of small spherical gymnospores (Fig.
6. 10, A), each of which is composed of radially arranged "merozoites."
When the cyst membrane finally ruptures, the gymnospores are released
into the sea water. Later stages develop in a molluscan host. After reach-
ing the mantle cavity of a lamellibranch, gymnospores may penetrate the
epithelium of a gill-filament or of the foot. In addition, phagocytes may
ingest gymnospores at the epithelial surface and take them into the
tissues. After passing through the epithelium, the gymnospores develop
to maturity in the tissue spaces. According to Hatt (42), the most plau-
sible interpretation of his observations is that the "merozoites" of the
gymnospores become differentiated into two types of gametes which un-
dergo anisogamy. Each zygote apparently develops directly into a young
sporozoite. The mature sporozoite of Nematopsis (Fig. 6. 10, D, E) is
enclosed in a spore membrane surrounded by a gelatinous sheath, whereas
the sporozoites of Porospora remain naked. In either case, a crustacean
host apparently becomes infected by eating infected molluscan tissue.

In terms of the usual cycle, it might be assumed that transfer from the
crustacean to the molluscan host interrupts the normal sequence of
gamogony and syngamy. Such an interpretation would imply that gymno-
spores contain immature gametes which mature in the molluscan host.
However, gymnospores are produced by single encysted gregarines, not
by pairs in syzygy, and two types of gametes seem to develop within each
gymnospore. Consequently, the mechanism of sexual differentiation in
the Porosporidae remains an interesting problem.

Taxonomy

The Gregarinidia are divided into two orders on the basis of a major
difference in life-cycles. In the Schizogregarinida merogony occurs in the
asexual phase; in the Eugregarinida, there is no merogony.

Order 1. Schizogregarinida. The trophozoite undergoes nuclear divi-
sion at maturity to produce a multinucleate schizont. Merogony then
occurs and the surviving merozoites repeat the cycle of growth and merog-
ony, perhaps several times before the merozoites develop into gamonts.
Merogony may occur either inside a tissue cell, or in a body cavity. Extra-
cellular types may be attached to an epithelium throughout much of the
growth period. Gametocysts and spores are developed as in gregarines
generally. The Schizogregarinida are parasites of various polychaetes,
gephyrean worms, insects, and (rarely) tunicates and Enteropneusta.

The order has been divided into two families (80), Ophryocystidae and

280 Sporozoa

Fig. 6. 6. Schizogregarinida: A. Schizocystis legeri, elongated schizont;
xl200 (after Leger). B, C. Meroselenidium keilini, mature form (B), me-
rogony of cytomeres (meroblasts) formed by division of schizont (C); x500
(after Mackinnon and Ray). D, E. Caulleryella pipientis, trophozoite and
spore; x900 (after Breslau and Bushkiel). F. Lipotropha macrospora, an
unusually small gregarine with intracellular development in fat body of
dipteran larvae; xl560 (after Keilin). G, H. Selenidium caulleryi, young
trophozoite and schizont; xl680 (after Ray). I-L. Lipocystis polyspora,
schizont in fat body of insect (I), syzygy in fat cell (J), merozoite (K), spore
(L); x2000 approx. (after Grell).

Schizocystidae. In the former, a single spore is produced within each
gametocyst (Fig. 6. 5, B, C); in the Schizocystidae, several to many spores
are produced (Fig. 6. 5, M, N). Genera assigned to the two families are
listed below:

Sporozoa 281

Fig. 6. 7. Ccphalina: A. Ancyrophora uncinata (intestine of beetles),
may reach length of 2 mm (after Leger). B. Acanthospora repelini, reaches
length of 1 mm (after Leger). C. Steinina rotundata (gut of dog-flea), x540
(after Ashworth and Rettie). D-G. Development of protomerite and epi-
merite in Actinncephahis pannis; x2560 (after Weschenfelder); compare with
Fig. 6. 2, A, B. H. Taeniocystis mira, young form (after Leger); compare
with Fig. 6. 1, A. I. Amphoroides calverti, xll7 (after Watson). J. Prismato-
spora evansi, x65 (after Ellis). K-M. Spores: Actinocephahis parvus (K),
x2880 (after Weschenfelder); Steinina rotundata (L), 11-12/* long (after
Ashworth and Rettie); Coelorhynchus heros (M), x330 (after Grell). N. C.
heros, young trophozoite attached to gut wall; x225 (after Grell). O.
Anthorhynchus sophiae, length up to 2 mm (after Schneider). P. Car-
cinoecetes hesperus (intestine of crabs), x306 (after Ball). Q. Cephaloido-
phora communis (intestine of barnacles), x546 (after Ball).

282 Sporozoa

Family 1. Ophryocystidae. Merogregarina Porter (Fig. 6. 5, G) Avith intracellular
merogony; Ophryocystis Schneider (78; Fig. 6. 5, A-F), with extracellular merogony; and
Spirocystis Leger and Duboscq (80), with intracellular merogony.

Family 2. Schizocystidae. CaiiUeryella Keilin (Fig. 6. 6, D, E), Lipocystis Grell (40;
Fig. 6. 6, I-L), Lipotropha Keilin (Fig. 6. 6, F), Machadoella Reichenow (117; Fig. 6.
5, H-N), Meroselenidium Mackinnon and Ray (87; Fig. 6. 6, B, C), Schizocystis Leger
(69; Fig. 6. 6, A), Selenidium Giard (109, 114; Fig. 6. 6, G, H), Selenocystis Dibb (28),
Siedleckia Caullery and Mesnil, and Syncystis Schneider.

Order 2. Eugregarinida. Each sporozoite which survives after reaching
the host develops directly into a mature trophozoite which may eventually
become a gamont. The Eugregarinida may be divided into two suborders,
Acephalina and Cephalina. The body of Cephalina is differentiated into
a protomerite and a deutomerite, and the typical protomerite is equipped
with an epimerite, at least in attached stages. The body of the Acephalina
is not differentiated into segments. The acephaline sporozoite commonly
enters a tissue cell and grows for some time as an intracellular parasite.
In cephaline gregarines, penetration of tissue cells is often incomplete
and may not occur at all.

Suborder 1. Cephalina

Division into families is based on such features as structure of the
epimerite, presence or absence of early syzygy, form of the trophozoites,
structure of the spore membrane and shape of the spore, and methods by
which the spores are released from the gametocyst.

Family 1. Acanthosporidae . The epimerite is usually knob-like or glob-
ular, with or without hooks, spines, or filaments in different genera. Early
syzygy is unknown. The spores, usually equipped with polar and equa-
torial spines (Fig. 6. 4, B), are released by rupture of the gametocyst.

The family includes the following genera (66): Acanthospora Leger (Fig. 6. 7, B),
Ancyrophora Leger (Fig. 6. 7, A), Cometoides Labbe (Fig. 6. 4, N), Corycella Leger
(Fig. 6. 1, E), Primatospora Ellis (Fig. 6. 7, J).

Family 2. Actinocephalidae. The epimerite may be short and button-
like, may lie at the end of a stalk, and may or may not be spiny. Early
syzygy is unknown. The often biconical but sometimes asymmetrical
spores (Fig. 6. 7, K-M) are released by rupture of the gametocyst.

The family contains the following genera (66): Actinocephalus Stein (140; Figs. 6.
2, A, B; 7, D-G), Amphorocephalus Ellis, Amphoroides Labbe (Fig. 6. 7, I), Anthorhyn-
chiis Labbe (Fig. 6. 7, O), Aslerophora Leger, Beloides Labbe, Bolhriopsis Schneider,
Coelorhynchus Labbe (40; Fig. 6. 7, M, N), Discorhynchus Labbe, Geneiorhynchus
Schneider, Legeria Labbe, Phialoides Labbe, Pileocephalus Schneider, Pyxiuia Hammer-
schmidt, Schneideria Leger, Sciadophora Labbe, Steinina Leger and Duboscq (Fig. 6.
7, C, L), Stictosopora Leger, Stylocystis Leger, Taeniocystis Leger (77; Figs. 6. 1, A; 7,
H).

Sporozoa 283

Fig. 6. 8. Cephalina: A. Lecythion thalassemae, trophozoite attached to
epithelial cell; x380 (after Mackinnon and Ray). B-D. Nirm gracilis, illus-
trating changes in form of the protomerite; arms extended (B), x60; partly
contracted in young specimen (C), and closed during movement through
intestinal debris (D), x400 (after Goodrich). E. Dactylophorus robustus,
xl20 approx. (after Leger). F, G. Hentschelia thalassemae, mature form with
anterior end embedded in tissue cell (F), x400 approx., and view of anterior
end (G), xl030 approx. (after Mackinnon and Ray). H. Pyxinoides puge-
tensis (from barnacles), x384 (after Henry). I. Hyalosporina cambolopsime
(from millipedes), mature form after losing small tongue-like epimerite;
cuticular striations not shown; x90 (after Chakravarty). J. Metamera reyn-
oldsi, attached to epithelium; xl40 (after Jones).

284 Sporozoa

Family 3. Cephaloidiphoridae. The epimerite is rudimentary. Early
syzygy occurs and association is sometimes multiple. Ovoid spores (Fig. 6.
4, K) are released by rupture of the gametocyst.

Two genera are included: Carci7ioecetes Ball (5; Fig. 6. 7, P) and Cephaloidophora
(5, 45; Fig. 6. 7, Q).

Fie 6 9 Cephalina: A, B. Stcnophorn shyamaprasadi (from centipede),
sporont, x365; spore, x3150 (after Chakravarty). C, D. Moywductus lunatus
trophozoite, x216; broad and narrow aspects of spore, x314y (after Ray and
Chakravarty). E. F. Colepismatophila ivalsonae, young trophozoite with sim-
ple epimerite, xllO; older form after loss of epimerite, x81 (after Adams
and Travis). G. Poiyrhabdina spionis, x298 (after Mackinnon and Ray = see
also Fig 6 ^ D H. 7.ygosoma glohosum. mature trophozoite, x74 (after
Noble); see epimerite of younger stage in Fig. 6. 2, G. I. Cystocephalus
aherianus. reaches length of 3-4 mm (after Schneider). J. Lecudma pclhinda
(from Kamm, after Kolliker). K. Menospora polyacantha, reaches length of
600-700^ (after Leger). L. Bulbocephalus elongatus, reaches length of about
1.5 mm (after Watson). M. Stylocephalus giganteus, reaches length of 1.8 mm
(after Ellis).

Sporozoa 285

Fajnily 4. Dactylophoridae. The protomerite is typically metabolic and
may serve as a sucker in attachment. In certain species, the "epimerites"
are slender temporary structures, tapering to delicate filaments. The
cuticle of the microgametocyte persists after gamogony as a "pseudocyst."
The spores (Fig. 6. 4, A), ellipsoidal, or cylindrical with rounded ends,
are released by rupture of the gametocyst. Members of the family have
been reported from the intestine of Chilopoda,

The following genera are included (66): Acutispora Crawley, Dactylophorus Balbiani
(Fig. 6. 8, E), Dendrorhynchus Keilin, Echinomera Labbe (37, 122), Hentschelia Mackin-
non and Ray (85; Fig. 6. 8, F, G), Lecythion Mackinnon and Ray (85; Fig. 6. 8, F,
G), Metamera Duke (65; Fig. 6. 8, J), Nina Grebnecki (37; Fig. 6. 8, B-D). Rhopalonia
Leger, Septicephalus Kamm, Trichorhynchus Schneider.

Family 5. Didymophyidae. The epimerite is small, resembling the mu-
cron of certain acephalines. Early syzygy occurs, in pairs or triplets, and
a septum may not be apparent in the satellites. Ellipsoidal spores are
released by rupture of the gametocyst.

The family includes the genus Didymophyes Stein (66).

Family 6. Gregarinidae. The epimerite is simple, knob-like, or some-
what elongated. Syzygy may occur early or late and is commonly multiple
in some genera. Spores emerge through sporoducts, often in chains (Fig.
6. 4, P-S), or else by rupture of the gametocyst.

The following genera are included (66): Anisolobiis Vincent, Gamocystis Schneider
Gregarina Dufour (2, 126; Fig. 6. 2, H), Hirmocystis Labbe, Hyalospora Schneider'
Hyalospanna Chakravarty (14; Fig. 6. 8, E), Leidyana Watson, Protomagalhaesia Pinto,
Pyxtnoides Tregouboff (45; Fig. 6. 8, H), Uradiophora Mercier.

Family 7. Lecudinidae. The epimerite may be knob-like, with or with-
out teeth and hooks, or an umbrella-like structure with lobate margin,
or cylindrical with a lobate tip. A septum is not evident although the
protomerite and deutomerite regions may differ in appearance. The
spores are usually ovoid.

The following genera have been referred to the family, which includes the Polyrhab-
dmidae of Kamm (66): Kofoidina Henry, Leucudina Mingazzini (Fig 6 9 J)
Polyrhabdina Mingazzini (85; Fig. 6. 9. G), Scyia L^ger, Ulivina Mingazzini, Zy^osoma
Labbe (96; Fig. 6. 9, H). "^^

Family 8. Menosporidae. The epimerite is cup-shaped, with marginal
hooks, and is borne on a stalk. Early syzygy is unknown. Crescent-shaped
spores (Fig. 6. 4, O) are released by rupture of the gametocyst.

A single genus is included: Menospora Lcger (66; Fig.. 6. 9, K).

286 Sporozoa

Family 9. Monoductidae. The epimerite is usually a small knob, with
or without prongs. Early syzygy is unknown. A single sporoduct is char-
acteristic of Monoductus but not of other genera. The spores usually
emerge in chains.

The following genera are included: Colepismatophila Adams and Travis (Fig. 6. 9,
E, F), Lepismatophila Adams and Travis, Monoductus Ray and Chakravarty (111; Fig.
6. 9, C, D), and Sphaerocystis Leger.

Fig. 6. 10. Porosporidae (after Halt): A. Gymnospore of Nematopsis
legeri, section, x3105. B. Later development of gymnospore (Porospora
gigantea) in molluscan tissue, x3240. C. Sporozoites of N. legeri in phagocyte,
molluscan gill, x2835. D. "Cyst" with spores, A', legeri, xl425. E. Sporozoite
(A^. legeri) escaping from spore in gut of crab; from living, xl425. F, G.
Young and older trophozoites (P. gigantea) attached to intestinal epithelium
in crustacean host; xl500. H. Young cephalin of N. legeri attached to epi-
thelium of crustacean intestine; xl425.

Family 10. Porosporidae. The epimerite is a simple disc or rudimentary.
Syzygy is often early and may be multiple, but a typical gametocyst is not
produced. Instead, gymnospores (blastula-like clusters of "merozoites"),
derived from individually encysted gregarines (42), leave the gut of the

Sporozoa 287

crustacean host. After gymnospores reach a suitable mollusc, development
results in sporozoites (Fig. 6. 10, A-D), one from each zygote, as described
above.

The family includes two genera (42): Nematopsis Schneider (Fig. 6. 10, H) with
monozoic spores, from crabs; and Porospora Schneider (Fig. 6. 10, F, G), with naked
sporozoites, from lobsters.

Family 11. Stenophoridae. The epimerite is rudimentary or absent.
Early syzygy is unknown. Ovoid spores are released by rupture of the
gametocyst.

Two genera are included (66): Fonsecaia Pinto and Stephanophora Labbe (15; Fig. 6.

Family 12. Stylocephalidae. The epimerite ranges from globular or dis-
coid to a complex elongated or conical organelle, sometimes lobate or
equipped with bristles. Early syzygy is unknown. A pseudocyst is reported
for some genera. Spores may be released in chains.

The following genera are included (66): Bulbocephalus Watson (Fig 6 9 L)
Cystocephalus Schneider (Fig. 6. 9, I), Lophocephalus Labbe, Oocephalm Schneider.
Sphaerorhy7ichiis Labbe, Stylocephalus Ellis (Fig. 6. 9, M).

Suborder 2. Acephalina

These are non-septate, mostly coelomic parasites, many of which occur
in the seminal vesicles of oligochaetes. Some of the exceptions are Hy-
pendion (86), a genus of uncertain taxonomic status, including intestinal
parasites of echiuroid worms; and Lankesteria and Allantocystis, from the
digestive tract of insects. A synoptic review of genera and families is
available (10).

Family 1. Aikinetocystidae. The family contains Aikinetocystis Gates
(Fig. 6. 1, F), in which the anterior end of the trophozoite is dichoto-
mously branched, with sucker-like organelles at the tips. Trophozoites
may reach lengths of 3-4 mm. Spores are similar to those of Monocystis.

Family 2. Allantocystidae. This family was established for Allantocystis
Keilm (67), in which the elongated trophozoites undergo head-to-head
syzygy. The gametocyst is much elongated (Fig. 6. 11, F). Spores are
spindle-shaped, not quite symmetrical.

Family 3. Diplocystidae. Early syzygy may or may not occur. Spores
(Fig. 6. 4, M) are ovoid to spherical. A small pseudopodial epimeritic
organ may be present. Species are known from flatworms, insects and
tunicates.

FijTfr Td)"'' '"'^"'^^''- ^'P^^^yf^' Kunstler (62) and Lankesteria Mingazzini (112;

288 Sporozoa

Fig. 6. 11. Acephalina: A-D. Lankesteria culicis Ross (after Ray), intra-
cellular stage (A), young trophozoite attached to intestinal epithelium (B),
xl332; mature trophozoite, cuticular striations omitted (C), x578; spore,
from living (D), x2280. E. Craterocystis papiia, schematic optical section show-
ing anterior "sucker," x70 approx. (after Cognetti). F. Elongated gametocyst
of Allantocyslis dasyhelei, x425 approx. (after Keilin). G. Rhynchocystis pilosa
Cuenot, young trophozoite, xl800 (after Troisi); see also Fig. 6. 2, C. H.
Apolocystis minuta, yoimg trophozoite, xl600 (after Troisi); compare with
Fig. 6. 1, C. I. Monocyslis agilis Stein, commonly 120-145/i (after Berlin).
J. Spore of Monocystis ventrosa (after Berlin). K. Ganymedes anapsides,
syzygy showing "ball-and-socket" junction, x515 approx. (after Huxley). L.
Beccaricystis loriai, "sucker" at anterior end; x630 approx. (after Cognetti).

Family 4. Ganymedidae. Syzygy in primite-satellite fashion is charac-
teristic. Gametocysts are spherical. Life-cycles are incompletely known for
the only genus, Ganymedes Huxley (Fig. 6. 11, K).

Family 5. Monocystidae. Mature trophozoites range from spheroid (Fig.

Sporozoa 289

6. 1, C) to much elongated types (Fig. 6. 1, B). A sucker-like epimeritic
organ or a mucron may or may not be present. Spores are typically
spindle-shaped. Various species of Monocystis and Nemotocystis have been
described in detail by Berlin (7). Many Monocystidae occur in the seminal
vesicles of earthworms. The sporozoites of some species enter germinal
cells and grow within the developing sperm-morulae. In other species
parasitizing the seminal vesicles, development is extracellular. Species
within a single genus, such as Apolocystis (133), may differ with respect
to intracellular or extracellular development.

The following genera have been assigned to the family: Apolocystis Cognetti (106,
133; Figs. 6. 1, C; 11, H), Echinocystis Bhatia and Chatterjee (11), Enterocystis Zvvetkow,

Fig. 6. 12. Acephalina. A, B. Stonmtophora simplex, view of mobile
anterior "sucker" with central mucron (A), x8U0; trophozoite, light anterior
area representmg region of sucker (B), x960 (after Bhatia). C.-E. Urospora
rliyacodrili. young trophozoite in gut wall (C), xl225; older trophozoite in
seminal vesicle (D), x216; syzygy (E), x216 (after Gabriel). F. Choatiocys-
toides costaricensis, anterior end of trophozoite showing "sucker," schematic,
730 approx. (after Cognetti). G, H. Gonospora varia Leger, mature stage
and a pair in syzygy; x96 approx. (after Hentschel). I. Heteropolar spore
of Lithocystis brachycercus, xl396 (after Goodrich). J. Zygocystis wenrichi,
young trophozoite, x700 (after Troisi); compare with Fig. 6. 2, E.

290 Sporozoa

Monocystis Stein (7, 41; Fig. 6. 11, I), Nematocystis Hesse (7; Fig. 6. 1, B), Rhabdocystis
Coldt.

Family 6. RJiynchocystidae. The family contains only the genus Rhyn-
chocystis Hesse (7, 133; Figs. 6. 2, C; 11, G). A mucron is more or less
evident. The body is usually elongated, sometimes with an anterior con-
ical or cylindrical "neck," and may be covered with cuticular "hairs."
Early syzygy does not occur and autotomy may precede the association.
Spores are typically spindle-shaped.

Family 7. Schaudinnelidae. The only genus is Schaudinnella Nusbaum
(101; Fig. 6. 1, D), showing two types of gametocytes and well-marked
anisogamy. Trophozoites may be free or attached, the latter stage with a
primitive epimerite.

Family 8. Stomatophoridae. A discoid sucker-like epimerite is charac-
teristic of the elongated to spheroid trophozoites. Early syzygy is unknown.
Spores are usually truncate spindles.

The following genera are included: Albertisella Cognetti, Astrocystella Cognetti,
Beccaricystis Cognetti (Fig. 6. 11, L), Choanocystella Cognetti, Choanocystoides Cog-
netti (Fig. 6. 12, F), Craterocystis Cognetti (Fig. 6. 11, E), Stomatophora Drzewecki (8;
Fig. 6. 12, A, B).

Family 9. Urosporidae. Form of the trophozoite varies in different
genera. Early syzygy is characteristic. The spore luembrane may be drawn
out into horns or flanges, and there is often a funnel-like depression at
one end.

The family contains the following genera: Ceratospora Leger, Cystohia Mingazzini,
Gonospora Schneider (46; Fig. 6. 12, G, H), Lithocystis Giard (36; Fig. 6. 12, I), Ptero-
spora Racovitza and Labbe, Urospora Schneider (31, 92; Fig. 6. 12, C-E).

Family 10. Zygocystidae. The trophozoites are commonly pyriform.
Early syzygy, sometimes with longitudinal pairing, is the rule. The spores
are spindle-shaped, with thickened poles. Species are known from the
seminal vesicles and coelom of oligochaetes.

The family contains the genera Pleurocystis Hesse and Zvgocystis Stein (7, 133; Figs. 6.
2, E; 12, J).

Subclass 2. Coccidia

The Coccidia are predominantly parasites of epithelial tissues in
invertebrates (Annelida, Arthropoda, Mollusca) and vertebrates, and are
typically intracellular throughout most of their life-cycles. Reproduction
occurs in both asexual and sexual phases of the cycle, as in Schizogreg-
arinida.

Sporozoa 291

Life-cycles

An infection is initiated when the host ingests oocysts or sporocysts
(spores), or in rare cases, when naked sporozoites are inoculated or in-
gested. Each surviving sporozoite enters a tissue cell and develops into a
multinucleate schizont. Merogony then occurs. The resulting merozoites
enter other cells and repeat the cycle. In typical Coccidia, merogony in-

_^.,.«flrr^®,

Fig. 6. 13. A. Merogony in Oi'ivora thalassemae; section of schizont, x500
(after Mackinnon and Ray). B-D. Merogony, involving formation of mero-
blasts, in Caryotropha mesnili (after Siedlecki): young trophozoite (B); mero-
blasts formed by division of a trophozoite (C); formation of merozoites from
meroblasts (D); x535. E-G. Production of microgametes in Ovivora thalas-
semae; x850 (after Mackinnon and Ray). H. Syzygy in Adelea ovata; xll40
(after Shellack and Reichenow). I. Syzygy in Adelina deronis, microgametocyte
with four nuclei; xl600 (after Hauschka). J. Zygote of Adelea ovata, oocyst
membrane formed, three microgametes left outside; xll40 (after Shellack and
Reichenow). K. Flagellate microgamete of Caryotropha mesnili; xl510 (after
Siedlecki).

292 Sporozoa

volves a preliminary arrangement of nuclei at the surface of the schizont
and then a superficial budding (Fig. 6. 13, A). However, the trophozoite
of Caryotropha (Fig. 6. 13, B-D) first divides into cytomeres (meroblasts)
and each meroblast then produces merozoites.

There appears to be a limited number of merogonic cycles, the exact
number varying with the species — usually two in Adelina deronis (43);
three in Eimeria separata and E. miyarii; and foin- in E. Jiieschidzi of rats
(118). The time required for completion of the first merogonic genera-
tion, as reported for different species, ranges from about 25 hours to 25
days.

The last generation of merozoites produces two types of gametocytes,
which vary in relative numbers. In Adelina deronis, in which the gameto-
cytes develop in syzygy and relatively few microgametes are produced,
there are about twice as many microgametocytes as macrogametocytes
(43). In Eimeria nieschuhi, which produces many microgametes, macro-
gametocytes outnumber microgametocytes about three to one (118). The
mechanism underlying the differentiation of two kinds of gametocytes is
not yet known. However, the development of a normal cycle in the host
after experimental introduction of one oocyst (135) suggests that the
basic sexual differentiation occurs early in development of the zygote,
although its expression may be delayed until gametocytes appear.

The gametocytes may be similar in size (Eimeriida) or the m.acroga-
metocyte may be distinctly the larger (Adeleida). The macrogametocyte,
during development, typically accumulates stored reserves such as the
glycogen in Eimeria tenella (30), Avhereas the microgametocyte contains
little stored food. The gametocytes of Eimeriida differentiate independ-
ently, and the microgametocyte typically produces many microgametes
(Fig. 6. 13, E-G). In the Adeleida the two types of gametocytes, sometimes
at an early stage of development, become associated in syzygy (Fig. 6.
13, H, I), which is correlated with the production of relatively few, often
2-4, microgametes (Fig. 6. 13, J). For the Coccidia as a group, the mor-
phological differentiation of macrogametes and microgametes is marked
and the production of small flagellate microgametes (Fig. 6. 13, K) is
typical. Microgametogenesis in Eimeriida generally resembles the process
described for Ovivora thalassemae (Fig. 6. 13, E-G). In Merocystis (105)
and Myriospora (82), however, the microgametocyte divides into gameto-
blasts, each of which produces a number of microgametes. The details of
syngamy seem to be similar throughout the group.

Either before or immediately after entrance of a microgamete the
macrogamete usually secretes an oocyst membrane, although the zygote
of the Haemogregarinina is at first a migratory ookinete which later
secretes a thin flexible membrane. In the majority of Coccidia, the oocyst
membrane is relatively thick and firm, and may be composed of two or

Sporozoa 293

Fig. 6. 14. A. Oocyst of the Eiiueria-type, with heavy ectocyst and thin
endocyst; thin area in the ectocyst is the region of the micropyle; the zygote
is undivided; diagrammatic (after Goodrich). B, C. Ovivora thalassemae,
x500 (after Mackinnon and Ray): development of sporoblasts (B); oocyst
containing sporoblasts (C). D. Oocyst of Aggregata eberthi, portion of a
section; nuclei are arranged at the surfaces of folds in the plasmodial mass,
shortly before the production of many sporoblasts; x305 (after Dobell). E.
Sporozoites formed within sporocysts in oocyst of Eimeria vison; xl620 (after
Levine). F-H. Oocysts containing sporozoites not enclosed in sporocysts:
Legerella parva (F), xl440 (after Noller); Haemogregarina stepanowi (G),
xl890 (after Reichenow); Pfeifjerinella impudica (H), xl560 (after Leger
and Holland).

more layers (Fig. 6. 14, A). The mature macrogamete of Eimeria stiedae
(Fig. 6. 18, E, F) contains a peripheral zone of globular inclusions which
are extruded to form an ectocyst, continuous except for a micropyle (a
minute opening through which the microgamete will enter). After syn-
gamy the micropyle is closed by the secretion of more material but the
closed area remains thinner than the rest of the ectocyst in certain species.
Before the zygote rounds up, a relatively thin endocyst (Fig. 6. 14, A) is
secreted within the ectocyst (38). Three layers have been described in
Eimeria intricata (44) — a thin transparent outer layer, thickened as a

294 Sporozoa

polar cap over the micropyle; a thick brownish intermediate layer which
becomes quite thin at the micropyle; and a thin colorless endocyst. Al-
though there are a number of exceptions, size and structure of the oocyst
are often rather characteristic of the species.

The zygote usually divides into sporoblasts, often leaving a residual
mass, and each sporoblast usually secretes a sporocyst membrane (Fig. 6.
14, B-E). Less commonly, sporocysts are not produced, sporozoites being
protected only by the oocyst membrane (Fig. 6. 14, F-H). The sporoblasts
of Karyolysus are unusual in that they are released from the ruptured
oocyst as motile elongated sporokinetes which invade the eggs of a mite.
The sporoblast then rounds up and secretes a sporocyst membrane. By
the time the egg has developed into a nymph, the sporoblast has produced
sporozoites. In the more typical Coccidia, both the number of sporocysts
and the number of sporozoites are features of taxonomic value.

Sporogony may or may not be completed within the host. The oocysts
of avian and mammalian parasites are typically eliminated with the zy-
gote undivided or in the process of forming sporoblasts. Under favorable
conditions, sporozoites are developed within a few days, and the oocyst
is then infective for a new host. At the other extreme, the production of
sporozoites is completed within the host. Development of the latter type
has led to an interesting modification of the coccidian life-cycle in
Shellackia (Fig. 6. 19, A-F). Sporozoites are developed within an asporo-
cystic oocyst, which eventually ruptures in the intestinal connective tissue
of the reptilian host. The liberated sporozoites enter erythrocytes. In-
vaded blood corpuscles are swallowed by a mite, in which cells of the gut
wall apparently ingest the sporozoites without destroying them. If such a
mite is eaten by a vertebrate host the sporozoites are released and invade
the intestinal epithelium. A comparable transfer by leeches occurs in
Lankesterella, in which there is a similar invasion of erythrocytes by
sporozoites.

In these cycles of Shellackia and Lankesterella the mite and the leech
are mechanical vectors in which the parasites do not undergo develop-
ment. A true intermediate host occurs in certain cases involving invasion
of erythrocytes or leucocytes by gametocytes. After ingestion of gameto-
cytes by a leech (Haemogregarina) or by a tick or mite {Hepatozoon,
Karyolysus), gametogenesis and syngamy occur and sporozoites are pro-
duced in the invertebrate. Transfer of sporozoites may be effected by
inoculation, as in the case of leeches feeding on a vertebrate, or by the
ingestion of infected mites. Aggregata eberthi (29) shows an unusual
cycle in which the "intermediate" host becomes infected by eating the
"final" host. Merogony occurs in the intestinal connective tissue of a crab
which has ingested sporocysts. If an infected crab is eaten by a squid,
some of the merozoites develop into gametocytes, and syngamy and
sporogony follow,

Sporozoa 295

Fig. 6. 15. A-F. Aclelina deronis Hauschka and Pennypacker (after
Hauschka): Early (A) and later syzygy (B), xl700; sporoblasts in oocyst,
remains of microgametocyte still attached (C), xl600; mature sporocyst (D),
xl900; young schizont in a peritoneal cell (E), xl600; merogony (F), xl600.
G. Oocyst of Adelea ovata Schneider, with sporocysts; x960 (after Shellack
and Reichenow). H, I. Chagasella hartmanni (Chagas) Machado: oocyst with
three developing sporocysts (H); sporocyst with four sporozoites (I); x750
(after C). J, K. Hepatozoon tntiris (Balfour) Miller (after M.): ookinete
penetrating intestinal epithelium (J): oocyst (K). L. Hepatozoon canis
Leger, portion of oocyst containing sporocysts; x630 (after Wenyon). M.
Hepatozoon adiei, gametocyte in leucocyte; x2100 (after Hoare).

Taxonomy

On the basis of life-histories, the Coccidia may be divided into two or-
ders, the Adeleida and the Eimeriida. In the Adeleida, the gametocytes are
associated in syzygy during differentiation and only a few microgametes

296 Sporozoa

are usually produced. In the Eimeriida, gametocytes develop independ-
ently and the microgametocyte typically produces many microgametes.
Order 1. Adeleida. On the basis of differences in the zygote and oocyst,
the Adeleida may be divided into the suborders Adeleina and Haemo-
gregarinina. The Adeleina form an inactive zygote which develops a
typical oocyst. The ookinete of the Haemogregarinina secretes a flexible
membrane which is stretched during development.

Suborder 1. Adeleina

Four families have been recognized. Sporocysts are developed in t'wo
families but not in the others.

Family 1. Adeleidae. Sporocysts are developed within an oocyst and the
life-cycle is typical of the suborder.

The following genera have been assigned to the family: Adelea Schneider (Figs. 6.
13, H, J; 15, G), with a large oocyst and a variable but fairly large number of discoidal
sporocysts. each containing two sporozoites; Adelina Hesse (43, 113; Fig. 6. 15, A-F),
oocyst containing relatively few spherical sporocysts, each with two sporozoites; Chaga-
sella Machado (Fig. 6. 15, H, I), oocyst containing three sporocysts, each with four
sporozoites; Klossia Schneider (91, 93), oocyst containing many spherical sporocysts,
each with four sporozoites.

Family 2. Dobelliidae. The only genus is Dobellia Ikeda (59), which is
unusual in that the microgametocyte produces a fairly large number of
microgametes in spite of the fact that syzygy occurs. Sporocysts are not
produced. A single species has been reported from sipunculids.

Family 3. Klossiellidae. The oocyst contains a number of sporocysts,
each with many sporozoites. Microgametogenesis yields two microgametes.
The family contains the genus, Klossiello Smith and Johnson (128), rep-
resented in mice and guinea pigs.

Family 4. LegerelUdae. The single genus, Legerella Mesnil (Fig. 6. 14,
F), produces an oocyst with many sporozoites but no sporocysts. The
known species occur in fleas and myriapods.

Suborder 2. Haem,ogregarinina

Members of this group differ from other Adeleida in that the life-cycle
involves two hosts and the zygote is an ookinete. Three families, each with
a single genus, are generally recognized.

Faynily 1. HaemogregarinJdae. In Haemogregarina Danilewsky (Fig. 6.
16, A-E), the small oocyst contains no sporocysts. The sexual phase of the
cycle occurs in leeches; asexual stages, in various turtles. Merozoites in-
vade erythrocytes of the vertebrate and develop into gametocytes.

Family 2. Hepatozoidae. The large oocysts contain many sporocysts,
each with a dozen or more sporozoites. The gametocytes appear in leuco-
cytes of the vertebrate host. Sexual stages occur in tsetse flies (50), lice,

Sporozoa 297

mites, and ticks. The type genus is Hepatozoon Miller (50; Fig. 6. 15,
J-M), represented by several species in birds and mammals.

Family 3. Karyolysidae. The sporoblasts become sporokinetes which
invade the egg of a mite before secreting sporocyst membranes. Gameto-
cytes appear in erythrocytes of the vertebrate host. Several species of
Karyolysus Labbe (115; Fig. 6. 16, F-K) have been described from lizards.

Fig. 6. 16. A-E. Haemogregarina stepanowi Danilewsky, xl890 (after
Reichenow): microgainetocyte (A); macrogametocyte (B); syzygy (C); schi-
zont in erythrocyte (D); merozoites in erytlirocyte (E). F-K. Karyolysus lacer-
tarum (Danilewsky) Labbe (after Reichenow): gametocyte in erythrocyte
(F), xl050; oocyst producing sporoblasts (G), x800; motile sporokinete (H)
and sporokinete in egg of mite (I), xl050; sporocyst in larval mite (J), x800;
merozoite in endothelial cell (K), xl050.

Order 2. Eimeriida. These Coccidia differ from the Adeleida in the
absence of syzygy. Certain species are economically important as parasites
of poultry, quail, pheasants, cattle, sheep, and such fur-bearing animals
as the fox and mink (6, 12). Problems of control are aggravated by the
survival of oocysts for prolonged periods on the soil. Six families of
Eimeriida are often recognized. However, Hoare (51) has suggested a
division of the group into only two families, the Selenococcidiidae and
the Eimeriidae, the latter containing six subfamilies.

298 Sporozoa

Family 1. Aggregatidae. Several to many sporocysts, in some species
several hundred, are developed within the oocyst. In Aggregata, merogony
occurs in crabs and syngamy and sporogony in cephalopods; in Ovivora
both phases of the cycle are completed in one host. Life-cycles are incom-
pletely known for various other members of the family.

Fig. 6. 17. A-E. Cryptosporidium parviim, x3200 (after Tyzzer): tropho-
zoites (A); schizont (B); merogony (C); microgametogenesis (D); oocyst with
four sporozoites (E); all stages extracellular, on surface of intestinal epithelium
(mice). F-H. Aggergata eberthi (Labbe): sporoblast before secretion of sporo-
cyst membrane (F), x2000; lateral and polar views of sporocyst containing
three sporozoites (G, H), x2200 (after Dobell). I. Sporocyst of Myriospora
trophoniae, Avith sporozoites (after Lermantoff). J. Oocyst of Caryotropha
mesnili, with several sporocysts; x535 (after Siedlecki). K, L. Ovivora thalas-
semae (after Mackinnon and Ray): egg of Thalassema containing two parasites
(K), x240; sporocyst with 10 nuclei (L), xl440.

The following genera have been included: Aggregata Frenzel (29; Figs. 6. 14, D; 17,
F-H), oocyst containing many sporocysts, each with 3-16 sporozoites in different species;
Angeiocystis Brasil, oocyst with four sporocysts, each containing about thirty sporozoites;
Merocyslis Dakin (21, 105), two sporozoites in each of many sporocysts; Myriospora
Lermantoff (82; Fig. 6. 17, I), oocyst with a few hundred sporocysts, each with 24-36
sporozoites; Ovivora Mackinnon and Ray (88; Figs. 6. 13, A, E-G; 14, B, C; 17, K, L),
many sporocysts, each with about twelve sporozoites; Pseudoklossia Leger and Duboscq
(81), oocyst with many dizoic sporocysts, sporogony in Pelecypoda, but merogonic cycle
unknown.

Sporozoa 299

Fig. 6. 18. A-J. Eimeria stiedae, stages in epithelium of bile ducts (rab-
bits); X580-600; diagrammatic: growth stage (A); multinucleate schizont (B);
merozoites (C, D); young and more mature macrogametocytes (E, F); oocyst
membrane formed (G); stage in microgametogenesis (H); surface view and
optical section, microgametes nearly mature (I, J). K. Oocyst with four
sporocysts, E. stiedae; x860 (after Kessel and Jankiewicz). L. Isospora
bigemina, oocyst with two sporocysts; x2400 (after Wenyon). M. Oocyst of
Echinospora Jabbei, xl950 (after Lcger). N, O. Barrouxia schneideri (after
Shellack and Reichenow): oocyst with sporocysts (N), xl565; sporocyst with
single sporozoite (O), xl895. P. Oocyst with collar, surface view, Jarrina
paludosa; xl560 (after L^ger and Hesse). Q-S. Dorisiella scolelepidis (after
Ray): division of zygote into two sporoblasts (Q, R), xl340; sporocyst with
eight sporozoites (S), xl650. T. Oocyst of Cyclospora caryolytica, two sporo-
cysts; xl680 (after Schaudinn).

300 Sporozoa

Family 2. Caryotrophidoe. The oocyst contains many sporocysts, each
with many sporozoites. The merogonic cycle involves division of the
schizont into a number of meroblasts, each of which produces merozoites.
Microgametogenesis involves a similar process. The only genus is Caryo-
tropha Siedlecki (Figs. 6. 13, B-D; 17, J).

Family 3. Crypiosporidiidae. Development is extracellular, the parasites
apparently being embedded in the mucus covering the epithelium of the
gut. The small oocyst contains four sporozoites but no sporocysts. Certain
parasites of mice have been assigned to the only genus, Cryptosporidium,
Tyzzer (134; Fig. 6. 17, A-E).

Family 4. Eimeriidae. The characteristics of the family are somewhat

Fig. 6. 19. A-F. Shellackia bolivari, x900 (after Reichenow): zygote in
subepithelial tissue, intestine of li/ard (A); development of sporozoites from
zygote (B, C); a sporozoite in an erythrocyte (D); sporozoites in cell of in-
testinal epithelium of mite (E); a young trophozoite and a schizont in
intestinal epithelium of lizard after ingestion of infected mites (F). G-L.
Selenococcidium intermedium, x765 (after Leger and Duboscq): schizont
with eight nuclei, before invasion of an intestinal cell in lobster (G); com-
pletion of merogony (H); a macrogamelocyte from the intestinal lumen (I);
an intracellular macrogametocyte (J); a microgametocyte before invading a
gut cell (K); oocyst (L). M. Oocyst of Lankesterella minima, with sporozoites
(no sporocysts arc formed); xll25 (after Noller).

Sporozoa 301

flexible. Sporocysts are lacking in a few cases; in others, the oocyst con-
tains one, two, four, or many sporocysts. The number of sporozoites within
each sporocyst also varies from one to many.

The following genera have been assigned to the family: Barrouxia Schneider (Fig.
6. 18, N, O), smooth oocyst containing many sporocysts, each with one sporozoite;
Caryospora Leger (51), oocyst containing one sporocyst with eight sporozoites; Cyclo-
spora Schneider (Fig. 6. 18, T), oocyst with two dizoic sporocysts; Dorisiella Ray (110;
Fig. 6. 18, Q-S), zygotes (apparently with a very delicate oocyst membrane) producing
two sporocysts, each with eight sporozoites; Ecfiinospora Leger (Fig. 6. 18, M), spiny
oocyst containing 4-8 bivalve sporocysts, each with one sporozoite; Eiineria Schneider
(6; Fig. 6. 18, A-K), oocysts containing four dizoic sporocysts, many species known from
mammals, birds, reptiles. Amphibia and fishes, and a few from Arthropoda; Isospora
Schneider (6; Fig. 6. 18, L), oocyst with two tetrazoic sporocysts; Jarrina Leger and
Hesse (Fig. 6. 18, P), oocyst with an elevated collar surrounding the micropyle, four
dizoic sporocysts; Pfeiffcriuella ^V'asieIewski (Fig. 6. 14, H), oocyst with eight sporozoites
but no sporocysts; Wenyonella Hoare (51), oocyst with four tetrazoic sporocysts.

Fainily 5. Lankesterellidae . Sporozoites, developed within the asporo-
cystic oocyst, are liberated in the vertebrate host and enter erythrocytes.
Invaded corpuscles are ingested and transferred mechanically by an in-
vertebrate vector (mite, leech). The merogonic cycle is then resumed in
the new vertebrate host. In Shellackia Reichenow (Fig. 6. 19, A-F), de-
velopment occurs in the intestinal epithelium of lizards. Sporozoites
which have entered red corpuscles are transferred mechanically by a mite.
In Lankesterella Labbe (100, 100a; Fig. 6. 19, M), development occurs in
the endothelial cells of capillaries in frogs. Sporozoites, after entering
erythrocytes, are transferred by a leech.

Family 6. Selenococcidiidae. The details of sporogony are unknown.
The reported phases of the life-cycle are unusual in that growth stages of
both schizonts and gametocytes are extracellular but enter tissue cells
to complete their development. The only genus, Selenococcidinyn Leger
and Duboscq (79a; Fig. 6. 19, G-L), contains a species reported from the
intestine of lobsters.

Subclass 3. Haemosporidia

These are the typical blood parasites whose gametocytes, and also
the merogonic cycle in some cases, occur in red blood cells. Syngamy and
sporogony occur in an invertebrate host. The zygote becomes a migratory
ookinete. Although an oocyst may be developed later, the membrane is
never a thick, resistant covering. Sporozoites are never enclosed in a
sporocyst membrane and inoculative transfer is the rule. In the Plasmo-
diida, the stages which invade red blood cells deposit pigment in their
cytoplasm, but this apparently is not true for the Babesiida. Malarial
parasites are known to split hemoglobin into globin and hematin, digest
the protein, and retain the hematin in characteristic pigment granules
(90).

302 Sporozoa

The Haemosporidia may be divided into the Plasmodiida and the
Babesiida. The life-cycle of the Plasmodiida resembles that of Coccidia,
with well-marked merogony and sporogony. The life-cycles of many
Babesiida are incompletely known. Some species apparently undergo
fission in red corpuscles of the vertebrate host. Merogony in lymphocytes
has been reported for certain others. The fusion of similar gametes has
been described in Babesia (27).

'Zi'

it..

J ■- ; -*^i^v ■■■2 »'■ />■'.'

Fig. 6. 20. Plasmodiuiyi circunifJexum, stages in mosquitoes (after Reiche-
now): macroganiete (A) and ookinete (B), x3640; ookinete in cell of gut wall
(C), xl950; young oocyst with four nuclei (D), x3640; portion of a section
through an older oocyst, remnant of gut cell shown (E), xl950; portion of a
section through a mature oocyst, just before sporogony (F), xl850; a sporozoite
(G), x3960.

Order 1. Plasmodiida. Throughout the order, merogony occurs in endo-
thelial or related tissue cells of the vertebrate host. In one family, me-
rogony is apparently restricted to parasites in such tissue cells. In the
malarial parasites, however, merozoites from the basic merogonic cycle
invade red blood cells and undergo a series of erythrocytic cycles of
merogony. Gametocytes develop from some of these erythrocytic mero-
zoites. In other Plasmodiida, only the gametocytes appear in blood cells.
In either case, gametocytes are ingested by an invertebrate host in which
syngamy and sporogony occur.

Sporozoa 303

Gametogenesis and syngamy of Plasmodiida resemble these processes
in Eimeriida. The gametocytes (Fig, 6. 21, F, G) are more or less similar
in size, and are not associated during development. At maturity, the
microgametocyte rounds up and produces a few slender microgametes
which are rapidly separated from the residual protoplasm in a process of
"exflagellation." Syngamy results in a migratory ookinete (Fig. 6. 20, A-C).
This ookinete may pass through the gut wall of the vector and come to
rest beneath the membrane covering the gut; or as in Plasmodium circum-
flexum of birds and moscpiitoes (116), it may invade an epithelial cell of
the gut and develop there. The zygote apparently secretes a thin oocyst
membrane which is stretched as the parasite grows. Repeated nuclear
division results in a multinucleate sporont, or "oocyst" (Fig. 6. 22, A),
which produces many sporozoites. Sporoblasts are not formed, although
the sporont in Plasmodium becomes extensively "vacuolated" before
sporogony (Fig. 6. 20, F). Some workers have interpreted this apparent
vacuolation as a series of infoldings from the surface. The liberated sporo-
zoites (Fig. 6. 20, G) migrate through the tissues, and some of them reach
the anterior part of the digestive tract from which they are inoculated
into a vertebrate.

After inoculation into a vertebrate, the sporozoites typically invade, or
are ingested by, phagocytic cells of the viscera within an hour or so. The
sporozoite becomes a trophozoite which develops into a multinucleate
schizont and undergoes merogony (Fig. 6. 21, A, B). The surviving mero-
zoites may enter other tissue cells, so that exoerythrocytic merogonic cycles
continue throughout the infection. In the Plasmodiidae, merozoites from
the first or a later exoerythrocytic merogony may enter red blood cells
and develop into schizonts, thus starting a series of cycles in the blood.
Sooner or later, merozoites develop directly into gametocytes which, at
maturity, are infective for the vector. In the Haemoproteidae, merogony
does not occur in erythrocytes, although gametocytes (Fig. 6. 22, E-J)
do invade blood cells after the infection has been in progress for some
time.

For many years the erythrocytic stages of Plasmodiidae were the only
stages known in vertebrates. As a result of investigations by Cotilston,
Garnham, Hawking, Huff, James, Porter, Raffaele, Shortt, and others
(reviews: 32, 54, 55, 108), the occurrence of exoerythrocytic merogony in
reptilian, avian, and mammalian parasites is now clearly established. In
the typical infection, a pre-erythrocytic phase results from the devel-
opment of sporozoites introduced by the vector and may include one
(primates) or several merogonic cycles (birds). Products of the first
pre-erythrocytic merogony have been referred to as cryptozoites; the
merozoites formed in later exoerythrocytic cycles, as metacryptozoites
(56). Exoerythrocytic stages in later stages of an infection are known also
as phanerozoites {bl). Merozoites from pre-erythrocytic merogony con-

304 Sporozoa

tinue the exoerythrocytic cycle after invasion of blood cells occurs. Exo-
erythrocytic schizonts, in such species as Plasmodium gnlUnaceum (56),
may be of two kinds, macroschizonts and microschizonts. The latter pro-
duce many micromerozoites (100-1000 or so) which enter erythrocytes.
The macroschizonts produce a smaller number (64 or less, in P. relictum)
of macromerozoites which enter cells other than erythrocytes. In experi-

Fig. 6. 21. Malarial parasites in birds: A, B. Exoerythrocytic stages of
Plasmodium relictinn and P. gallhiaceum in phagocytes (after Coulston and
Huff). C-G. Erythrocytic stages of Plasmodium elongatum, Feulgen prep-
arations (after Chen): trophozoite (C), schizont with 14 nuclei and a small
pigment granule (D), and merozoites (E), x4050; microgametocvte with
elongated nucleus and pigment (F), macrogametocyte (G), x3375.

mental avian infections, exoerythrocytic stages often appear after inocu-
lation of erythrocytic forms.

Exoerythrocytic stages of Plasmodiida develop mainly in lymphoid-
macrophage cells (cells of the "reticulo-endothelial system"), although
their localization varies from species to species. In Haemoproteus, the
parasites occur mostly in endothelial cells of visceral capillaries, especially
in the kidney, liver, lungs, and sjjleen. Plasrnodiutn elongatum. has been

Sporozoa 305

found in a variety of blood-forming cells, while other avian parasites
apparently prefer lymphoid-macrophage cells (55).

The erythrocytic cycle in Plasmodiidae is initiated by merozoites from
an exoerythrocytic merogony. Growth into a schizont is followed by
merogony (Fig. 6. 21, C-E), and the surviving merozoites enter other
erythrocytes to repeat the process. The periodicity of erythrocytic merog-
ony varies with the species, or even strains within a species, and the
cycle covers a period of one to several days in different malarial parasites.

Fig. 6. 22. AH. Hacmoproteus cnlumhae: oocysts on portion of s^ut in
Lyncliia luaura (A), xl05; and sporozoite (B), xl740 (after Adie); tropho-
zoite in leucocyte (C); merohlasts, produced by division of a trophozoite,
have undergone nuclear division in preparation for merogony (D); young
and approximately niatme macrogametocytes (E, F); young and mature
microgametocytes (G, H); C-H, xl320 (after Aragao). I, J. Macro- and
microgametocytes of Leucocytozoon coccyzus, x2136 (after Coatney and
West)'.

Sooner or later, gametocytes (Fig. 6. 21, F, G) are developed, and the
blood of the host is then infective for the vector.

The Order Plasmodiida may be divided into two families, the Haemo-
proteidae and the Plasmodiidae, differentiated by the absence of erythro-
cytic merogony in the former. Only the gametocytes of Haemoproteidae
are to be expected in erythrocytes.

Family 1. Haemoproteidae. These are blood parasites of birds and
reptiles. Merogony is exoerythrocytic, primarily in endothelial cells of
visceral caj^illaries {Haemoprotens), or in lymphoid-macrophage cells of
the viscera (e.g., spleen, liver) as in Leucocytozoon simondi (53). Schizonts

306 Sporozoa

of Haemoproteus often divide into meroblasts, each of which grows be-
fore producing merozoites. However, the formation of meroblasts may
be skipped. Gametocytes of Haemoproteus develop in erythrocytes and
deposit cytoplasmic pigment comparable to that of malarial parasites.
Young gametocytes of L. simondi appear in lymphocytes, monocytes,
myelocytes, and late polychromatophil erythroblasts (53); only those in
the red cells deposit pigment in their cytoplasm. The Haemoproteidae
of birds undergo syngamy and sporogony in blood-sucking flies (Lynchia,
Simulium, and related genera) which ingest gametocytes from the blood
(1, 48, 102, 103).

The family includes Haemoproteus Kruse (52, 102; Fig. 6. 22, A-H) and Leucocyto-
zoon Danilewsky (53, 138; Fig. 6. 22, I, J). Checklists of species and host indices are
available for Leiicocytozoon (19), Haemoproteus (18), and for species of both genera
found in North American birds (47).

Family 2. Plasm odiidae. This family includes one genus, Plasmodium
Marchiafava and Celli (Figs. 6. 20, 21), which includes malarial parasites
of reptiles (130, 131), birds (49), and mammals. Check-lists and host-
indices are available for the genus (20) and for species parasitic in North
American birds (47). Species causing malaria in man are discussed in
Chapter XIII.

Order 2. Babesiida. The life-cycles are not yet completely known. Non-
pigmented stages, in the red corpuscles of cattle and certain other mam-
mals, are ingested by ticks and establish infections in these invertebrate
hosts. With the demonstration by Smith and Kilbourne (123), that ticks
transmit Babesia bigemina, arthropods were identified for the first time
as vectors of protozoan parasites.

One of the most completely known life-cycles is that of Theileria pai~ua
(21, 22; Fig. 6. 23), which causes African Coast fever of cattle. Erythro-
cytic stages are ingested by a tick and liberated in the gut. Small parasites
of two sizes are soon observed, usually in clumps, and syngamy is be-
lieved to occur. Larger parasites (Fig. 6. 23, A), believed to be zygotes,
now replace the ones which first appeared in the tick. After preliminary
growth, an elongated ookinete is developed within the zygote. Such
ookinetes appear later in the body cavity near the salivary glands and
some of them enter gland cells (Fig. 6. 23, B-D). The ookinete now rounds
up and produces a number of sporoblasts, each of which divides into
sporozoites (Fig. 6. 23, E, F). The sporozoites escape into the salivary
ducts and are inoculated into the mammalian host when the tick begins
to feed. Sporozoites pass by way of lymph vessels to a lymph gland, and
the survivors invade lymphocytes where they develop into multinucleate
schizonts (Fig. 6. 23, G), or "agamonts" (21). As the infection progresses,
so-called gamonts appear. These stages stain less intensely and have
smaller nuclei than those of the agamonts. Multinucleate gamonts (Fig.

Sporozoa 307

6. 23, H) divide into uninucleate forms, some of which enter the blood
stream and invade red corpuscles. The erythrocytic forms (Fig. 6. 23,
I-S) are infective for ticks.

The life-cycle of Babesia bigemina (26, 27), the causative organism of
Texas cattle fever, is similar to that of Theileria parva. Erythrocytic

Fig. 6. 23. A-F. Theileria parva, stages in ticks (after Cowdry and Ham):
A. Group of three "zygotes," x4550. B. Ookinete in gland cell. C, D. Growth
of ookinete. E. Multinucleate sporoblast. F. Sporozoites surrounding a resid-
ual inass; B-F, x2925. G-S. Theileria pama, stages in cattle (after Cowdry
and Banks): G. Multinucleate "agamonts" in a lymphocyte, x2925. H. A
multinucleate "gamont" in a lymphocyte, x2600. I-P. Stages suggesting re-
production of T. parva in red corpuscles; x2925. Q-S. Corpuscles containing
two, three and ten parasites; x2925.

Stages are ingested by the tick and liberated from the corpuscles (Fig. 6.
24, K-M). Elongated "isogametes" later appear and undergo apparent
isogamy (Fig. 6. 24, N-R). The zygotes become ookinetes which migrate
through the gut wall. Those which invade ova continue their develop-
ment in the resulting young ticks, j^roducing sporoblasts (Fig. 6. 24,
S-W) which become sporokinetes. Some of these sporokinetes invade cells

308 Sporozoa

of the developing salivary glands and give rise to sporozoites (Fig. 6. 24,
X, Y). A variety of forms (Fig. 6. 24, A-J), similar to those for Theileria,
finally appear in the red corpuscles of cattle after sporozoites are inocu-
lated by a tick. These erythrocytic stages are infective for ticks.

The life-cycles of the two genera — Babesia Starcovici and Theileria
Bettencourt, Franca and Borges — seem to differ primarily in the verte-
brate phase. Multiplication in lymphocytes, as established for Theileria

Fig. 6. 24. Babesia bigemina (after Dennis). A-J. Stages in red corpuscles
of cattle; reproduction by fission is suggested by stages F-J; x4795. KM.
Parasites from the gut of ticks. N-P. Development of an "isogamete." Q,
R. Supposed stages in isogamy. S. Ookinete from gut of a tick. T. Ookinete
in ovum of tick; yolk globules outlined; portion of a section. U. Oocyst. V.
Small oocyst with five nuclei; K-V, x2818. W. Developing sporoblasts, x3270.
X. Sporokinetes, smear preparation from tick; x3270. Y. Group of sporozoites
from embryonic salivary gland of tick; xl940.

Sporozoa 309

parva (21), is not known to occur in Babesia. The supposed absence, in
Theileria, of erythrocytic reproduction — a process generally accepted for
Babesia — may not be a completely valid distinction. Erythrocytic stages
of Theileria parva (21) may undergo a certain amount of growth and
possibly reproduction as well (Fig. 6. 23, I-P). Such apparent differences
in life-cycles form the basis for the usual recognition of two families, the
Babesiidae and Theileriidae, with the general characteristics of their
type genera.

Genera of uncertain status. Two additional genera, Dactylosorna Labbe
and Toxoplasma Nicolle and Manceaux, may or may not belong in the

Fig. 6. 25. A-D. Dactylosoina ranaruin ('Kvuse) Labbe. x2835 (after
Mathis and Leger): multinucleate schizont (A), merogony (B), microgame-
tocyte (C), niacrogametocyte (D). E-K. Dactylosorna jahni, xl985 (after
Nigrelli): parasite entering erythrocyte of a newt (E); stages in reproduction
(F-I); microgametocyte (J); niacrogametocyte (K). L-O. Toxoplasma canis,
x2875 (after Ray): extraccllidar forms seen in smears from liver and spleen
(L, M); binucleate form (X); stage in nuclear division (O). P-U. Toxoplasma,
a strain of human origin maintained in mice (after Cross): P. Two parasites
in a polymorphonuclear cell, x2500. Q. Group of parasites in a large mono-
nuclear leucocyte, x2500. R. Extracellular stage showing nucleus, thick para-
style, and cytoplasmic granules. S, T. Nuclear division, Feulgen preparations.
U. Binucleate stage with two parastyles; R-U, x4500.

3 1 0 Sporozoa

Babesiida; the question cannot be decided without further knowledge
of the life-cycles.

Dactylosoma Labbe. This genus (95, 100) includes little known non-
pigmented forms occurring in erythrocytes of frogs, urodeles, and lizards.
Merogony in red cells and the development of supposed gametocytes
(Fig. 6. 25, A-K) have been described but the rest of the life-cycle is
unknown.

Toxoplasma Nicolle and Manceaux. Organisms assigned to this genus
have been found in various tissue cells of vertebrates — several types of
leucocytes, lymphoid-macrophage (reticulo-endothelial) cells, cells of the
central nervous system, and in erythrocytes of experimentally inoculated
birds (144). In addition, extracellular stages have been observed by
various workers. Several strains have been maintained in chick embryos
by serial transfers (83). Life-cycles have not yet been worked out and the
taxonomic status of the genus is uncertain. However, the protozoan
nature of Toxoplasma has been affirmed in some of the more recent in-
vestigations (23, 89).

Individual parasites range from almost spherical to elongated forms
(Fig. 6. 25, L-U). The larger stages are usually not more than 5-6pt long,
while small forms may measure only 2-3[jl in diameter. In addition to the
nucleus and sometimes cytoplasmic globules. Cross (23) occasionally noted
a longitudinal axostyle-like band ("cytostyle") just beneath the pellicle
(Fig. 6. 25, R, U). Supposed flagella have been described by several
workers, although more evidence is needed for any definite conclusion
(23). Within invaded tissue cells the parasites occur singly or in groups
(Fig. 6. 25, Q) in one or more vacuoles, or "pseudocysts." These pseudo-
cysts have been interpreted in some instances as the results of schizogony,
but most workers are agreed that reproduction by longitudinal binary
fission is the rule.

The type species, Toxoplasjna gondii Nicolle and Manceaux (16), was
described from a North African rodent. Infections with Toxoplasma
have been reported subsequently from a variety of birds (143) and mam-
mals, including man, but there is much uncertainty in regard to the
specific status of described types. The problem of differentiating species
is complicated by the apparent lack of host-specificity (89). For instance,
strains isolated from cases of human toxoplasmosis have been found in-
fective for monkeys, rabbits, mice, guinea-pigs, hamsters, cotton rats,
white rats, and chickens.

Infection with Toxoplasma seems to be responsible for several patho-
logical conditions in man: (1) a type of congenital encephalomyelitis
which appears in infants shortly after birth or even in utero (141, 142);
(2) a type of acute encephalomyelitis in children (119); (3) a syndrome
resembling spotted fever and associated with inflammation of the lungs
(107); and (4) mild cases, in which the mothers of congenitally infected

Sporozoa 311

infants may show no history of previous toxoplasmosis. Human infections
have been diagnosed microscopically and by inoculation of laboratory
animals. A complement-fixation test also shows some promise for diagnosis
of active toxoplasmosis (136).

CLASS 2. CNIDOSPORIDEA

A general characteristic of this group is the production of spores
which differ distinctly from those of Telosporidea. Each spore (Fig. 6.
26, A) typically contains one or more polar capsules and also one or more
sporoplasms analogous to the sporozoites of Telosporidea. Each polar
capsule contains a coiled polar filament (Fig. 6. 26, C) which is extruded
under certain conditions. This filament has been considered an organelle
of attachment which prevents passage of the spore through the gut of
the host before the sporoplasm can emerge. Another view is that the
polar filament is a tube through which the sporoplasm travels from the
spore directly into a tissue cell (104). The membrane of the spore may
be apparently continuous, or it may consist of two or three sections, or
valves (Fig. 6. 26, B, E). In many Cnidosporidea each spore appears to
be multicellular in origin, in contrast to the sporocysts and oocysts of
Telosporidea and the cysts of other Protozoa. Another distinction between
the two classes is that the zygote of the Telosporidea undergoes sporogony,
while that of the Cnidosporidea gives rise to one or more trophozoites.
The young trophozoite is a small amoeboid organism which typically
develops into a plasmodium (Fig. 6. 26, D, F). However, the trophozoites
of Microsporida, which are almost exclusively intracellular parasites, are
very small and the nuclei are few in number. The trophozoites of other
Cnidosporidea typically invade body cavities of the host or else grow as
tissue parasites (intercellular rather than intracellular). Reproduction of
the trophozoite — by a so-called schizogony in certain Microsporida, or
by fission, budding, or plasmotomy in other cases — has been reported.
However, this phase of the cycle seems to have been eliminated com-
pletely in many Cnidosporidea.

The Cnidosporidea have been divided into four orders: Myxosporida,
Microsporida, Actinomyxida, and Helicosporida. The spores of Myxo-
sporida are bivalve, usually with two, but sometimes one or four polar
capsules. The spore of the Actinomyxida contains three valves, three
polar capsules, and one to many sporoplasms. The spores of Microsporida
are small, usually with only one polar capsule, and the presence of
separate valves is doubtful in most species. The spore of Helicosporida
contains a single coiled filament but no polar capsules.

Order 1. Myxosporida. The Myxosporida are mostly parasites of fishes,
less commonly of Amphibia and Reptilia. The supposedly more primitive
types are coelozoic, invading the gall-bladder, kidney tubules, and urinary
bladder. Others have been found in most tissues and organs of fishes.

3 1 2 Sporozoa

although a given species may be limited to a particular tissue. Some of
these infections are frequently fatal to the hosts.

The life-cycles show certain general features, although details vary to
some extent. The zygote is formed by the fusion of two haploid sporoplas-
mic nuclei, commonly during the dispersal of spores to new hosts (99).

Fig. 6. 26. A. Spore of Myxobolus osburni Herrick, showing two polar
filaments (coiled in capsules) and sporoplasm containing an iodinophilous
vacuole; x2250 approx. (after Otto and Jahn). B. Spore of M. osburni,
sutural view shov.'ing the two valves and the sutural ridges (after Otto
and Jahn). C. Extruded polar filaments, spore of Leptotheca ohhnacheri
(Gurley) Labbe; xll75 (after Kudo). D. Amoeboid trophozoite of L.
ohlmacheri containing two spores; xl880 (after Kudo). E. Spore of Sphaer-
actinomyxon gigas Granata, polar view showing the three valves charac-
teristic of spore membranes in the Actinomyxida; the three polar capsules
are indicated in solid black; x850 (after G.). F. Trophozoite of Myxobilatus
asymmetricus Davis, attached to epithelium of urinary bladder (fish);
several developing sporoblasts are present; the free end of the trophozoite
is covered with delicate bristles of uncertain significance; xSOO (after D.).

After ingestion, the zygote escapes from the spore membrane and migrates
to the tissue or body cavity in which development will occur. The growth
phase includes both nuclear division and cytoplasmic growth. Depending
upon the species, reproduction by plasmotomy (13), or by endogenous or
exogenous budding may occur, particularly in monosporous and dispo-
rous species (99). If buds are produced, they may repeat the reproductive

Sporozoa 3 1 3

cycle or may develop directly into sporoblasts. The trophozoite of the
large polysporous Myxosporida usually becomes a plasmodium (Fig. 6.
26, F) without intervening reproduction. The size of the mature tropho-
zoite varies considerably in different species but lengths of 100-500pi. are
not uncommon.

frS

Fig. 6. 27. A. Young trophozoite with a single nucleus, Leptotheca ohlma-
cheri; xl880 (after Kudo). B. Binucleate trophozoite, Ceratomyxa shasta
Noble; the more heavily stained nucleus will gi\e rise to sporoblastic nuclei,
the other will become the somatic residual nucleus; x2160 (after N.). C-F.
Leptotheca nhlmacheri (after Kudo): C. Trophozoite with eight sporoblastic
nuclei and a lightly stained somatic residual nucleus. D. Trophozoite with
two sporoblasts; in each, two cystogenous "cells" lie at the periphery in a
dense zone of cytoplasm; somatic residual nucleus lies outside the sporoblasts.
E. Later stage in the development of spores; the development of polar
capsules from each pair of capsulogenous "cells" is under way; nuclei of the
cystogenous "cells" lie in the denser peripheral cytoplasm; the nuclei of the
sporoplasms are surrounded by vacuolated cytoplasm; C-E, xl880. F. Mature
spore with two polar filaments and two sporoplasms; xl200.

The growth phase often ends with the appearance of sporoblasts. How-
ever, growth continues during the production of spores in polysporous
genera such as Myxidium (98). Although there are reports to the contrary,
the most conclusive recent evidence (97, 98, 99) indicates that the sporo-
blast or pansporoblast is not the result of syngamy or plasmogamy. In
typical sporulation, certain "cells" in the trophozoite become differen-

314 Sporozoa

tiated from the rest of the protoplasm. Each such "cell" is the initial
stage of a sporoblast, or of a disporous pansporoblast if it is to produce
two spores. In at least some species, the differentiation of a sporoblastic
and a somatic nucleus is already apparent in the young binucleate tropho-
zoite (Fig. 6. 27, B). Division of the sporoblastic nucleus during the
growth phase results in a sporoblast with 6-8 nuclei, or a pansporoblast
with a larger number of nuclei (Fig. 6. 27, C-E), the number varying
with the number of polar capsules to be produced. If a pansporoblast is
developed, it later divides into two sporoblasts.

Six nuclei appear in each developing spore of Leptotheca ohlmacheri
(70; Fig. 6. 27, D, E), Two acquire differentiated zones of cytoplasm and
become the cystogenous cells which gradually enclose the rest of the
young spore and produce the valves of the spore membrane. Two other
nuclei and their surrounding masses of cytoplasm become the capsulog-
enous cells which produce the polar capsules. The remaining two nuclei
become the haploid nuclei of the sporoplasm. As a rule, however, young
spores of species with two polar capsules contain eight nuclei (99). Two
of these, the so-called residual nuclei, degenerate during later develop-
ment. The development of the other six follows the course outlined for
Leptotheca.

The spore membrane is composed of two valves united in a suture
which may be either straight (Fig. 6. 26, B) or irregular (Fig. 6. 28, O),
and is often marked by a sutural ridge formed by the thickened edges of
the valves. The valves may be smooth or may be decorated with striations,
ridges, or papillae. The spores of some species are ovoid, those of others
may be spindle-shaped or somewhat asymmetrical, and the valves are
sometimes drawn out into horns or spines (Fig. 6. 28). Each polar capsule
lies near a pore which opens through the spore membrane in or near
the sutural plane; or sometimes two adjacent capsules share a common
pore. The mature spore usually contains a single sporoplasm, although
two may be present as in Leptlwtheca oJilmacheri (70). The single sporo-
plasm usually contains two nuclei, and in either case the sporoplasmic
nuclei are haploid (74, 97, 99). A fairly large inclusion ("iodinophilous
vacuole"), which is stained reddish-brown with iodine, is characteristic
of the sporoplasm in certain genera (Family Myxobolidae), but not in
the majority. The number of spores produced by each trophozoite varies
in different cases, and a given species may be typically monosporous, dis-
porous, or polysporous. However, this is not a rigidly fixed characteristic
and all three types of sporulation are sometimes observed within a single
species.

Although the occurrence of meiosis is well established, the stage at
which this process apparently occurs varies in the descriptions of different
species. In some cases (97, 98), meiosis occurs in one of the last nuclear
divisions in development of the sporoblast, the products being the hap-

Sporozoa 3 1 5

Fig. 6. 28. Spores of various Myxosporida: A. Unicapsula muscularis
Davis, showing suture and the single polar capsule; x3125 (after D.). B.
Splmeromyxa balbiani, siitmal view, one polar capsule at each pole; xl575
(after Kudo). C, D. Sphaerospoia polymorpJm Davis, optical section and
sutural view; x2200 (after Kudo). E. Myxidium melum (11-12/i long), valve
view (after Otto and Jahn). F. Wardia ovinocua, sutural view; xI575 (after
Kudo). G. Myxoproteus cornutus, sutural view; xl200 (after Kudo). H.
Mitraspora nprini, sutural view; xl350 (after Kudo). I. Coccomyxa morovi,
one polar capside; xI065 (after Leger and Hesse). J, K. Chloromyxum triju-
gum Kudo, sutural and valve views; four polar capsules (after Otto and
Jahn). L. Myxosoma okobojiensis (16.3xl3.2ju), two large polar capsules (after
Rice and Jahn). M. Ceratomyxa shasta, sutural view; x3040 (after Noble). N.
Henjieguya magna, overall length 87^^ (after Rice and Jahn). O. Sinuolinea
capsularis, sutural view; xl575 (after Kudo). P. Zschokkella hildae, sutural
view; x630 (after Auerbach). Q, R. Thelolianellus notatus, sutural and valve
views; xl605 (after Kudo). S. Agarella gracilis, four polar capsules; xl495
(after Dunkerly).

3 1 6 Sporozoa

loid nuclei of the sporoplasm. According to certain other reports, nieiovsis
occurs at an earlier stage so that all nuclei of the sporoblast are haploid,
and a haploid cycle with the zygote as the only diploid stage also has been
reported (99).

According to Kudo, who has published a check-list of species (72), the
Myxosporida may be divided into three suborders on the basis of form
and structure of the spores. In the Eurysporina the sutural plane is ap-
proximately perpendicular to the major axis of the spore, there are two
polar capsides, one on each side of the sutural plane, and there is no
iodinophilous vacuole. The Sphaerosporina have spherical spores with
one, two, or four polar capsules and no iodinophilous vacuole. In the
Platysporina, the sutural plane coincides with, or approximates, the major
axis of the spore, there are one, two, or four polar capsules, and an
iodinophilous vacuole may or may not be present. More recently, Tripathi
(132) has suggested division of the Myxosporida into a "Suborder Uni-
polaria," with the polar capsules at or near one end of the spore, and a
"Suborder Bipolaria" with one polar capsule at each end of the spore. The
"Bipolaria" would include the Myxidiidae.

Suborder 1. Eurysporina

Family 1. Ceratomyxidae. Most known species are coelozoic parasites
of marine fishes and are assigned to three genera: Ceratomyxa Thelohan
(Fig. 6. 28, M), Leptotheca Thelohan (70; Fig. 6. 27, A, C-F), and Myxo-
proteus Doflein (Fig. 6. 28, G).

Family 2. Wardiidae. Histozoic or coelomic parasites of fresh-water
fishes are included in this group, which contains two genera, Mitraspora
Fujita (Fig. 6. 28, H) and Wardia Kudo (Fig. 6. 28, F).

Suborder 2. Sphaerosporijia

Fatnily 1. Chloromyxidae. In the only known genus, Chloromyxum
Mingazzini (Fig. 6. 28, J, K), the spore contains four polar capsules.

Family 2. Sphaerosporidae. Spores with two polar capsules are found in
Sijiuolinea Davis (Fig. 6. 28, O) and Sphaerospora Thelohan (Fig. 6. 28,
C, D).

Family 3. Unicapsulidae. A single polar capsule is characteristic of the
only genus, Unicapsula Davis (Fig. 6. 28, A).

Suborder 3. Platysporina

Family 1. Coccomyxidae. In the only genus, Coccomyxa Leger and
Hesse (Fig. 6. 28, I), the spore contains one polar capsule and no iodino-
philous vacuole.

Family 2. Myxidiidae. The spores contain one polar capsule at each end.

Sporozoa 317

Three genera are included: Myxidium Biitschli (Fig. 6. 28, E), Sphaeroniyxa
Thelohan (Fig. 6. 28, B), and Zschokkella Auerbach (Fig. 6. 28, P).

Family 3. Myxobolidae. The spores contain two polar capsules at one
end and an iodinophilous vacuole.

The family includes the following genera: Henneguya Thelohan (Fig. 6. 28, N),
Myxobilatiis Davis (25; Fig. 6. 26, F), Myxobolus Thelohan (Fig. 6. 26, A, B), Thelo-
hanellus Kudo (Fig. 6. 28, Q, R), and Unicattda Davis (25).

Family 4. Myxosomatidae. The spores contain two or four polar cap-
sules and no iodinophilous vacuole. There are only two genera: Agarella
Dunkerly (Fig. 6. 28, S) and Myxosoma Thelohan (Fig. 6. 28, L).

Order 2. Actinomyxida. This group includes organisms whose discov-
erer, Stole, believed that they should be considered Mesozoa rather than
Protozoa, in view of their complexity. The pansporoblast, or pansporo-
cyst, typically develops eight spores, each with a membrane composed of
three valves (Fig. 6. 26, E). In some species, each valve is drawn out into
a horn, or spine, which may or may not be bifurcated. There are also
three polar capsules, but the number of sporoplasms ranges from one to
a hundred or more in different species. Species are known from sipuncu-
lids and tubificid annelids.

The life-cycle of Triactinomyxon legeri (84) is probably representative.
The mature spore (Fig. 6. 30, D) contains a sporoplasmic mass in which
lie 24 uninucleate sporoplasms and three or more residual somatic nuclei.
The sporoplasms eventually fuse in pairs to produce 12 binucleate stages.
Subsequently, the mass breaks up into several fragments which leave the
spore membrane separately (Fig. 6. 29, A, B) and liberate the binucleate
stages as small amoebae. Eachamoebula (Fig. 6. 29, C) grows for a time
and then undergoes nuclear division. Two of the nuclei, with associated
cytoplasm, become peripheral in position and, as the cystogenous cells,
produce a cyst around the remaining protoplasm (Fig. 6. 29, D). This
stage is called a jDansporoblast, or after the membrane is completed, a
pansporocyst (84). The central binucleate mass next divides into two cells,
one of which reproduces more rapidly than the other, so that a number of
small cells and a few larger ones are produced (Fig. 6. 29, E, F). Ani-
sogamy involves fusion of a large cell with a small one.

In development of sporoblasts within the pansporocyst, the nucleus of
each zygote gives rise to seven nuclei (Fig. 6. 29, G). Three of these, with
associated cytoplasm, migrate to one end of the developing spore as cap-
sulogenous cells which later produce polar capsules (Fig. 6. 29, H). Three
other cells, which are to produce the valves of the spore membrane, mi-
grate to the opposite pole of the sporoblast (Fig. 6. 29, I, J). The seventh
nucleus initiates a series of mitoses resulting in 27 nuclei. Three of these
are residual somatic nuclei; the other 24 become sporoplasmic nuclei.

318 Sporozoa

Fig. 6. 29. Triactinomyxon legeri Mackinnon and Adam (after M. & A.):
A. Sporoplasmic fragment approaching point of exit from spore membrane;
nuclei of valve cells shown below the sporoplasmic fragment. B. Sporoplasmic
fragment after leaving the spore. C. Growing biniicleate amoebula after re-
lease from a sporoplasmic fragment. D. Young pansporocyst, nuclei of cys-
togenous cells at the periphery. E. Pansporocyst containing two binucleate
stages. F. Pansporocyst containing two large cells and a number of smaller
ones; additional large and small cells are produced by division of each type.
G. Two sporoblasts derived from two zygotes within a pansporocyst. H. A
sporoblast in which the three capsulogenous cells have migrated to the upper
pole. I. Developing spore; three cystogenous cells (valve-cells) have migrated
to the lower pole; the seventh sporoblastic nucleus has produced a number
of daughter nuclei in the sporoplasmic mass. J. Young spore with developing
polar capsules and spore membrane; the seventh sporoblastic nucleus has not
yet divided in this case. A, B, xHOO; C-J, xl800.

Sporozoa 319

Taxonomy

The taxonomy of the order has been discussed by Granata (39). Two
families have been recognized. The spores of the Tetractinomyxidae have
a continuous endocyst and an outer membrane composed of three valves.
The endocyst is lacking in the Triactinomyxidae.

Fig. 6. 30. Spores of Actinomyxida: A. Sphaeractinomyxon gigas Granata,
lateral view, x850 (after G.); compare with Fig. 6. 26, E. B, C. Neoactino-
myxum globosum Granata, from stained (B) and living (C) material; x2470
(after G.). D. Triactinoinyxon legeri Mackinnon and Adam, two horns ex-
panded and the third partly expanded; sporoplasmic mass lies near the
upper pole; x260 approx. (after M. & A.).

The following genera have been included in the two families:

Family 1. Tetractinomyxidae: Tetractinomyxon Ikeda (58).

Family 2. Triactinomyxidae: Guyenotia Naville (94), Hexactinomyxon Stole (39),
Neoactinomyxon Granata (39; Fig. 6. 30, B, C), Sphaeractinomyxon Caullery and
Mesnil (39; Fig. 6. 30, A), Synactinomyxon Stole (39), and Triactinomyxon Stole (39,
84).

Order 3. Microsporida. These are small and generally intracellular
parasites, mostly of arthropods and fishes, although a few have been
reported from annelids and other hosts. As parasites of insects, Micro-
sporida are commonly found in the epithelium of the gut and in the
fat body or other tissues. Species invading fishes are commonly found in
the skin and muscles.

The characteristic spores (Fig. 6. 31), the smallest of which may resem-
ble yeasts or large bacteria, range from about 2.0 to more than 20[jl in
length in different species. It is doubtful that the spore membrane is
composed of separate valves. A single polar filament is the rule, although
two are present in Telomyxa. The polar filament, when extended, is

320 Sporozoa

strikingly long in proportion to size of the spore and may measure 25-
500[j.. The sporoplasm is binucleate in some Microsporida and uninucleate
in others. As seen in invaded tissues, the spores often lie in groups within
the sporont membrane, the number of spores being more or less charac-
teristic of certain species.

After a spore is ingested by a new host the sporoplasm emerges as an
amoeboid trophozoite (Fig. 6. 32, A), which may pass through the gut

Fig. 6. 31. Spores of Microsporida: A, B. Coccomyxa slavinae, mature
spore and one with extruded filament; x3700 (from Kudo, after Leger and
Hesse). C. MrazckJa lumbricidi, binucleate sporoplasm; xl820 (after Jirovec).
D, E. Octosporea bayeri, from living (D), and Feulgen preparation showing
two nuclei (E); x2340 (after Jirovec). F. Spirogliigea octospora, x3900 (from
Kudo, after Leger and Hesse). G. Toxoglugea vibrio, x3900 (from Kudo,
after Leger and Hesse). H. Nosema termitis, x2990 (after Kudo). L Duboscqia
legeri, x3120 (after Kudo). J, K. Plistophora intestinalis , from Giemsa and
Feulgen preparations; x2700 (after Jirovec). L. Glugea acerinae, Giemsa
stain; x2700 (after Jirovec). M. Thelohania cladocera, two nuclei, Feulgen
stain; x2700 (after Jirovec). N, O. Telomyxa glugeiformis, unstained and
stained; x5400 (from Kudo, after Leger and Hesse). P, Q. Racillidiinn argoisi,
hematoxylin preparation (P); Feulgen preparation showing spiral nucleus
(Q); xl950 (after Jirovec), R. Cougourdella magna, x2600 (after Hesse). S.
Stempellia magna, x2070 (after Kudo). T. Xosema elongatum, Feulgen stain;
x3300 (after Jirovec). U. Gurleya richardi. x3240 (from Kudo, after CepMe).

Sporozoa 321

wall into the tissue spaces or blood stream, and thence into some particu-
lar type of tissue cell; or as in a number of species, the trophozoite re-
mains in the epithelium of the gut. In any case the young trophozoite
grows and reproduces by binary fission (Fig. 6. 32, B, C), as in Nosema
termitis, or else by "schizogony" (Fig. 6. 32, F-H), as in Duboscqia legeri

Fig. 6. 32. A-E. Nosema termitis, x2990 (after Kudo): A. Young amoeboid
trophozoite. B. C. Stages in fission of trophozoites. D. Young sporont. E.
Immature spore; compare with Fig. 6. 31, H. F-K. Duboscqia legeri, x2760
(after Kudo): F-H. Stages in schizogony. I. Sporont with four nuclei. J.
Sporoblasts have developed within the sporont membrane. K. Two immature
spores which have developed from sporoblasts within the sporont membrane.

(73). The products of reproduction may repeat the reproductive cycle, or
they may become sporonts which produce spores (Fig. 6. 32, D, I).

A sporont may develop directly into one sporoblast, as in Noseina
termitis (lb), or may undergo nuclear division and produce a number
of sporoblasts within the original sporont membrane (Fig. 6. 32, J, K).
Sporoblasts are either uninucleate or binucleate, depending upon the
species. It has been impossible to determine with certainty the occurrence
of nuclear division during development of the sporoblast. The best

322 Sporozoa

modern evidence indicates that, as in Nosema (75), Duboscqia (73), Bacil-
lidium, and Mrazekia (63), the single nucleus or the two nuclei of the
sporoblasts become the corresponding nuclei of uninucleate and binu-
cleate spores. Accordingly, it appears that somatic differentiation and
division of labor, as seen in the Myxosporida for example, are lacking in
the Microsporida.

Taxonomy

Classification of the Microsporida is based primarily upon the form
and structure of the spores and to a lesser degree upon differences in the
details of sporogenesis. Four families have been recognized (71).

Family 1. Coccosporidae. The spores are spherical, or approximately
so, and contain one polar filament. The family contains the genus Cocco-
spora Kudo (71; Fig. 6. 31, A, B).

Family 2. Mrazekiidae. The spores have a single polar filament and are
cylindrical, or tubular and curved. The ratio of length to thickness is
greater than 5:1.

The family includes the following genera: Bacillidium Janda (64; Fig. 6. 31, P, Q),
Cougourdella Hesse (71; Fig. 6. 31, R), Mrazekia Leger and Hesse (64; Fig. 6. 31, C),
Octosporea Flu (64; Fig. 6. 31, D, E), Spiroglugea Leger and Hesse (71; Fig. 6. 31, F),
and Toxoglugea Leger and Hesse (71; Fig. 6. 31, G).

Family 3. Nosematidae. The spores are usually ovoid or pyriform; if
more elongated, the ratio of length to thickness is less than 4:1. There
is only one polar filament.

The following genera have been assigned to the family. Duboscqia Perez (73; Fig. 6.
31. I), Glugea Thelohan (137; Fig. 6. 31, L), Gurleya Doflein (71; Fig. 6. 31, U),
Nosema Nageli (75, 76; Fig. 6. 31, H, T), Plistophora Gurley (Fig. 6. 31, J, K), Pyrotheca
Hesse (71), Stempellia Leger and Hesse (71; Fig. 6. 31, S), Thelohania Henneguy (Fig.
6. 31, M) and Trichoduboscqia Leger (71).

Family 4. Telomyxidae. The single genus, Telomyxa Leger and Hesse
(71; Fig. 6. 31, N, O), is characterized by spores with two polar filaments.

Order 4. Helicosporida. This order was erected by Kudo for the genus
Helicosporidium Keilin (68). The single known species, H. parasiticum,
was found in larvae of a ceratopogonid dipteran, Dasyhelea obsciira, from
sap in wounds of elm and horse-chestnut trees. All stages of development
occur in the body cavity of the host. Occasionally, however, parasites were
found in fat bodies and in nerve ganglia, the invasions resulting in
destruction of the fat bodies and reduction of the ganglia to neurilemma.

Young trophozoites grow and divide, frequently producing groups of
eight (Fig. 6. 33, A-D). Sporulation is preceded by a period of rapid
multiplication, and each spore apparently is developed from a group of

Sporozoa 323

Fig. 6. 33. Helicosporidium parasitkum; A-H, x3720; I, x2325; J, xl400
(after Keilin): A. Young growth stage. B. Binucleate growth stage. C. Four-
cell stage. D. Eight-cell stage. EG. Stages in development of the spore; a
young spore with a central mass surrounded by a protoplasmic rim (E);
lateral and end views of a later stage with three central cells, spiral filament
not yet differentiated (F, G). H. Mature spore showing peripheral spiral
filament and the three central cells. I. Spore membrane ruptured and spiral
filament protruding. J. Unravelled filament after rupture of the spore mem-
brane; these filaments are 60-65,u long and contain one nucleus.

four cells. One of the four is believed to spread over the others to form
a capsule (Fig. 6. 33, E-G). A spiral filament develops later but its exact
origin is unknown. The mature spore (Fig. 6. 33, H) contains three cells
("sporozoites"), supposedly infective stages, and the spiral filament, be-
lieved to play the role of an elater when the membrane is ruptured. When
spores are placed in water the spore membrane is ruptured, the filament
is extruded (Fig. 6. 33, I, J), and the three central cells are expelled.

CLASS 3. ACNIDOSPORIDEA

The organisms usually assigned to this class do not produce spores
containing polar filaments and are thus unlike the Cnidosporidea. Their
life-cycles fail to suggest any close relationship to the Telosporidea. Fur-
thermore, the interrelationships of the groups included in the Acnido-
sporidea are somewhat obscure, and as now constituted, the class may be
largely a taxonomic convenience. As a result, the conventional division
of the Acnidosporidea into the Subclasses Sarcosporidia and Haplospo-
ridia indicates no firm belief that the two groups are as closely related
as this arrangement might imply.

324 Sporozoa

Subclass 1. Sarcoporidia

The characteristic "cysts" of these organisms^ have been reported
mainly from striated muscles of reptiles, birds, and mammals, while forms
believed to be infective or developmental stages have been found in
the blood, in the intestinal epithelium and submucosa, and in the feces
of infected animals. Experimental infections have been produced by
feeding infected muscle tissue and also by the intra-muscular injection of
"spores."

Much of the evidence indicates that infective stages are ingested by a
new host and that an intermediate host or mechanical vector is not neces-
sary. There are some reports that infection may occur in utero, but the
evidence is not entirely conclusive. The so-called "spores" of Sarcocystis
have been found in feces of infected sheep (121), and after experimental
feeding of laboratory animals, in the lumen of the intestine. In the intes-
tine of mice fed infected muscle, "spores" penetrate the mucosa and ap-
pear in the blood stream after 5-6 hours (3). Circulatory distribution of
the parasites is followed by invasion of muscle tissue and the eventual
development of sarcocysts (Fig. 6. 34, E-I). The time required for develop-
ment of typical sarcocysts has ranged from four to seven weeks after
feeding infective stages.

Although this outline of the cycle seems to be based upon sound evi-
dence, the details of development are incompletely known. One important
gap is the scarcity of information concerning the "spores" after their
production in the sarcocyst. There is some evidence that sarcocysts occa-
sionally are ruptured and that the released parasites invade fresh tissue,
but the route followed in reaching a new host is yet to be determined.
The fact that "spores" have been recovered from the feces of various
hosts (121) might suggest that after rupture of a sarcocyst, some of the
parasites are transported through the circulatory system to the wall of
the intestine and from there migrate into the lumen. This would involve
retracing the route apparently followed in initial invasion. The obvious
difficulties in tracing such parasites through the tissues account for the
present lack of adequate information.

The parasites may be found in the oesophagus, heart, diaphragm,
tongue muscles, and occasionally in other parts of the body. Invasion of
smooth muscle apparently is rare, if it occurs at all. The earliest stages
reported from muscles are single "sporoblasts" (Fig. 6. 34, E, F) and
groups of several such forms. Development of the mature cyst from these
stages has not been traced completely. The stage usually found in muscle
is the sarcocyst (Fig. 6. 34, I-K), the size and shape of which vary. The
larger sarcocysts are typically spindle-shaped in the diaphragm but are

^Literature on the Sarcosporidia has been reviewed by Badudieri (4) and Scott (120,
12n. •

Sporozoa 325

more nearly ovoid in cardiac muscle. Mature cysts may reach a length of
25-50 mm and the larger ones contain several million "spores." The sarco-
cyst membrane (Fig. 6. 34, J, K) is composed of two or three layers, and
is enclosed in a zone of loose connective tissue and sometimes a layer

Fig. 6. 34. AD. Spores of Sarcosporidia: A. Sarcocystis platydactyli, from
lizards; nucleus near blunt (posterior?) end, a central vacuole, and a granule
at the pointed end; x4750 (after Ball). B. S. lacertae, from lizards; x3120
(after Badudieri). C. 5, tenella, x3120 (after Badudieri). D. S. muris, x3120
(after Badudieri). E-I. Sarcocystis muris in muscle of rat; E-H, x3120; I, x48
approx. (after Badudieri). E, F. Transverse and longitudinal sections, initial
stage of development. G, H. Successive stages in early development of a
sarcocyst. I. Mature sarcocyst. J. Sarcocystis miescheriana, from pig; section
showing outer striated "membrane" and portion of a sarcocyst containing
spores; xl440 (after Badudieri). K. Sarcocystis tenella, portion of a sarcocyst
membrane and adjacent developing spores; xl440 (after Badudieri).

of fibrous tissue. From the membrane, trabeculae extend inward to
form numerous compartments, many of which are filled with "spores"
(Rainey's corpuscles) in the mature sarcocyst. The striations of the so-
called striated membrane at the periphery of the sarcocyst seem to be
continuous with the connective tissue of the adjacent muscle fibres.

326 Sporozoa

Therefore, the striated membrane may be a product of the host rather
than of the parasite.

The term, "spore," is applied rather loosely to the elongated stages
which develop in the sarcocyst, since there is no apparent spore mem-
brane. The visible structures include a nucleus and more or less promi-
nent granules (Fig. 6. 34, A-D). Several workers have noted that these
"spores" can undergo twisting movements, rotation on the long axis,
longitudinal contraction and elongation, or even locomotion (120).

Although the Sarcosporidia are commonly considered Protozoa and
placed in the Acnidosporidea for lack of a more appropriate place, their
protozoan nature has been questioned. One suggestion is that Sarcocystis
from hogs is a fungus, identified as a species of Aspergillus. The re-
ported evidence involves: (1) recovery of such a mold from cultures
inoculated with sarcocysts removed from muscles of hogs; (2) recovery of
sarcocysts from muscles of young pigs inoculated with conidia from such
cultures; (3) recovery of a similar mold from experimentally infected
animals (124). If this report can be confirmed, it should be possible to
transfer to the mycologists the puzzling problems involved in taxonomy
of the Sarcoporidia.

Aside from their interest as unusual parasites of uncertain relation-
ships, the Sarcosporidia are of some importance in veterinary medicine
as parasites of cattle, horses, sheep, and hogs. Sarcoporidiosis of man is
apparently rare, although cases are reported occasionally (33).

Subclass 2. Haplosporidia

These organisms show certain similarities to the Cnidosporidea,
although they produce cysts without polar filaments. Species have been
reported from fishes, tunicates, insects, molluscs, annelids, nemertines,
trematodes, and rotifers. They have been found in the coelom or other
body cavities and also in tissues and individual cells in different cases.

The life-cycles are incompletely known. In some species, a small amoe-
boid stage emerges after a spore is ingested by the host. This uninucleate
or binucleate trophozoite (Fig. 6. 35, A) may invade a tissue cell or some
tissue of the host, or else make its way into a body cavity, where develop-
ment is continued. Growth is usually accompanied by nuclear divisions
(Fig. 6. 35, B), and the plasmodia of certain species contain many nuclei
(Fig. 6. 35, C). In Coelosporidium periplanetae, however, occasional fis-
sion of binucleate trophozoites occurs, in addition to the development of
Plasmodia (60). Division of plasmodia into uninucleate stages (Fig. 6. 35,
D, E) also occurs in Coelosporidium (60) but has not been reported in
Haplosporidium (63).

The development of spores resembles that in certain Microsporidia.
Uninucleate sporoblasts are formed within a mature plasmodium and
each sporoblast apparently develops directly into a spore (Fig. 6. 35, F-H).

Sporozoa 327

r^'

/•»?vV':

...■-•"■ .'»»■

Fig. 6. 35. A. Young trophozoite of Coelosporidium periplanetae (aftei-
Ivani^). B. Nuclear division in binucleate trophozoite of Haplosporidium
cernosvitovi, x2000 (after Jirovec). C. Phismothum of Coelosporidium peri-
planetae (after Ivanit). D, E. Division of plasmodium (C. periplanetae)
into uninucleate trophozoites (after Ivanic). F. Sporoblasts in C. peri-
planetae (after Ivanic). G. Young spores in plasmodiiun of C. periplanetae,
x2290 (after Sprague). H. Spores within a pansporocyst in Haplosporidium
cernosvitovi, xI375 (after Jirovec).

In Haplosporidium cernosvitovi, a histozoic species from oligochaetes,
nearly mature spores generally lie within a distinct membrane (Fig. 6.
35, H).

Shape of the spore (Fig. 6. 36) and structure of the membrane vary in
different species. The membrane apparently is bivalved in Coelospori-
dium periplanetae (Fig. 6. 36, B) and seems to be operculate in certain
other species. In some cases, the membrane is extended into horns or may
show adherent filaments, while the "tail" in Urosporidiiim possibly rep-

328 Sporozoa

Fig. 6. 36. Spores of Haplosporidia: A. Haplosporidium heterocirri,
xl540 (after Caullery and Mesnil). B-D. Coelosporidium periplanetae, from
hematoxylin (B) and Feidgen preparations (C, D); x2910 (after Sprague).
E. Vrosporidium fuliginosum, x2800 (after Caullery and Mesnil). F. Hap-
Josporidium chitonis, x3150 (after Goodrich). G. Haplosporidium caulleryi,
xI540 (after Mercier and Poisson). H. Anurosporidiuni pelseneeri, x2660
(after Caullery and Chappellier). I. Haplosporidium cernosvitovi, x2000
(after Jirovec).

resents an outer membrane enclosing an operculate endocyst. Nuclear
division within the spore, and spores with two nuclei, have been reported
for Coelosporidhirn periplanetae (60, 125). The nuclei of a binucleate
spore apparently fuse as the spore approaches maturity (125). In Ichthyo-
sporidiuni giganteum, the two sporic nuclei are said to undergo meiosis.
Two haploid nuclei then fuse to form a synkaryon while the other two
degenerate (129). In addition, it has been reported that an encysted Plas-
modium of /. hertwigi may divide into amoeboid gametes which undergo
syngamy within the cyst, the zygotes producing the pansporoblasts (129).

The following genera have been assigned to the Haplosporidia: Anurosporidium
Caullery and Chappellier (Fig. 6. 36, H), Bertramia Caullery and Mesnil, Coelospo-
ridium Mesnil and Marchoux (125), Haplosporidium Caullery and Mesnil (63), Ichthy-
osporidium Caullery and Mesnil (129), Nephridiophaga Ivanid (61), and Urosporidium
Caullery and Mesnil (Fig. 6. 36, E).

LITERATURE CITED

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2. Allegre, C. F. 1948. Trans. Amer. Micr. Soc. 67: 211.

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Sporozoa 329

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25. Davis, H. S. 1944. Trans. Amer. Micr. Soc. 63: 31!.

26. Dennis, E. W. 1930. Unix\ Calif. Publ. Zool. 33: 179.

27. 1932. Univ. Calif. Publ. Zool. 36: 263.

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32. Garnham, P. C. G. 1948. Prop. Dis. Bull. 10: 831.

33. Gilmore, H. R., B. H. Kean and F. M. Posey 1942. Amer. J. Trop. Med. 22: 121.

34. Gohre, E. 1943. Arch. f. Protistenk. 96: 295.

35. Goodrich, E. A. and H. L. M. P. Goodrich 1921. Quart. J. Micr. Sci. 65: 157.

36. Goodrich, H. P. 1925. Quart. J. Micr. Sci. 69: 619.

37. 1938. Quart. J. Micr. Sci. 81: 107.

38. 1944. Parasitol. 36: 72.

39. Granata, L. 1925. Arch. f. Protistenk. 50: 139.

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41. Hahn, J. 1928. Arch. f. Protistenk. 62: I.

42. Hatt, P. 1931. Arch. Zool. Exp. Gen. 72: 341.

43. Hauschka, T. S. 1943. /• Morph. 73: 529.

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46. Hentschel, C. C. 1926. Parasitol. 18: 137.

47. Herman, C. M. 1944. Bird Banding 15: 89.

48. Herms, W. B. and C. G. Kadner 1937. /. Parasit. 23: 296.

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51. 1933. Paraiitol. 25: 359.

52. Huff, C. G. 1932. Amer. J. Hyg. 16: 618.

53. 1942. J. Inf. Dis. 71: 18.

54. 1947. Aim. Rev. Microbiol. 1: 43.

55. 1948. /. Parasit. 34: 261.

56. and F. Coulston 1944. /. Inf. Dis. 75: 231.

57. and F. Coulston 1946. /. Inf. Dis. 78: 99.

58. Ikcda, I. 1912. Arch. f. Protistenk. 25: 240.

59. 1914. Arch. f. Protistenk. 33: 205.

60. Ivanic, M. 1926. Arch. f. Protistenk. 56: 63.

61. 1937. La Cellule 45: 291.

62. Jameson, A. P. 1920. Quart. J. Micr. Sci. 64: 207.

63. Jirovcc, O. 1936. Arcluf. Protistenk. 86: 500.

330 Sporozoa

64. 1936. Arch. f. Protistenk. 87: 314.

65. Jones, A. W. 1943. Trans. Amer. Micr. Soc. 62: 254.

66. Kamm, M. W. 1922. ///. Biol. Monogr. 7, No 1.

67. Kcilin, D. 1920. Parasitol. 12: 154.
68. 1921. Parasitol. 13: 97.

69. 1923. Parasitol. 15: 103.

70. Kudo, R. R. 1922. Parasitol. 14: 221.

71. 1924. III. Biol. Monogr. 9, Nos. 2, 3.

72. 1933. Trans. Amer. Micr. Soc. 52: 195.

73. 1942. /. Morph. 71: 307.

74. 1943. /. Morph. 72: 263.

75. 1943. 7. Morph. 73: 265.

76. 1944. ///. Biol. Monogr. 20, No. 1.

77. Lcger, L. 1906. Arch. f. Protistenk. 7: 307.

78. 1906. Arch. f. Protistenk. 8: 159.

79. and O. Duboscq 1909. Arch. f. Protistenk. 17: 19.

79a. and 1910. Arch. Zool. Exp. Gen. 45: 187.

80. and 1915. Arch. f. Protistenk. 35: 199.

81. and 1915. Arch. Zool. Exp. Gen. 55 (N. & R.): 7.

82. Lermantoff, E. 1913. Arch. f. Protistenk. 32: 205.

83. MacFailane, J. O. and I. Ruchman 1948. Proc. Soc. Exp. Biol. Med. 67: 1.

84. Mackinnon, D. L. and I. Adam 1924. Quart. J. Micr. Sci. 68: 187.

85. and H. N. Ray 1931. Quart. J. Micr. Sci. 74: 439.

86. and 1931. Quart. J. Micr. Sci. 74: 467.

87. and 1933. Parasitol. 25: 143.

88. and 1937. Parasitol. 29: 457.

89. Manwell, R. D., F. Coulston, E. C. Bnnkley and V. P. Jones 1945. /. Inf. Dis. 76: 1.

90. Moulder, J. W. and E. A. Evans 1946. /. Biol. Chem. 164: 145.

91. Nabih, A. 1938. Arch. f. Protistenk. 91: 474.

92. Naville, A. 1927. Parasitol. 19: 100.

93. 1927. Arch. f. Protistenk. 57: 427.

94. 1930. Quart. J. Micr. Sci. 73: 547.

95. Nigrelli, R. F."l930. Ann. Protistol. 3: 13.

96. Noble, E. R. 1938. Univ. Calif. Publ. Zool. 43: 41.

97. 1941. /. Morph. 69: 455.

98. 1943. J. Morph. 73: 281.

99. 1944. Quart. Rev. Biol. 19: 213.

100. Noller, W. 1913. Arch. f. Protistenk. 31: 169.
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101. Nusbaum, J. 1903. Ztschr. wiss. Zool. 75: 281.

102. O'Roke, E. C. 1930. Univ. Calif. Publ. Zool. 30: 1.

103. 1934. School. Forest. Conserv. Univ. Mich., Bull. No. 4.

104. Oshinia, K. 1937. Parasitol. 29: 220.

105. Patten, R. 1935. Parasitol. 27: 399.

106. Phillips, N. E. and D. L. Mackinnon 1946. Parasitol. 37: 65.

107. Pinkeiton, H. and R. G. Henderson 1941. J. Amer. Med. Assoc. 116: 807.

108. Porter, R. J. and C. G. Huff 1940. Amer. J. Trop. Med. 20: 869.

109. Ray, H. 1930. Parasitol. 22: 370.

110. 1930. Parasitol. 22: 471.

111. 1933. Arch. f. Protistenk. 81: 352.

112. 1933. Parasitol. 25: 392.

113. and M. Das-Gupta 1940. Parasitol. 32: 392.

114. Reed, N. 1933. Parasitol. 25: 402.

115. Reichenow, E. 1921. Arch. f. Protistenk. 42: 180.

116. 1932. Jen. Ztschr. Naturwiss. 67: 434.

117. 1935. Arch. f. Protistenk. 84: 431.

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Sporozoa 331

121. • 1943. Bull. Wyoming Agr. Exp. Sta., No. 259.

122. Shellack, C. 1907. Arch. f. Protlstenk. 9: 297.

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125. Sprague, V. 1940. Trmis. Amer. Micr. Soc. 59: 460.

126. 1941. ///. Biol. Monogr. 18, No. 2.

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144. 1941. Amer. J. Trap. Med. 21: 653.

VII

Cilioph

ora

Class 1. Ciliatea

Subclass 1. Protociliatia
Taxonomy

Geographical distribution
Subclass 2. Euciliatia
Order 1. Holotrichida

Suborder 1. Gymnostomina

Fam
Fam
Fam
Fam
Fam
Fam
Fam
Fam
Fam
Fam
Fam
Fam
Fam
Fam
Fam

Suborder 2. Trichostomina

Fam
Fam
Fam
Fam

ly 1. Actinobolinidae

ly 2. Amphibotrellidae

ly 3. Amphileptidae

ly 4. Biitschliidae

ly 5. Chlamydodontidae

ly 6. Colepidae

ly 7. Didiniidae

ly 8. Dysteriidae

ly 9. Holophryidae

ly 10. Loxodidae

ly 11. Metacystidae

ly 12. Nassulidae

ly 13. Pycnothricidae

ly 14. Spathidiidae

ly 15. Tracheliidae

ly 1. Blepharocoridae
ly 2. Clathrostomidae
ly 3. Colpodidae
ly 4. Conidiophryidae
ly 5. Cyathodiniidae
ly 6. Entorhipidiidae
ly 7. Isotrichidae
ly 8. Marynidae
ly 9. Paraisotrichidae
ly 10. Parameciidae
ly 11. Plagiopylidac
ly 12. Spirozonidae
ly 13. Trichopelmidae
ly 14. Trichospiridae
ly 15. Trimyemidae

ly 1. Cohnilembidae
ly 2. Frontoniidae
ly 3. Tetrahymenidae
ly 4. Hysterocinetidae

Family 5. Ophryoglenidae
Family 6. Philasteridae
Family 7. Pleuronematidae

Suborder 4. Thigmotrichina
Family 1. Ancistrocomidae
Family 2. Conchophthiriidae
Family 3. Hemispeiridae
Family 4. Hypocomidae
Family 5. Sphenophryidae
Family 6. Thigmophryidae

Suborder 5. Apostomina

Suborder 6. Astomina

Family 1. Anoplophryidae
Family 2. Haptophryidae
Family 3. Hoplitophryidae
Family 4. Intoshellinidae
Family 5. Maupasellidae
Order 2. Spirotrichida

Suborders of the Spirotrichida

Suborder 1. Heterotrichina
Family 1. Balantidiidae
Family 2. Bursariidae
Family 3. Chattonidiidae
Family 4. Clevelandellidae
Family 5. Condylostomidae
Family 6. Folliculinidae
Family 7. Lichnophoridae
Family 8. Metopidae
Family 9. Pcritromidae
Family 10. Plagiotomidae
Family 11. Reichenowellidae
Family 12. Spirostomidae
Family 13. Stentoridae

Suborder 2. Oligotrichina
Family 1. Halteriidae
Family 2. Strobilidiidae

Suborder 3. Tintinnina

Suborder 4. Entodiniomorphina
Family 1. Ophryoscolecidae
Family 2. Cycloposthiidae

Suborder 5. Hypotrichina
Family 1. Aspidiscidae
Family 2. Euplotidae

332

Ciliophora 333

Family 3. Oxytrichidae
Family 4. Paraeuplotidae
Suborder 6. Ctenostomina
Family 1. Epalcidae
Family 2. Mylestomidae
Family 3. Discomorphidae
Order 3. Peritrichida
Family 1. Astylozoonidac
Family 2. Epistylidae
Family 3. Lagenophryidae
Family 4. Ophrydiidae
Family 5. Scyphidiidae
Family 6. Urceolariidae
Family 7. Vaginicolidae
Family 8. Vorticellidae

Order 4. Chonotrichida
Family 1. Chilodochonidae
Family 2. Stylochonidae
Family 3. Spirochonidae

Class 2. Suctorea
Taxonomy

Family 1. Acinetidae
Family 2. Dendrocoraetidae
Family 3. Dendrosomidae
Family 4. Discophryidae
Family 5. Ephelotidae
Family 6. Ophryodendridae
Family 7. Podophryidae

Literature cited

.EMBERS OF THIS suBPHYLUM posscss cilia or ciliary derivatives
in some stage of the life-cycle. The equipment ranges from a complete
covering of simple cilia to a relatively few membranelles more or less
completely restricted to the peristomial area. Within this range, types of
ciliary specialization and patterns of distribution form a major basis for
differentiating taxonomic subdivisions. The Ciliophora are usually di-
vided into two classes, Ciliatea and Suctorea. In the Ciliatea, cilia or their
compound derivatives are present in the dominant phase of the cycle.
Suctorea are non-ciliated as adults and have developed peculiar tentacles
which function in feeding. The ciliated larval stages characteristic of
most species establish the relationship of this group to the Ciliatea and
larval ciliary patterns imply that Suctorea^ are more closely related to the
Holotrichida than to the more specialized ciliates (82, 103).

CLASS 1. CILIATEA

Cilia or compound ciliary organelles are present in active stages of
the life-cycle. The ciliates show a variety of trends in specialization of the
ciliature, and on this basis may be divided logically into a number of
groups. The class includes the Subclasses Protociliatia and Euciliatia.
The nuclei of Protociliatia are apparently similar in structure and func-

^ In fact, some workers believe that the "Class Suctorea," as well as the "Order
Peritrichida" and the "Order Chonotrichida," would be more appropriately placed as
subdivisions of the Holotrichida, as suggested by Faur6-Fremiet (62). Rapidly accumu-
lating data tend to support these proposals, and it now appears that such a taxonomic
revision of the ciliates can be expected in the near future. The older system, as followed
in the present chapter, presinnably will be replaced by two major subdivisions of the
ciliates, (1) a group corresponding to the Holotrichida plus the Suctorea, Chonotrichida,
and Peritrichida; and (2) a group probably including the Spirotrichida as now con-
stituted.

334 Ciliophora

tion, whereas nuclear dimorphism (macronucleus and micronucleus) is
characteristic of the Euciliatia.

Subclass 1. Protociliatia

These are the opalinid ciliates which, except for a few species from
fishes and snakes, are parasitic in the large intestine of Amphibia. The
opalinids have no cytostome, although this is not a feature exclusive to
them among the Ciliatea. The distribution of cilia is practically uniform
and in this respect the opalinids resemble many holotrichous ciliates with
which they have sometimes been classified.

Ciliary patterns are rather simple. As described in Opalina obtrigo-
noidea (43), the dorsal rows follow sigmoid paths while the ventral rows
are relatively straight (Fig. 7. 1, A). About half of the rows pass com-
pletely around the body. The rest, intercalary rows which extend from the
anterior end toward one margin of the body, possibly represent stages in
the development of new rows (43). Similar intercalary rows have been
described in Zelleriella elliptica (38) and other species (152). Along the
anteroventral surface in O. obtrigonoidea, a number of stout falcular
cilia arise from the falcular fibrils (Fig. 7. 1, C, E). The latter are two
subpellicular fibrils which extend along the anteroventral margin and
then fuse into a single fibril which continues for some distance along the
left margin of the body. The falcate fibril is connected with the first basal
granule in each row of somatic cilia (Fig. 7. 1, E). Although longitudinal
fibrils joining basal granules could not be detected, oblique fibrils, per-
pendicular to the rows of cilia, connect basal granules of different longi-
tudinal rows (Fig. 7. 1, D). In addition to the fibrils parallel to the body
surface, dorso-ventral fibrils extend inward from the basal granules, usu-
ally passing to granules on the other side of the body (Fig. 1. 11, G).
In contrast to an earlier report for Opalina rananun (73), no connections
between the fibrils and the endoplasmic spherules could be detected in
O. obtrigonoidea. The absence of such connections also has been reported
for Cepedea metcalfi, Opalina coracoidea, and O. ranarian (8).

The pellicle of opalinids (8, 43, 152) shows ninnerous grooves, parallel
to the rows of cilia, and each row apparently arises in such a groove. In
O. obtrigonoidea the grooves are produced by pellicular folds (Fig. 7. 1,
B) which may be a factor in maintenance of body form, "functioning
much like the corrugations in corrugated cardboard" (43). Myonemes
have not been demonstrated in the cortex.

The endoplasm typically contains Feulgen-negative (177) endoplasmic
spherules (endospherules, endosarc bodies) which have been interpreted
variously as Golgi bodies, parabasal bodies comparable to those of certain
flagellates, masses of stored food, and even as stages in the development
of nuclei. There is no evidence that these endoplasmic spherules are
homologues of the macronuclear derivatives in Dileptiis (Chapter I). Al-

Ciliophora 335

'-^* '. i i « '. I-r-T^

Fig. 7. 1. A-E. Opalina obtrigonoidea Metcalf (after Cosgrove): A. Dia-
gram showing about half the actual number of ciliary rows. B. Cross-section
showing pellicular grooves and basal granules; cilia omitted; x2160. C.
Lateral view, falcular cilia, the fused falcular fibrils, basal granules of
somatic cilia; semidiagrammatic. D. Surface view of basal granules and
oblique fibrils; pellicular grooves indicated by dotted lines; x2160. E.
Anterior end, longitudinal section; two falcular eilia arismg from the
falcular fibrils; somatic cilia and their basal granules; xl800. F. Proto-
opalina intestinalis (Stein) Metcalf; endoplasmic spherules not shown; x270
(after M.). G. Zelleriella truncata Carini, x35 (after C.)- H. Opalina rana-
rum (Ehrbg.); length, 62-232/x (after Bhatia and Gulati). I. Protoopalina
montana Metcalf, x390 (after M.). J. Cepedea punjabensis Bhatia and
Gulati; average, 82x35/x (after B. & G.).

though the spherules may be involved in synthesis and storage of reserve
food (164), the nature and significance of these inclusions are undeter-
mined. The presence of an "excretory" canal or vacuoles, as described in
Protoopalina intestinalis (151), has not been demonstrated in other
species.

336 Ciliophora

Binary fission is unusual in that the plane of division is oblique, or
almost longitudinal, instead of transverse as in typical ciliates. In the
binucleate Zelleriella elliptica (38), fission often precedes the completion
of mitosis and produces temporarily uninucleate daughter organisms.
Although nuclear behavior is not easily traced in multinucleate species,
series of nuclear divisions apparently may precede and follow fission.
Behavior of the ciliature and fibrillar system in fission of Opalina ra-
narum has been described (21).

Little is known about the life-cycles of Protociliatia. However, the
cycle of Opalina ranariim, according to one account (123), is fairly com-
plicated. Throughout most of the year large multinucleate forms are
present in the host. In the spring, plasmotomy produces small ciliates
containing only a few nuclei. These stages encyst and are eliminated by
the host. Such cysts are ingested by tadpoles and liberate "gametocytes."
Repeated division of the "gametocytes" results in "gametes" of two sizes.
According to another version (160), some ingested cysts give rise to
gametes while others hatch into ciliates which merely grow to maturity.
Gametes similar to those reported for O. rananun have been described in
Protoopalina intestinalis (151). Gametogenesis is said to be followed by
anisogamy. The resulting zygotes encyst and are eliminated from the
intestine. After ingestion by another tadpole, each zygote excysts and
develops into a multinucleate ciliate. Encysted adults, as well as small
cysts containing 1-4 nuclei, have been observed in Opalina chattoni (217).

Unfortunately, the behavior of chromosomes has not yet been traced
throughout the life-cycle and meiosis remains to be described. However,
a few apparently haploid specimens have been noted in Zelleriella louisi-
anensis (38). These ciliates were approximately normal in size but con-
tained four relatively small nuclei, each with 12 chromosomes, instead of
the usual two nuclei with 24 chromosomes each.

Taxonomy

Flagellate affinities have been suggested for the Protociliatia (73, 118).
On the other hand, nuclear structure and mitosis in Opalina ranarum
and O. obtrigona show features characteristic of ciliates (92) and the
fibrillar system of O. ohtrigonoides resembles that of holotrichous ciliates
(43). Accordingly, retention of the opalinid ciliates in the Ciliatea appears
to be sound practice at present. The evident lack of macronuclei is a
logical basis for continued recognition of Metcalf's Subclass Protociliatia.

The most extensive taxonomic work on the group is that of Metcalf
(152, 153), who established two families on the basis of nuclear number.
The Protoopalinidae include binucleate types assigned to two genera:
Protoopalina Metcalf (8, 152; Fig. 7. 1, F, I), with cylindrical or slightly
flattened bodies; and Zelleriella Metcalf (7, 37, 52; Fig. 7. 1, G), with dis-
tinctly flattened bodies. The multinucleate types are placed in the Opa-

Ciliophora 337

linidae, which also include two genera: Cepedea Metcalf (8, 152; Fig.
7. 1, J), with cylindrical or slightly flattened bodies; and Opalina Purkinje
and Valentin (43, 152, 153, 160; Fig. 7. 1, A-E, H), with much flattened
bodies.

Geographical distribution

The geographical distribution of the Protociliatia is interesting (152,
153). The genus Protoopalina, supposedly the most primitive, is widely
distributed and seems to be excluded only from the northeastern United
States and from southern India and neighboring islands. The genus
Zelleriella, represented in Central America, South America and southern
North America, apparently does not extend north of Australia in the
eastern hemisphere. Cepedea, although not represented in Australia, is
otherwise widely distributed, whereas the genus Opalina apparently has
not become established in Australia or South America. Metcalf (153) has
attempted to correlate these peculiarities in distribution of the ciliates
with the phylogeny of their hosts.

Subclass 2. Euciliatia

These are the typical ciliates with macronuclei and micronuclei.
Subdivision into orders and suborders is based largely upon the distribu-
tion of cilia and their derivatives and upon the differentiation of such
structures in the peristomial area. Following the practice of Kahl (100,
102, 104, 106), the subclass is now commonly divided into four orders —
Holotrichida, Spirotrichida, Peritrichida, and Chonotrichida — but taxo-
nomic treatment of the group has varied in different systems of classifica-
tion (Chapter III). One of the more recent proposals would divide the
Euciliatia into two groups: the "Spirotricha," the Spirotrichida as defined
below; and the "Holotricha," including the rest of the Euciliatia and the
Suctorea (62).

The Holotrichida lack the strongly developed adoral zone of mem-
branelles so characteristic of the peristome in Spirotrichida. A rather uni-
form covering of somatic cilia is typical, although there are some genera
in which the cilia are restricted to certain zones or to one surface of the
body.

The Spirotrichida show an extensive development of membranelles
and cirri which, in certain groups, have completely replaced simple cilia.
An adoral zone of membranelles arises at the left of the cytostome and
extends anteriorly, often winding around the anterior end of the body.
The group as a whole shows a strong trend toward reduction of the total
ciliated area.

In the Peritrichida the epistome (peristomial area) is commonly a dis-
coid region bounded by two or more rows of cilia which, as viewed from
the oral end of the body, pass counter-clockwise around the epistome and

338 Ciliophora

through the cytostome into the "vestibule" (pharynx). The majority are
sessile and are commonly equipped with stalks.

The Chonotrichida are ectocommensals attached to their hosts by a
basal disc or a short stalk. The peristome, at the distal pole, is usually
surrounded by a funnel-like prolongation of the body, or sometimes by
two concentric funnels. The wall of the funnel may or may not be rolled
into a spiral. Cilia may be restricted to the peristome and funnel in the
adult stage. Reproduction by budding is characteristic.

Order 1. Holotrichida. This large order, usually considered more primi-
tive than the rest of the Euciliatia, shows considerable diversification of
the peristomial area and in one group the cytostome has disappeared.
Such specializations furnish a basis for dividing the Holotrichida into
suborders.

Suborder 1. Gymnostomina. The cytostome opens directly at the sur-
face or else into a slight depression, or oral groove, which lacks a well
developed peristomial ciliature. In many genera the cytostome lies at or
near the anterior end of the body. In others, the mouth has shifted
posteriorly to either a compressed or a broad flattened oral ("ventral")
surface.

Suborder 2. Trichostomina. The cytostome usually lies on the ventral
surface at the base of a rather well defined oral groove, typically equipped
with one or more fields of densely set cilia. Fusion of peristomial cilia
into simple membranes or membranelles, or both, occurs in a few species.

Suborder 3. Hymenostomina. The peristomial ciliature has become
modified into several membranes, perhaps derived phylogenetically from
the peristomial cilia of Trichostomina.

Suborder 4. Thigmotrichina. The most characteristic feature of these
commensals is an anterior group of thigmotactic cilia serving for attach-
ment to the host. The cytostome is shifted to a position at or near the
posterior end of the body. In some families there is an anterior sucker,
a new organelle.

Suborder 5. Apostomina. The ventral cytostome is so reduced in size
that ingestion is probably limited to very small particles. Beneath the
cytostome there is a pecidiar "rosette" (Fig. 7. 25, A-F) of uncertain func-
tion. The somatic ciliation includes less than 22 complete rows of cilia.
Life-cycles are often fairly complex.

Suborder 6. Astomina. These are endoparasitic holotrichs without a
cytostome. The body is rather uniformly ciliated as a rule, but there may
be a small cilia-free area at the anterior end.

Suborder 1. Gymnostomina

Form of the body varies considerably. Ovoid, pear-shaped, spindle-
shaped, and long vermiform types are common, and laterally compressed
and dorso-ventrally compressed species are not unusual. The ciliation is

Ciliophora 339

commonly uniform except for the frequent occurrence of large cilia
around the cytostome. In some genera, however, somatic cilia are limited
to a few transverse bands or to one surface of the body. The cytostome
usually opens at the surface. There is no well defined oral groove or
peristome equipped with specialized cilia. Even if a rudimentary peri-
stome is present, or if there are distinct preoral and postoral fields of
cilia, the organization of the peristomial area is primitive as compared
with that in more specialized groups. There is often a circumoral zone
of cilia, somewhat longer and sometimes stouter than the somatic cilia.
In addition, the pharynx is commonly surrounded by a ring of rod-like
trichites, which are sometimes partially fused to form a pharyngeal basket.

The position of the cytostome varies in different families and on this
basis, Kahl (100) has divided the suborder into three tribes. In one group
("Tribe Prostomata") the cytostome is anterior. Among the families listed
below, this is the situation in the Actinobolinidae, Biitschliidae, Cole-
pidae, Didiniidae, Holophryidae, Metacystidae, and Spathidiidae. In a
second group ("Tribe Hypostomata") the cytostome lies on the flattened
ventral surface and in the anterior half of the body. This is the case in
the Chlamydodontidae, Dysteriidae, and Nassulidae. The cytostome of a
third group ("Tribe Pleurostomata") lies on a compressed margin of the
body — the narrow ventral surface, according to Kahl, although others
have considered the cytostome lateral in position. This is the condition
in the Amphibotrellidae, Amphileptidae, Loxodidae, and Tracheliidae.

Family 1. Actiyiobolinidae. These prostomatous ciliates possess exten-
sible tentacles in addition to the usual ciliature. The tentacles in Dactylo-
chlamys pisciformis (103) are similar to those of many Suctorea (Fig. 7.
50, C). The tentacles in ActinoboUna vorax (221), which are slender struc-
tures emerging in the ciliary meridians (Fig. 7. 2, J), may be extended for
lengths of lOOpi or more, but are usually retracted is swimming ciliates.
The tip of each tentacle is said to contain a toxicyst (Chapter I). In
stained preparations the proximal ends of the tentacles are continuous
with a system of cytoplasmic fibrils (Fig. 7. 2, I). On the basis of such
tentacular equipment, Kahl (103) has suggested that the Actinobolinidae
are related to the ancestral holotrichs from which the Suctorea were
evolved and that Dactylochlamys may even represent a primitive type of
Suctorea which has not developed a sessile stage.

Only three geneia have been assigned to the family: ActinoboUna Strand (103, 221;
Fig. 7. 2, I, J), Dactylochlamys Lauterborn (103; Fig. 7. 2, B), and Enchelyoniorpha Kahl
(103; Fig. 7. 2, A). ActinoboUna is the only genus in which a cytostome has been
described. The cytostome of A. vorax (221) opens into a pharynx surrounded by a
double ring of fibrils. These apparently converge in the rim of the cytostome (Fig.
7. 2, I).

Family 2. Amphibotrellidae. This family contains the genus Amphi-
botrella R. and L. Grandori (106; Fig. 7. 2, K), characterized by location

340 Ciliophora

Fig. 7. 2. A. Enchelyomorpha vermicularis (Smith) Kahl, length about
35^; knobbed tentacles; cytostome not described (after K.). B. Dacty-
lochlamys piscijormis Lauterborn (after Kahl), about lOO/x long; knobbed
tentacles, firm pellicle with rugose ribs (stippled). C. Cross-section of Liono-
tus brancJriarum (Wenrich) Kahl, showing distribution of cilia (after W.).
D. Ampliileptus clapnredei Stein, 120-150^ long (after Entz). E. Lionotus
fasciola (O.F.M.) Wrzesniowsky, usually 300-450/^ long (after De Morgan).
F, G. Loxophyllum rostratum Cohn, 300-400/^; lateral view showing naked
dorsal surface, and dorsal view (after De Morgan). H. Centrophorella fascia-
lata (Sauerbrey) Kahl, x80 (after Noland). I, J. Actinobolina vorax (Wen-
rich): anterior part of body showing circumpharyngeal fibrils and retracted
tentacles connected with inner bundle of fibrils (I); general organization of
the ciliate (J); x300 (after W.). K. AmpJiibotrella enigmatica R. and L.
Grandori, about 250/^ (from Kahl, after R. & L. G.). L. BryophyUum carina-
tum Gelei; sensory bristles indicated near the cytostome and in several ciliary
meridians; x375 (after G.).

Ciliophora 341

of the cytostome in a non-ciliated anterior furrow which extends ahnost
to the tip of the body. Near the posterior end of the body there is also
a short ciliated groove of uncertain significance.

Family 3. Amphileptidae. In this pleurostomatous group the body is

Fig. 7. 3. A. Bundleia postciliata (Bundle) da Cunha and Muniz, x464
(afler Hsiung). B. Polymorpha ampulla Dogiel, x958 (after Hsiung). C. Atn-
pullacula ampulla (Fiorentini) Hsiung, x363 (after H.). D. Didesmis ovalis
Fiorentini, x484 (after Hsiung). E. Blepharoprosthium pireum Bundle, x484
(after Hsiung). F. Paraisotrichopsis composita Gassovsky, x484 (from Hsiung,
after G.). G. Blepharoconus cervicalis Hsiung, x383 (after H.). H. Biitschlia
nana Dogiel, x725 (after D.). I-L. Concretion-vacuoles of Biitschliidae, sche-
matic (after Dogiel): I. Didesmis quadrata; J. Biitschlia sp., cross-section
througli the vacuole; K. Polymorpha ampulla; L. Paraisotricha colpoidea, an-
terior end of body. M. Prorodonopsis coli Gassovsky, x443 (after Hsiung). N.
Sulcoarcus pellucidulus Hsiung, x533 (after H.). O. Blepharosphaera intesti-
nalis Bundle, x443 (after Hsiung). P. Alloiozona trizona Hsiung, x363 (after H.).

342 Ciliophora

laterally compressed, slightly or extensively in different genera. The slit-
like cytostome lies on the typically convex "ventral" surface and is usually
bordered by a zone of trichocysts. Ciliation may be uniform or may be
reduced or lacking on one surface (Fig. 7. 2, C). There are usually two
or more macronuclei. Carnivorous habits are characteristic, other ciliates
and rotifers being common prey of various Amphileptidae.

The family includes the following genera: Ampliileptits Ehrbg. (102; Fig. 7. 2, D),
Bryophylliim Kahl (102; Fig. 7. 2, L), Centrophorella Kahl (106, 158; Fig. 7. 2, H),
Lionotus Wrzesniowsky (102, 165; Fig. 7. 2, C, E), Loxophyllum Dujardin (102, 165,
167; Fig. 7. 2, F, G).

Family 4. Butsdiliidae. These ciliates occur in the digestive tract of
such herbivores as horses and camels. The body is more or less ovoid or
pear-shaped, with the cytostome usually at the anterior end. An anterior
concretion-vacuole (Fig. 7. 3, I-L) — which has been considered a statocyst
(52) — and one or more contractile vacuoles are characteristic. A posterior
cytopyge is typical. The cilia may be uniformly distributed or else re-
stricted to certain areas. In fission, the concretion-vacuole is retained by
the anterior daughter and a new organelle is developed by the posterior
one.

Hsiung (91) has published a key to most of the following genera: Alloiozona Hsiung
(91; Fig. 7. 3, P), AmpuUaciila Hsiung (91; Fig. 7. 3, C), Blepharoconus Gassovsky (91;
Fig. 7. 3, G), Blepharoprosthium Bundle (91; Fig. 7. 3, E), Blepharosphaera Bundle
(91; Fig. 7. 3, O), Blepharozoum Gassovsky (91), Bundleia da Cunha and Mimiz (91;
Fig. 7. 3, A), BiUschlia Schuberg (51; Fig. 7. 3, H). Didesmis Fiorentini (91; Fig. 7. 3,
D), Holophryoides Gassovsky (91), Paraisotrichopsis Gassovsky (91; Fig. 7. 3, F), Poly-
morpha Dogiel (91; Fig. 7. 3, B), Prorodonopsis Gassovsky (91; Fig. 7. 3, M), and
Sulcoarcus Hsiung (91; Fig. 7. 3, N).

Family 5, Chlamydodontidae. The cilia are restricted essentially to the
ventral surface (Fig. 7. 4, D). A narrow transversely striated band borders
the ciliated area in Chlamydodon. The cytostome is antero-ventral and
the pharynx is surrounded by a pharyngeal-basket (Fig. 7. 4, J). Adoral
membranes are sometimes present but are always small and poorly de-
veloped and lie anterior to the cytostome. There is no ventral proto-
plasmic stylus such as is found in the Dysteriidae (Fig. 7. 5, I). The
Chlamydodontidae commonly feed on diatoms and other algae, phyto-
flagellates, and bacteria.

The family includes the following genera: Chilodonella Strand (102, 141, 165; Fig. 7.
4, J), Chlamydodon Ehrbg. (102, 140; Fig. 7. 4, E), Cryptopharynx Kirby (113; Fig. 7.
4, K), and Phascolodon Stein (102; Fig. 7. 4, C, D).

Family 6. Colepidae. These are somewhat barrel-shaped forms with an
armored cortex. The armor is composed of plates (Fig. 7. 4, I), the form
and arrangement of which vary with the species. The armor of Coleps

Ciliophora 343

Fig. 7. 4. A. Tiari7ia fusus (Clapaiede and Lachmann), 1 10^ (after Faure-
Fremiet). B. Coleps amphacanthus Ehrbg. (70-90^), longitudinal optical sec-
tion showing circiimpharyngeal trichites, macronucleus, contractile vacuole
(after Kahl). C, D. Phascolodon vorticella Stein, 90-110^; ventral view and
schematic cross-section (after Kahl). E. Chlamydodon triquetrus (O.F.M.),
80-120yti; ventral view, striated band, ventral ciliary pattern, cytostome,
macronucleus, contractile vacuoles (after Kahl). F. Chilodonella cucullus
(Ehrbg.), cross-section of ventral cortex; xlOOO (after Wetzel). G-I. Coleps
hirtus O. F. M.: C, H. Cross-sections at level of cytostome, showing trichites,
and near the equator, showing macronucleus and large mass of food; xlOOO
(after Wetzel). I. Diagram showing three longitudinal rows of cortical plates
(after Faure-Fremiet and Hamard). J. Chilodonella caudata Stokes, 40-50^,
ventral view, pharyngeal basket, basal granules, macro-, and micronucleus
in outline (after MacDougall). K. Cryptopharynx setigerus Kahl, ventral
view showing cytostome, longitudinal ribs, cilia, and marginal spines; x485
(after Kirby).

344 Ciliophora

hirtus contains calcium carbonate and apparently is covered by an organic
pellicle (65). Calcification of the plates is inhibited by exposure of the
ciliates to benzenesulfamid (67). A ring of circumpharyngeal trichites
(Fig. 7. 4, B, G) is characteristic. Although the Colepidae sometimes ingest
small algae, they are primarily carnivorous.

Fig. 7. 5. A, B. Mesodinium acarus Stein (after Noland), lateral view,
xl400; anterior end, showing oral tentacles, x2800. C-E. Didinhim nasutum
O. F. M., 80-150^: C. General organization (after Kahl). D. Freshly excysted
specimen, ciliation more extensive than in the adult; x450 (after Beers).
E. Schematic longitudinal section; endoplasmic fibrils, circumpharyngeal
trichites, macronucleus, contractile vacuole (after ten Kate). F, G. Askenasia
volvox (Claparede and Lachniann) Kahl, anterior and lateral views, x630
approx. (after Wang and Nie). H. Cyclotrichium gigns Faurc-Fremiet, speci-
men 160x250^; schematic (after F-F.). I. Trochilia marina Mereschkowsky,
x850 (after Kahl). J. Dysteria navicula Kahl, x650 (after Wang and Nie).
K. Hartmannula entzi Kahl, x460 (after Wang).

Ciliophora 345

The family includes two genera: Coleps Nitzsch (74, 99, 100, 157; Fig. 7. 4, B, G-I) and
Tiarina Bergh (100; Fig. 7. 4, A).

Family 7. Didiniidae. These ciliates are radially symmetrical with re-
spect to the longitudinal axis. The anterior cytostome (Fig. 7. 5, B, F) is
not surrounded by cilia although there is a ring of circumoral tentacles
in Mesodiy-iiiim. A circlet of pectinellae (slender membranelles) usually
lies at or near the rim of the anterior pole (Fig. 7. 5, C, E, G), and there
may be one to several similar rings of pectinellae located more posteriorly.
The rest of the body may be either naked or ciliated in different species.
A band of circumpharyngeal trichites is characteristic. Although a few
species apparently eat algae, a carnivorous diet is the usual one. Didiiiium
nasutum is noted for its habit of capturing and ingesting such ciliates as
Paraynecium (150).

The family includes the following genera: Askenasia Blochmann (100, 165; Fig. 7.
5, F, G), Cyclotrichiuw Meunier (100, 171; Fig. 7. 5, H), Didinium Stein (97, 100; Fig.
7. 5, C-E), and Mesodiniuin Stein (100, 158; Fig. 7. 5, A, B). Mnnodiniiitu Fabre-
Domergue (58), with one anterior ring of pectinellae. has been considered a separate
genus by some workers; others include such ciliates in Didinium Stein. The family
includes both fresh-water and marine species, and Cyclotricliiurn rneunieri has been
recorded as a cause of red water in the Gidf of Maine (171).

Family 8. Dysteriidae. These hypostomes differ from the Chlamydodon-
tidae and Nassulidae in the presence of a ventral protoplasmic stylus
(Fig. 7. 5, I-K). In Dysteria this structure may adhere to a solid surface
and serve as a temporary anchor (46), whereas the stylus in Trochilioides
secretes a slender filament which serves the same purpose (64). The dorsal
surface is not ciliated and a reduction of the ventral ciliation is often
noticeable. A pharyngeal rod-apparatus is characteristic. The Dysteriidae
are mainly marine ciliates which feed mostly on diatoms, other algae, or
bacteria.

The family includes five genera: Dysteria Huxley (46, 102; Fig. 7. 5, J), Hartmannula
Poche (102; Fig. 7. 5, K), Scaphidion Stem (102), Trochilia Dujardin (102; Fig. 7. 5,
I), and Trochilioides Kahl (64, 102).

Family 9. Holophryidae. These are rather uniformly ciliated species in
which the cytostome lies at or near the anterior pole and often opens on
a rounded elevation. There is generally a circumpharyngeal zone of
trichites (Fig. 7. 6, A), and an adoral row of fused cilia (syncilia) is some-
times present (Fig. 7. 6, F). Form of the body ranges from plump ovoid or
cylindrical shapes to long slender types, the latter sometimes possessing
a very extensible and mobile "neck" several times as long as the rest of
the body. Feeding habits vary widely. Some species are rapacious carni-
vores, pursuing and capturing other ciliates or rotifers. Some usually eat
small flagellates, others feed mainly on bacteria and small algae, while

346 Ciliophora

Fig. 7. 6. A. Prorodon teres Ehrbg., longitudinal section showing circum-
pharyngeal trichites; x240 (after Wetzel). B. Platophrya spumacola Kahl,
x450 (after K.). C. Lagynophrya simplex Kahl, 35-40,u (after K.). D. Placiis
socialis (Fabre-Domergue), slit-like cytostome, macronucleus, contractile
vacuole; x680 (after Noland). E. Plagiocampa longis Kahl, 70-80;x (after K.).
F. P. marina Kahl, syncilia along cytostome; x850 (after Noland). G.
Holophrya obloiiga Maupas, macronuclear chain, contractile vacuole and
accessory canals; x60 (after De Morgan). H. Holophrya (Trachelocerca})
coronata De Morgan, anterior end of body; schematic (after De M.). I, J.
Helicoprorodon gigas (Kahl) Faure-Fremiet; lateral view, xl650; anterior end
of body, schematic (after F-F.). K, L. Trachelocerca enlzi Kahl, length
reaches 270/x; extended specimen and longitudinal optical section of the
anterior end (after K.). M. Spasmostoma viride Kahl, 50-60/^ (after K.). N.
Prorodon parafarctus Wang and Nie, x300 (after W. k N.).

Ciliophora 347

the diet of certain species includes such a variety as bacteria, diatoms,
and small nematodes.

The family includes the following genera, some of which are represented in both
fresh and salt water: Bursella Schmidt (100), Chaenea Quennerstedt (46, 98, 100; Fig.
7. 7, G, H), Chilophrya Kahl (100), Crobylura Andre (100), Enchelyodon Clap'arMe
and Lachmann (100; Fig. 7. 7, D), Enchelys Hill (100; Fig. 7. 7, I), Helicoprorodon
Faure-Fremiet (61a; Fig. 7. 6, I, J). Holophrya Ehrbg. (46, 98; Fig. 7. 6, G, H),
Ileonema Stokes (100), Lacrymaria Ehrbg. (46, 100, 165; Fig. 7. 7, B, C), Lagynophrya
Kahl (100; Fig. 7. 6, C), Microregma Kahl (100), Nannophrya Kahl (100), Pithothorax

Fig. 7. 7. A. Urotricha armata Kahl, AOAbu (after Kahl). B, C. Lacry-
maria olor (O.F.M.) Biitschli, usually 110-160/x, sometimes extended to ISOO^uj
general organization (B); anterior end (C), schematic (after De Morgan).
D. Enchelyodon elegans Kahl, x400 (after K.). E. Remanella rnargaritifera
Kahl, 100-200^; ciliary pattern similar to that of Loxodes rostrum (after K.).
F. Pseudoprorodon emmae (Bergh) Kahl. 100-200^ (after K.). G. Chaenea
limicola Levander, length reaches 300^ (after Kahl). H. Chaenea teres Du-
jardin, anterior end of contracted specimen; schematic (after Kahl). I.
Enchelys gasterosteus Kahl, xl200 (after K.). J. Loxodes striatus Penard, two
macronuclei, Midler's vesicles; x298 (after Wang and Nie).

348 Ciliophora

Kahl (100), Placus Cohn (158; Fig. 7. 6, D), Plagiocampa Schewiakoff (100; Fig. 7. 6,
E, F), Platophrya Kahl (100; Fig. 7. 6, B), Prorodon Ehrbg. (100, 165, 210; Fig. 7. 6, A,
N), Pseudoprorodon Blochmann (100; Fig. 7. 7, F), Rhopalophrya Kahl (100), Spasmo-
stoma Kahl (98; Fig. 7. 6, M), Stephcniopogon Entz (100), Trachelocerca Ehrbg. (13,
100, 188; Fig. 7. 6, K. L), Trachelophyllum Claparede and Lachmann (100, 165), Uro-
tricha Claparede and Lachmann (100, 165, 188; Fig. 7. 7, A).

Family 10. Loxodidae. As in the Amphileptidae, the body is compressed
laterally but the "ventral" margin, on which the cytostome lies, tends to
be slightly concave. 1 he right surface is ciliated, the left naked. The
presence of Midler's bodies (Fig. 1. 16, D; 7. 7, J) is characteristic. Algae
and bacteria are the usual food.

Two genera are recognized: Loxodes Ehrbg. (102, 165; Fig. 7. 7, J), which includes
fresh-water species; and Remanella Kahl (102; Fig. 7. 7, E), which includes only marine
ciliates.

Family 11. Metacystidae. This family (97) is characterized by a terminal
cytostome and a firm cortical layer enclosing a peculiar alveolar zone. The
cytostome may be rounded or slit-like, and in certain species, opens into
an endoplasmic cavity ("receptacle"). These ciliates apparently feed
mainly on bacteria. A pseudochitinous lorica is characteristic, although
a gelatinous lorica has been reported in rare cases.

The family includes only three genera: Metacystis Cohn (100, 165; Fig. 7. 8, H),
Pelatractus Kahl (100; Fig. 7. 8, I, J), and Vasicola Tatem (100, 165; Fig. 7. 8, K).

Family 12. Nassiilidae. In this hypostomatous family the body is com-
pletely ciliated, although the dorsal ciliation is less dense than that of the
ventral surface. A pharyngeal basket is typical but there is no ventral
stylus like that of the Dysteriidae. The family contains marine and fresh-
water ciliates which feed mainly on diatoms and other algae.

The following genera are included: Chilodontopsis Blochmann (102; Fig. 7. 8, B),
Cyclogramma Perty (102), Eucainptocera da Cunha (102), Nas.sula Ehrbg. (102, 165;
Fig. 7. 8, C-E), Orthodon Gruber (102; Fig. 7. 8, A), and Paranassula Kahl (102; Fig.
7. 8, F, G).

Family 13. Pycnothricidae. These ciliates occur in the cecum and large
intestine of various mammals. Species of Collinella and Pycnothrix are
known from Procavia (Hyrax); Biixtonella, from cattle; Nicollella, and
also Collinella, from Cteiiodactylus. The body is completely ciliated and
a long groove usually leads to the cytostome, which may lie near the
middle or at the posterior end of the body. In Pycnothrix monocystoides
(which reaches a length of 2-3 mm), there is no single cytostome. Instead,
food apparently is ingested through pits in the unusually long groove.
A thick layer of ectoplasm in the anterior part of the body (Fig. 7. 9,
E, F) is a striking feature in Collinella, Nicollella, and Pycnothrix.

Ciliophora 349

Fig. 7. 8. A. Orlhodon hamatus Gruber, 90-260/ri (after Kahl). B. Chilo-
dontopsis muscorum Kahl, 65-80yn (after K.). C, D. Transverse and longitu-
dinal sections showing circumpharyngeal trichites of Nassula aurea Ehrbg.,
xloOO and x240 (after Wetzel). E. Nassula gracilis Kahl, x360 (after K.).
F, G. Paia7iassula microstoma (Claparede and Lachmann) Kahl; ventral view
of preoral suture and cytostome (F), x600; pharyngeal apparatus, macro-
nucleus, contractile vacuole, trichocysts (G), x450 (after Noland). H. Meta-
cystis elongata Kahl, without lorica, xOOO approx. (after K.). I, J. Pelatractus
constractus Wang and Nie; extended specimen (I) showing macronucleus and
catial extending posteriorly from contractile vacuole, xlyO; contracted speci-
men (J), schematic (after W. & N.). K. Vasicola parvula Kahl, 30-50;a, spec-
imen in lorica (after K.).

The family includes the following genera: Buxtonella Jameson (94, 175; Fig. 7. 9,
\, B), Collinella Chatton and Perard (33; Fig. 7. 9, C, D), Nicollella Chatton and
Perard (33; Fig. 7. 9, E-G), and Pycnothrix Schubotz (33; Fig. 7. 9, H).

Family 14. Spathidiidae. These prostomatous ciliates have a slit-like
cytostome generally lying in a non-ciliated ridge (Fig. 7. 10, B-D). In
some genera, this ridge is continued posteriorly and spirally for some
distance (Fig. 7. 10, H; 11, B, C). In others, the ridge does not extend

350 Ciliophora

Fig. 7. 9. A. BuxtoneUa sulcata Jameson (after Rees): somatic ciliation,
nuclei, groove leading to the posterior cytostome; x225. B. Longitudinal sec-
tion of posterior end of B. sulcata, showing cytostome and gullet; x5I0
(after Rees). C, D. ColUnella gundii Chatton and Perard (after C. & P.): C.
Ventral view showing preoral groove, nuclei, ectoplasmic and endoplasmic
zones separated by a layer of myonemes, xl25. D. Optical parasagittal sec-
tion, posterior end of body, showing cytostome, gullet, and contractile vacu-
ole, x275. E-G. Nicollella ctenodactyli Chatton and Perard (after C. & P.):
E. Ventral view showing preoral groove ending at the cytostome, the nuclei,
contractile vacuole, thick anterior zone of ectoplasm, and layer of myonemes;
xl25. F. Cross-section between cytostome and anterior pole, showing preoral
groove and layer of myonemes separating ectoplasm and endoplasm; x240.
G. Cross-section through cytostome and pharynx, x240. H. Pycnothrix mono-
cystoides Schubotz: the groove follows a slightly spiral course down one side
to the posterior pole and then up on the other side almost to the anterior
pole; in each groove, a number of pits which serve as cytostomes; x35
(after Chatton and Perard).

Ciliophora 351

Fig. 7. 10. AD. Perispira ovum Stein: A. Specimen showing spiral ridge
and anterior cytostome; x513 (after Wang and Nie). B. Anterior end of ridge
showing the closed cytostome (a slit ending anteriorly in a small pore);
schematic (after Dewey and Kidder). C, D. Stages in ingestion of a flagellate;
schematic (after Dewey and Kidder). E. Spathidium amphorijorme Greef,
IQQfx or smaller (after Kahl). F, G. Spathidioides exsecata Kahl, length about
60/i; lateral \iew (F); ventral view (G), showing slit-like cytostome opened
inider pressure from a coverslip (after K.). H. Diceras bicornis Kahl, length
about 260/^; spiral ridge, posterior contractile vacuole (after K.). I, J. Teuto-
plirys trisitica Chatton and de Beauchamp, x250 (after Wenrich): specimen
showing proboscidial arms, macronucleus, contractile vacuole, zoochlorellae
(indicated along one margin); longitudinal section through cytostome, show-
ing circumpharyngeal fibrils and the trichocysts in one arm.

352 Ciliophora

much beyond the posterior end of the cytostome (Fig. 7. 10, E-G). In any
case, the oral ridge and its extension, if present, may be armed with
trichites or trichocysts. In Legendrea, the posterior part of the body bears
a number of tentacles, each equipped with trichocysts (Fig. 7. 11, D).
UnUke the usual condition, the cytostome in Teiitoplirys lies at the base
of three proboscis-like extensions of the body (Fig. 7. 10, I, J). Although
the somatic ciliation is usually complete and uniform, except where in-
terrupted by the oral ridge or its extension, a row of flattened "cilia"
(slender membranelles?) extends along each side of the cytostome in
Spathidioides. In addition, a more or less complete loss of cilia on the

Fig. 7. 11. A. Enchelydiuiii ampliora Kahl, 30-45/^ (after K.). B, C. Pen-
ardiella undulata Kahl, 90-1 30/i, ventral and lateral views of spiral ridge and
other features (after K.). D. Legendrea loyczae Faur^-Fremiet, showing
tentacles, macronucleus, pharyngeal fibrils (from Kahl, after F-F.). E. Trach-
elius ovum Ehrbg., 200-400/x; cytostome, circumpharyngeal trichites, macro-
nucleus, contractile vacuoles (after Kahl). F. Dilrptus anser (O.F.M.),
250-600^ but usually 250-400yLt (after Kahl). G. Paradileptus conicus Wenrich,
cytostome, circumpharyngeal trichites, zone of trichocysts, macronuclear
chain, nuinerous contractile vacuoles; x250 (after W.).

Ciliophora 353

left surface has occurred in Homalozoon. The Spathidiidae are typically
foraging carnivores which commonly prey on ciliates and flagellates.

The family contains the following genera: Cranotheridium Schewiakoff (99), Diceras
Eberhard (99; Fig. 7. 10, H), Enchelydium Kahl (99; Fig. 7. 11, A), Homalozoon Stokes
(99), Legendrea Faure-Fremiet (99, 165; Fig. 7. II, D), Paraspatbidium Noland (158),
Penardiella Kahl (99; Fig. 7. II, B, C), Perispira Stein (48, 99; Fig. 7. 10, A-D). Spathi-
dioides Biodsky (99; Fig. 7. 10, F, G), Spathidiurn Dnjardin (99, 165, 226; Fig. 7. 10,
E), Teittopluys Chatton and Beauchamp (219; Fig. 7. 10, I, J).

Family 75. Tracheliidae. The approximately circular cytostome, located
some distance from the anterior pole and at the end of a ventral row of
trichocysts, is surrounded by trichites or trichocysts and sometimes by
both (102). The body is completely ciliated. The Tracheliidae occur in
fresh and salt water and are typically carnivorous, feeding on other cili-
ates and on flagellates.

Four genera have been assigned to the family: Branchioecetcs Kahl (102), Dileptus
Dujardin (102, 167, 215; Fig. 7. 11, F), Paradileptus Wenrich (220; Fig. 7. II, G), and
Trachelius Schrank (102. 165; Fig. 7. II, E).

Suborder 2. TricJiostomina

The cytostome usually lies at the base of a well-defined oral groove or
pit, the wall of Avhich bears one or more dense fields of adoral cilia. Such
fields often contain free cilia. However, both adoral membranelles and
an undulating membrane (or possibly a "pseudo-membrane") have been
reported in certain genera, such as Woodruffja (96) and Colpoda (211).
In some primitive Trichostomina, the cytostome lies almost at the an-
terior pole. More often, the mouth is shifted posteriorly on the ventral
surface. Spiral torsion of the body, tending to complicate peristomial
ciliary patterns, is characteristic of certain genera. Fifteen families have
been recognized.

Family 1. Blepharocoridae. These ciliates occur in the digestive tract of
horses and ruminants. Somatic ciliation (Fig. 7. 12, A-C) is reduced to a
few anterior and posterior fields. One {Blepharocorys) or two {Charon)
groups of anal cilia lie near the posterior cytopyge, and there are two or
three distinct anterior groups. A band of slender adoral membranelles
has been reported in Blepharocorys (189). The antero-ventral cytostome
opens into a long ciliated pharynx.

Two genera are referred to the family: Blepharocorys Bundle (91, 189; Fig. 7. 12,
A, B) and Charon Jameson (91, 93; Fig. 7. 12, C).

Family 2. Clathrostomidae. The peristome is a shallow, uniformly cili-
ated ventral groove, with an oval cytostome lying in the anterior half.
The rim of the cytostome is a differentiated band from which circum-
pharyngeal fibrils extend into the endoplasm.

354 Ciliophora

Fig. 7. 12. A. Blepharocorys curvigiila Gassovsky, x480 (after Hsiung). B.
Blepharocorys equi Schumacher, x940 (after S.). C. Charon equi Hsiung,
xlOOO (after Hsiung). D-F. Clathrostoma viininale Penard (after P.), US-
125/i: ventral view (D), lateral view (E), ventrally protruded cytostome (F).
G, H. Colpoda steinii Maupas, ciliary meridians (G), ciliature (H); x750
(after Burt). I. Tillina coTialifera Turner, 150-200yLt, left lateral view showing
oral groove and ventral lip of cytostome (after T.). J, K. Woodruffia meta-
bolica Johnson and Larson: ventral view of peristomial area (J), x450;
cross-section of oral groove (K) showing undulating membrane {um), a
membranelle {m) and two somatic cilia (c), schematic (after J. & L.).

The family contains a single genus, Clathrostoma Penard (102, 165; Fig. 7. 12, D-F),
to which three fresh-water species have been assigned.

Family 3. Colpodidae. The mouth, in the anterior half of the broad oral
surface, may be funnel-shaped, approximately triangular, or sometimes
elongated. The wall of the oral groove is often more or less perpendicular

Ciliophora 355

to the body surface, while the left wall tends to slope more gradually.
The somatic ciliary rows commonly form a somewhat concentric series
around the right margin of the peristome (Fig. 7. 12, G, J), while
the organization of the adoral ciliature varies to some extent within the
family. In Woodruffia metabolica (Fig. 7. 12, K), the left margin of the
oral groove bears a row of membranelles, each composed of two or three
fused cilia, while a delicate undulating membrane extends along the right
(96). Although the undulating membrane and the membranelles are
simple in structure, this type of adoral ciliation is similar to that found
in Heterotrichina. Fission within a reproductive cyst (Fig. 7. 13, B-D) is
typical, although it has been possible to obtain fission in the active stage
in Colpoda under experimental conditions (198). The usual diets range
from small ciliates to algae and bacteria.

The following genera have been included in the family: Bresslau Kahl (39, 102; Fig.
7. 13, A), Bryophrya Kahl (102), Colpoda Miiller (14; Fig. 7. 12, G, H), Tilli?ia Gruber
(102, 213; Fig. 7. 12, I) and Woodruffia Kahl (96, 102; Fig. 7. 12, J, K).

Family 4. Conidiophryidae. These are ectoparasites which live attached
to exoskeletal hairs of amphipod and isopod Crustacea. The adult (Fig.
7. 13, E), a non-ciliated stage in a secreted membrane, produces by ter-
minal budding a series of small ciliated stages (Fig. 7. 13, F, G), or
"tomites." The migratory stage swims about until it reaches a host and
becomes impaled upon an exoskeletal hair which passes into the pharynx
(Fig. 7. 13, H). The cilia then disappear and the young parasite secretes
a membrane around itself and the distal portion of the hair upon which
it is mounted (Fig. 7. 13, I). During growth, nourishment apparently is
furnished by a secretion of the exoskeletal hair.

The family includes the genus Conidiophrys Chatton and LwofE (29; Fig. 7. 13, E-I).

Family 5. Cyathodiyiiidae. This group, from the intestine of guinea pigs,
contains several species of Cyathodinium da Cunha (131, 132; Fig. 7. 13,
J). Cilia are limited to approximately the anterior half of the body. The
non-ciliated peristome is a rather long triangular groove. From a row of
papillae along the left rim of the peristome, slender trichites ("endosprits"
of Lucas) extend into the endoplasm. Externally, an adoral cilium arises
from each papilla.

These ciliates are unusual in that fission (Fig. 2. 4, D, F) involves re-
organization with a change in polarity so that the plane of division sep-
arates the posterior ends of the two daughter organisms. Furthermore,
the parental ciliature is discarded in fission and the primordial ciliature
of each daughter develops in the endoplasm and then passes to the sur-
face, where it becomes differentiated into the ciliation of the adult (132).

Family 6. Entorhipidiidae. These ciliates occur in the digestive tract of

356 Ciliophora

Fii;. 7. 13. A. Bnsslaun sicaria Claff, Dewey and Kidder, aboial surface,
buccal region indicated in outline; x230 (after C, D. & K.). B-D. Repro-
ductive cysts in Tillina magna; macionuclei shown; xlI2 approx. (after
Beers). E-I. ConJdiophrys pilisuctor Chatton and LwofE (after C. & L.): E.
Adult stage, secreted envelope enclosing the ciliate and distal portion of an
exoskeletal "hair" of the host; contractile vacuole and field of basal granules
shown; xlOOO. F. Adult undergoing budding; xlOOO. G. Ciliated larva emerg-
ing from the parental membrane; x2()00. H. Larva impaled upon an exo-
skeletal hair which extends into the pharynx; x2000. I. Young sessile form,
cilia discarded and envelope completely formctl; xlOOO. J. Cyathodinium
piriforme da Ciniha, from guinea pig; ventro-lateral view showing peristome,
macronucleus. micronucleus, contractile vacuole; x965 (after Lucas).

sea-urchins. The body is much flattened and the anterior end forms a
frontal lobe which overhangs the cytostome (Fig. 7. 14, A, F). The somatic
ciliation is complete and essentially uniform.

The family includes Entodiscus Madsen (172, 173; Fig. 7. 14, E, F) and Entorhipidium
Lynch (136; Fig. 7. 14, A-D).

Ciliophora 357

Family 7. Isotrichidae. This family is characteristic of the ungulate
rumen, although a species of Isotricha is known from cockroaches (217).
The moiuh is terminal or subterminal and a ciliated pharynx has been
reported (Fig. 7. 15, I, L). Longitudinal striations also have been de-
scribed in the wall of the pharynx. Whether these represent trichites is
uncertain, and they may be merely fibrils of the pharyngeal ciliature. The
somatic ciliation is complete and practically uniform. A cytopyge lies at

Fig. 7. 14, A. Entorhipidium echini Lynch, upper surface; macronu-
cleus, micronucleus, trichocysts, and pharynx indicated; x203 (after L.).
B-D. Other species of Entorltipidium, xllO (after Lynch): E. tenue Lynch
(B), E. multimicnmucleatiuu Lynch (C), E. pilatum Lynch (D). E, F. Erito-
discus borealis (Hentschel), surface view (E), x270; longitudinal section (F)
through oral cavity, showing adoral cilia, the parastyle (a rod-like structure
along one margin of the peristome), and the pharynx in outline, x335
(after Powers).

the aboral end in certain species. Although the cytostome is usually con-
sidered anterior, several species swim with this end of the body directed
posteriorly (4).

Two genera have been referred to the family: Dasytricha Schuberg (4, 12; Fig. 7.
15, I, J) and Isotricha Stein (4, 51; Fig. 7. 15, K. L).

Family S. Marynidae. These are solitary or colonial ciliates with a
gelatinous lorica. The peristome partially or completely encircles the free
end of the body and extends posteriorly for some distance on the ventral
surface (Fig. 7. 15, C). The organisms swim with the aboral end forward.

358 Ciliophora

Fig. 7. 15. A, B. Paraisotricha minula Hsiung, dorsal and ventral sur-
faces; concretion-vacuole, contractile vacuole, nuclei, and cytopharynx are
shown; x443 (after H.). C, D. Maryna socialis Gruber, single specimen (150^
long) and portion of a colony (from Kahl, after G.). E. Mycterotlirix
erlangeri Lauterborn, ciliate {50fi long) enclosed in lorica (after Kahl). F.
Spirozona caudata Kahl, specimen 80/^ long (after K.). G, H. Trichospira
inversa (Clapar^de and Lachmann), ventral view of ciliate 90;^ long, periph-
eral zone of trichocysts not shown (G), and longitudinal optical section of
oral region (after Kahl). I, J. Dasytricha ruminantium Schuberg: schematic
longitudinal section (I), showing nuclei, pharynx, cytopyge, and endoplasmic
fibrils (after ten Kate); surface view (J), x465 (after Becker and Talbott)
K. Isotriclia buhali Dogiel, from camel; x325 (after D.). L. Isotricha intes-
tinalis Stein, schematic sagittal section showing pharynx, nuclei, karyophore,
and endoplasmic fibrils (after ten Kate).

Ciliophora 359

Fig. 7. 16. AC. Transverse sections of Paramecium caudatum, anterior
part of oral groove, posterior part of groove, and near the cytostome; x380
(after Wetzel). D. Vestibule and cytopharynx of Paramecium bursaria, seen
from the right side, showing the dorsal "quadripartite membrane" and the
penniculus; vestibular cilia shown only on the outer margin; xlOOO (after
V. Gelei). E-L. Various species of Paramecium, showing general form, oral
groove, position of cytopharynx, macro- and micronuclei; x500 approx.,
schematic (after Wenrich): E. P. aurelia Ehrbg.; F. P. bursaria (Ehrbg.)
Focke; G. P. calkinsi Woodruff; H. P. caudatum Ehrbg.; I. P. multimicro-
nucleatnm Powers and Mitchell; J. P. polycaryum Woodruff and Spencer; K.
P. trichium Stokes; L. P. u'oodruffi Wenrich. M. Transverse section through
Paramecium caudatum showing trichocysts, somatic cilia, double vestibular
cilia, penniculus, quadripartite membrane; pharynx is not ciliated except
for the quadripartite membrane; schematic (after v. Gelei).

360 Ciliophora

Only two genera have been recognized; Maryna Gruber (102; Fig. 7. 15, C, D) and
Mycterothrix Lauterborn (102; Fig. 7. 15, E).

Family 9. Paraisotrichidae. The mouth is subterminal, opening just
posterior to the concretion-vacuole. The somatic ciHation is complete and,
except for an anterior tuft of longer cilia, is uniform.

This family was erected by Hsiung (91) for Paraisotricha Fiorentini (Fig. 7. 15, A;
B), several species of which have been reported from the cecum and colon of horses.

Family 10. Parameciidae. An oral groove (Fig. 7. 16, A-L) extends from
the anterior end toward the middle of the body. The somatic ciliation is
complete and essentially uniform. The adoral ciliature includes a differ-
entiated dorsal zone of long cilia ("quadripartite membrane" of von
Gelei) and a penniculus (76, I3-i), a dense band of cilia which extends
in a shallow spiral toward the cytostome (Fig. 7. 16, D, M).

In addition to the genus Paramecium Hill (107. 218; Fig. 7. 16, E-L), Kahl (102) has
referred his genus Physalophrya to this family. On the basis of general similarities in
the adoral ciliature of Paramecitini and Espejoia, Faine-Fremiet (62) has suggested the
possible transfer of the genus Paramecium to the Hymenostomina.

Family 11. Plagiopylidae. These are dorso-ventrally flattened ciliates
with a central peristomial groove which lies in the anterior half of the
body and extends more or less transversely from the right margin toward
or past the sagittal plane. A dorsal non-ciliated striated band, represent-
ing a thickened strip of the pellicle, occurs in Lechriopyla and Plagiopyla
(Fig. 7. 17, F, J). The functional significance of this band is unknown.
The somatic ciliation is otherwise complete. A ciliated cytoproct has been
reported in Lechriopyla (137).

The family includes the following genera: Lechriopyla Lynch (137; Fig. 7. 17, J),
containing an intestinal parasite of sea-urchins; Plagiopyla Stein (101, 102, 137; Fig.
7. 17, F-H), including ciliates from fresh and salt water; Sonderia Kahl (101, 102, 113;
Fig. 7. 17, I) and Sonderiella Kahl (102), both represented in salt water.

Family 12. Spirozonidae.The family includes Spirozona Kahl (102, Fig.
7. 15, F). A band of closely set cilia extends from the peristome posteriorly
and spirally to the right surface of the body. In addition, the tapering
posterior end bears a tuft of caudal bristles. Otherwise, the somatic cilia-
tion is uniform. The only described species occurs in fresh water and
feeds on bacteria.

Fatnily 13. Trichopelmidae. These laterally compressed ciliates have a
firm pellicle which usually shows a few longitudinal ribs and grooves, the
latter sometimes limited to the left surface. The semicircular or crescentic
dorsal margin is smooth in outline. The mouth may lie near the anterior

Ciliophora 36:

't/TTTTV'

Fig. 7. 17. A. Trichopelma sphagnetorum Levander, x800 (after Wang
and Nie). B. Drepanomouas dentata Fresenius, 40-65/^; lateral view of com-
pressed body (after Penard). C. Microthorax viridis Penard, 35-45/x (after
P.). D. Pscudomicrothorax agilis Mermod, longitudinal cuticular ribs, peri-
stomial area with undulating membrane on left; cilia shown at margin of
body; x630 (after \Vang and Nie). E. Trimyema compressa Lackey, x640
(after Wang and Nie). F-H. Plagiopyla nasuta Stein: dorsal view (F) show-
ing striated band, trichocysts, nuclei, cytopharynx, cytopyge anterior to
contractile vacuole, x450 (after Lynch); ventral view (G) showing ciliary
pattern, x450 (after Lynch); cross-section (H) at level of cytostome, schematic
(after Wetzel). L Sonderia pharyngea Kirby, adoral ciliature, pharynx, macro-
nucleus, surface ridges from which cilia arise; neither bristles overhanging
the margin of the peristome nor a gelatinous covering of the body are
shown; x440 (after K..). J. Lechriopyla mystax Lynch, dorsal surface; tricho-
cysts, striated band, nuclei, contractile vacuole, ciliary pattern, and ciliated
cytoproct are shown; x250 (after L.).

362 Ciliophora

or the posterior end or near the equator. There are only a few rows of
somatic cilia. The Trichopelmidae resemble the Ctenostomina but lack
the adoral membranelles characteristic of these heterotrichs.

The family includes the following genera from fresh water: Drepanomonas Fresenius
(102), Microthorax Engelmann (102, 165; Fig. 7. 17, C), Pseudomicrothorax Mermod
(102; Fig. 7. 17, D), and Trichopelma Levander (102; Fig. 7. 17, A).

Family 14. Trichospiridae. This family contains the genus Trichospira
Roux (102; Fig. 7. 15, G, H). A band of densely set cilia, comparable to
that in Spirozona, extends posteriorly from the peristome but spirals to
the right instead of the left. The body does not taper to a point poste-
riorly and there are no caudal bristles. The only known species occurs in
fresh water.

Fainily 13. Trimyemidae. The only known genus is Trimyema Lackey
(Sciadostoma Kahl). Except for a caudal bristle, somatic cilia are limited
to three or four spiral rows in the anterior half of the body (Fig. 7. 17,
E). The cytostome is subterminal. Species are known from fresh and salt
water.

Suborder 3. Hymenostomina.

Adoral cilia are fused into membranes, the number, size, and arrange-
ment of which vary in different genera. The peristomial area also shows
a certain amount of variation. In some genera there is a sort of oral pouch
containing the adoral membranes (Fig. 7. 19, B, D) and opening onto the
surface of the body. In others, the adoral ciliature arises in a groove which
may be fairly long (Fig. 7. 18, L) or may even extend throughout most of
the body (Fig. 7. 18, J). Although detailed information is not available
for a number of genera, modern investigations have demonstrated sev-
eral patterns of adoral organelles as well as differences in stomatogenesis
during fission. Eventually, the accumulation of such data should lead to
needed revisions in classification.

Kahl (102) divided the suborder into five families: Cohnilembidae
(Lembidae), Frontoniidae, Ophryoglenidae, Philasteridae and Pleurone-
matidae. Of these, the Frontoniidae seem to be a particularly hetero-
geneous group which would be less so if Tetrahymena and certain related
genera were removed. Such an improvement has been effected by Mugard
(155) in transferring these ciliates to a 'Tamily Leucophrydae." Since
Corliss (42b) has concluded that none of Midler's species of Leucophra
("Leucophrys" Ehrenberg) is congeneric with any species subsequently re-
ferred to "Leucophrys," and also that Leucophra should become a genus
dubium, the "Leucophrydae" shovild be replaced by the Tetrahymen-
idae- with Tetrahymena Furgason as the type genus. This procedure

''The Family Tetrahymenidae has recently been erected by Corliss (1952. Proc. Soc.
Protozool. 3: 4).

Ciliophora 363

Fig. 7. 18. A. Frontonia lencas Ehrenberg, specimen 300/i long, showing
oral region, anterior striated band, contractile vacuole, nuclei; ciliary pattern
shown schematically at anterior and posterior ends; trichocysts indicated
along one margin (after Kahl). B. Homalogastra setosa Kahl, about SOytt long
(after K.). C. Balanoyiema dubium (Penard) Kahl, about 50/^ long (after K.).
D. Platynematum hyalinuni Kahl, specimen 60;n long (after K.). E. Cohni-
leiubus (Letnbus) punctatus Kahl, 70-120;u, (after K.). F. Saprophilus putrinus
Kahl, specimen iO/jL long (after K.). G. Contractile vacuole and accessory
canals in Urocentrum turbo; silver impregnation; x250 (after Gelei). H.
Uronema pluricaudatum Noland, left lateral view, xll90 (after N.). I.
Cryptochilidium echini (Maupas), ventral view, x460 (after Powers). J.
Lembadion bullinum Perty, ventral view; large membrane at left of oral
groove; so-called "gullet-fibrils," just beneath the wall of the oral groove, are
not shown; specimen IbOfi long (after Kahl). K. Colpidium colpoda (Ehrbg.)
Stein, specimen 150/i long (after Kahl). L. Anophrys salmicida Mugard,
showing oral ciliature and somatic pattern; x830 approx. (after M.).

364 Ciliophora

would add a sixth family to Kahl's original five, but without eliminating
the need for further study of the remaining Frontoniidae.

Family 1. CoJmilembidae. This family was erected for Cohnilembus
Kahl (102, 106; Fig. 7. 18, E). Lembadionella Kahl (106) was subsequently
referred to the family, and more recently, Anophrys Cohn (Fig. 7. 18, L)
has been added (155). The somatic cilia tion of these ciliates is complete
and rather vuiiform and the adoral ciliature consists of four membranes.
A paroral, or lateral, membrane extends along the right margin of the
elongated peristome. Three adoral membranes, which lie to the left in
the oral pouch of Tetrahymenidae (Fig. 7. 19, C), are shifted to the right
in Anophrys as a linear series parallel to the paroral membrane (Fig. 7.
18, L). There appears to be no row of somatic cilia ending at the poste-
rior margin of the oral cavity. In stomatogenesis during fission of
Anophrys (155), the adoral organelles of the posterior daughter are de-
rived from basal granules which undergo mvdtiplication at the base of the
paroral membrane. This type of stomatogenesis differs from that in the
Tetrahymenidae, as described below.

Family 2. Frontoniidae. Although defining the family as one in which
the oral cavity does not open onto a clearly defined peristome, Kahl (102)
pointed out that the lack of information concerning adoral organelles
was responsible for much uncertainty in regard to the generic composi-
tion of the Frontoniidae. Later investigations have shown that Kahl's
uncertainty was justified. Removal of the Tetrahymenidae still leaves the
residual Frontoniidae a group probably in need of further subdivision.

After elimination of certain ciliates more or less closely related to Tetrahymena
Furgason, the family includes the following genera: Aristerostoma Kahl (102), Bala-
nonema Kahl (102; Fig. 7. 18, C), Bizone Lepsi (102), Cardiostoma Kahl (102), Chas-
matostoma Engelmann (102), Cinetochilum Perty (102), Cryptochilidium Schouteden
(172; Fig. 7. 18, I), Cyrtolophosis Stokes (102), Dexiotrichides Kahl (102), Dichilum
Schewiakoff (102), Disematostoma Lauterborn (102; Fig. 7. 19, A), Epimecophrya Kahl
(106), Espejoia Biirger (66; Fig. 7. 19, I, J), Eurychilum Andre (102), Frontonia Ehrbg.
(102; Figs. 7. 18, A; 19, K, L), Frontoniella Wet/el (102). Homalogustra Kahl (102; Fig.
7. 18, B), Lanihornella Keilin (102), Leucophrydium Roux (102), Lembadion Perty
(102; Figs. 7. 18, J; 19, E), Malacophrys Kahl (102). Moiwrhihim Schewiakoff (102),
Platynematum Kahl (102; Fig. 7. 18, D), Pseudoglaucoma Kahl (102), Rhinodisculus
Mansfield (102), Saprophilus Stokes (102; Fig. 7. 18, F), Stegochilum Schewiakoff (102),
Stokesia Wenrich (219), Turaiiia Brodsky (102), Uvocentrum Nitzsch (77. 102; Figs. 7.
18, G; 19, F, G), Urojiema Dujardin (162; Fig. 7. 18, H), Uronemopsis Kahl (102),
Uropedalium Kahl (102), and Urozona Schewiakoff (102).

Family 3. Tetrahymenidae. As shown by Furgason (69), the adoral
ciliature is composed of four membranes, three adoral ones lying to the
left in the oral pouch and a paroral membrane extending along the right
margin (Fig. 7. 19, C). Another feature is the presence of one or more
ciliary rows ("stomatogenous rows") which end at the posterior margin
of the oral pouch (Fig. 7. 20, H-L). In stomatogenesis the adoral mem-

Ciliophora 365

Fig. 7. 19. A. Oral region of Disematnstoma biltschlii Lauterborn, show-
ing striated preoral band; schematic (after Kahl). B. Oral region of Loxo-
cephahis colpidiopsis; silver Impregnation, showing basal granules of three
adoral membranes in oral pouch; schematic (after Gelei). C. Oral ciliature
of Tetrahyruena pyriformis (Ehrbg.) Lwoff; silver impregnation, showing
bases of three adoral membranes (solid black) and the paroral membrane;
xl25 approx. (after Corliss). D. Oral region of Deltopylum rhabdoides Faure-
Fremiet and Miigard, showing tetrahymenal organization; schematic (after
F-F. & M.). E. Cross-section through Lembadion bullijiiim at level of cyto-
stome, showing broad and narrow membranes (compare with Fig. 7. 18, J);
x240 approx. (after Wetzel). F, G. Cross-section of Urocentrum turbo
through oral groove (F) and near level of cytostome (G); x460 (after
^V'etzel). H. Storaatogenesis in fission of Tetraliymena pyriformis. differentia-
tion of adoral organelles about completed in posterior daughter; schematic
(after Corliss). I, J. Espejoia mucicola (Penard), oral regions of microstorae
and macrostome forms, showing differences in ciliature; schematic (after
Faure-Fremiet and Mugard). K. Oral region of Frontonia leucas, silver im-
pregnation; schematic (after Klein). L. Oral region of Frontonia parva
(after Klein).

^^^ Ciliophora

Fig. 7. 20. A-G. Changes in form observed in Tetrahymena {Letico-
phrys) patula (Ehrbg.) Corliss: mature macrostome (A); reproduction in
cyst (B); excysted microstome (C); growth of mouth (D, E); young macro-
stome with ingested ciliate (F); larger macrostome (G); x325 approx.
(after Corliss). H, I. Ciliary patterns in Glaucoma scintillans and Tetra-
hymena pyriformh; x540 approx. (after Corliss). J, K. Paraglaucoma
rostrata Kahl (?), slender non-feeding stage ("theronte") and larger feeding
stage ("trophonte"), x500 approx. (after Mugard). L. Colpidium-lype of
ciliary pattern, x540 approx. (after Corliss).

branes of the posterior daughter are derived from a field of basal granules
which originate by multiplication of granules in one or more stomatog-
enous rows (Figs. 2. 4, G, H; 7. 19, H).

Certain species exhibit two morphological phases, the macrostome and
the microstome (Fig. 7. 20, A-G). The former, equipped with a large oral
pouch, is carnivorous. Both T. patula and T. vorax appear to be dimor-
phic in this sense.

The Family Tetrahymenidae is particularly important because several
species — including Tetrahymena patula (Ehrbg.) Corliss, T. pyrijorinis
(Ehrbg.) Lwoff and T. vorax (Kidder, Lilly, and Claff) Kidder — have been
established in bacteria-free cultures and are being used in physiological

Ciliophora 367

and biochemical investigations. More than twenty strains of T. pyrijormis,
whose history has been traced by Corliss (42c), are being maintained in
various laboratories.

It is not yet certain just how many of the genera in Kahl's (102) Family Frontoniidae
should be transferred to the Tetrahymenidae, but the available data suggest the fol-
lowing list: Colpidium Stein (102; Figs. 7. 18, K; 20, L), Deltopylum Faure-Fremiet and
Mugard (155; Fig. 7. 19, D), Glaucoma Ehrbg. (102, 155; Fig. 7. 20, H), possibly
Loxocephalus Eberhard (78, 102; Fig. 7. 19, B), Paraglaucoma Kahl 3 (102, 155; Fig. 7.
20, J, K), and Tetrahymena Furgason (42a, 42b, 42c. 69; Figs. 7. 19, C, H; 20, A-G, I).
Leucophra Midler ("Leucophrys" Ehrenbcrg) is a genus of very uncertain status, and
as suggested by Corliss (42b), probably will become a genus dubium. Accordingly,
Corliss (42a) has transferred L. patula, the only remaining species of "Leucophrys," to
the genus Tetrahymena. In view of the similarity of this ciliate to T. vorax, this ar-
rangement woidd seem to be a sound one imless it can be shown that T. vorax and
T. patula are not congeneric with T. pyrijormis.

Family 4. Hysterodnetidae. These are uniformly and densely ciliated,
dorso-ventrally flattened ciliates with a posterior cytostome. The peri-
stome may be merely a transverse terminal groove, along which an undu-
lating membrane extends into the short pharynx. An anterior non-ciliated
"sucker" is characteristic. These ciliates have been reported from the in-
testine of certain snails and fresh-water oligochaetes.

The family includes Hysterocineta Diesing (6; Fig. 7. 21, G-I) and Ptychostomum
Stein (84, 181, 199; Fig. 9^ 21, F).

Family 5. Ophryoglenidae. There is a ciliated vestibule (peristome), an
invagination of the body wall, and a pharynx which opens into the ves-
tibule. The vestibule is relatively deep in Ophryoglena, less so in Proto-
phryoglena, and much reduced in Ichthyophthirius (155). A spiral ridge
extends from the opening of the vestibule into the pharynx in Ophryo-
glena. According to Mugard (155), a tetrahymenal organization of the
oral membranes is characteristic. The membranes curve along the spiral
ridge before entering the pharynx in Ophryoglena but follow a less curved
path in Protophryoglena and almost a straight course in Ichthyophthirius.
In stoma togenesis, the oral membranes are derived from basal granules
in a number of stomatogenous rows. A refractile "body of Lieberkiihn"
("watch-glass body"), usually accompanied by a mass of pigment, lies just
to the left of the vestibule.

The life-cycles follow a general pattern (155). Reproduction occurs
within a cyst (Fig. 7. 21, A). The young ciliates ("tomites"), after excyst-
ment, develop into the trophic stage ("theronte") which, in free-living
species, grows into a large slowly swimming stage ("trophonte") with
much stored food. This mature stage then encysts in preparation for re-

^ The status of the genus Paraglaucoma is rather confused at present, since Corliss
(1952. Proc. Soc. Protozool. 3: 3) has concluded that Paraglaucoma rostrata (the type
species) is congeneric with Tetrahymena pyrijormis.

368 Ciliophora

Fig. 7. 21. A. Reproductive cyst of Ichthyophthirius multifiliis Fouquet,
cross-section, nuclei indicated but cilia not shown; x450 (after MacLennan).
B. Ciliated larva of /. multifiliis just after excystnient; "perforatoriinn" at
anterior end, cytostome not differentiated; x750 (after MacLennan). C.
Longitudinal section through oral region of matme specimen of /. multifiliis,
embedded in epithelium of fish; x370 (after MacLennan). D, E. Ophryoglena
atra Lieberkiikn: ventral view (D) of specimen 400/i long; schematic repre-
sentation (E) of vestibular ciliatuie and body of Lieberkiihn (after Rahl).
F. Ptychostomum (Lada) pygostoma (Rossolimo); length reaches 200^; un-
dulating membrane extends through posterior cytostome into pharynx
(after R.). G-L Hysterocineta eiseniae Beers (after B.): ventral view of
posterior end (G), showing undulating membrane extending along the
peristomial groove and into the pharynx, x600; dorsal view (H), the ventral
sucker shown in outline at the anterior end, x285; lateral view (I), x285.

production. In Ichthyophthirius (145), fission produces many (100-1,000)
small ciliates which are set free with an incompletely developed mouth.
These active stages (Fig. 7. 21, B) swim about until they either starve to
death or encounter a suitable fish. In the latter case, the ciliate apparently
bores its way into the tissue as a result of strong ciliary action. After pene-

Ciliophora 369

Fig. 7. 22. A. Pleuronema setigerum Calkins; large paroral membrane
extends around posterior end of peristome; cilia shown at margin of body;
x450 (after Noland). B. Cristigera setosa Kahl, specimen iOfi long (after K.).
C. Cross-section of Pleuronema chrysalis through posterior end of peri-
stomial gioove; x510 (after Wetzel). D. Histiobalantium semisetatuin Noland,
inclusions omitted; x200 (after N.). E. Cledoctema acanthocrypta Stokes,
specimen 35yu, long (after Kahl). F. Cyclidium glaucoma O. F. M., cilia
shown only at margin; xl200 (after Parducz). G, H. Porpostoina notatum
Mobius: nuclear apparatus (G), xlUO; pcristomial area (H), anterior ciliary
field broken up into a linear series of pseudomembranelles, schematic, x500
(after Mugard). I. Peristomial area of Philasterides armata Kahl, showing
deltoid, trapezoid, and falciform ciliary fields; x500 (after Mugard). J.
Philaster digitiformis Fabre-Domergue, somatic and adoral ciliary patterns;
x500 (after Mugard).

370 Ciliophora

tration, the young parasite develops a functional cytostome, feeds partly
on fragments of epithelial cells, and grows to a diameter of 100-I,000[jl.
The mature ciliate then drops off the host and encysts.

Three genera have been referred to this family: Ichthyophthirius Fouquet (83, 145,
155; Fig. 7. 21, A-C), Ophryoglena Ehrenberg (155; Fig. 7. 21, D, E), and Proto-
phryoglena Mugard (155).

Family 6. Philasteridae. Members of this family are elongated ciliates
with a long and approximately triangular peristomial groove. Although
the adoral ciliature is basically "tetrahymenal," the three adoral mem-
branes are replaced by three ciliary fields — the deltoid, trapezoid, and
falciform fields (Fig. 7. 22, H-J) of Mugard (155). However, the paroral
membrane persists as such, extending part way along the right margin
of the peristome. Stomatogenesis involves multiplication of basal granules
at the jDOsterior end of the paroral membrane.

The following genera, represented by species in salt or brackish water, are referred
to the family: Helicostoma Cohn (102), Paralembus (Lentboides) Kahl (102), Philaster
Fabre-Domergue (155; Fig. 7. 22, J), Philasterides Kahl (155; Fig. 7. 22, I), and Porpo-
stoma Mcibius (155; Fig. 7. 22, G, H).

Family 7. Pleuronematidae. The paroral membrane is much enlarged
and may extend around the posterior margin of the peristome (Fig. 7.
22, A-F). The rest of the adoral ciliature is less uniformly developed and
may be represented by a single membrane at the left of the peristome or
by a field of cilia in this region (102). In Balantiophorus (216) a con-
tinuous membrane extends along the right, posterior, and left margins of
the peristome. When fully extended, the membrane forms a sac-like struc-
ture covering the peristome except at the anterior end. In certain Pleuro-
nematidae, some of the dorso-lateral cilia are thigmotactic and these
ciliates often become attached momentarily to a solid surface. One or
more long caudal cilia also are often present.

The Pleuronematidae, which are represented in fresh and salt water, include the
following genera: Balantiophorus Schewiakoff (216), Calyptotricha Phillips (102),
Cristigera Roux (102; Fig. 7. 22, B), Ctedoctema Stokes (102; Fig. 7. 22, E), Cyclidium
Midler (163; Fig. 7. 22, F), Histiobalantium Stokes (102, 158; Fig. 7. 22, D), LarvuUna
Penard (102, 165), Pleurocoptes Wallengren (102), Pleuronema Dujardin (102, 158;
Fig. 7. 22, A, C).

Suborder 4. Thigmotrichina

These ciliates occur mostly in the mantle cavity or on the gills and
palps of bivalve molluscs, although species are known also from the
mantle cavity of pulmonale snails and from the tentacles of Fhoronopsis.
An anterior field of thigmotactic cilia is a general characteristic. A cyto-
stome and the adoral ciliature, if present at all, lie in the posterior half

Ciliophora 371

of the body, and in certain species, the cytostome lies at the posterior
pole. Some specialized genera have lost the cytostome and developed an
anterior suctorial tentacle. Except in primitive types, there is reduction
of the somatic ciliature and this trend reaches a climax in genera which
retain only the thigmotactic cilia.

Chatton and Lwoff (30) have listed six families. In three ("Tribe
Stomodea") — Conchophthiriidae, Hemispeiridae, Thigmophryidae — the
cytostome is functional and the adoral ciliature is recognizable. In the
others ("Tribe Rhynchodea") — Ancistrocomidae, Hypocomidae, Spheno-
phryidae — the adoral ciliature has undergone regression and there is no
functional cytostome, although a suctorial tentacle serves for the ingestion
of food much as in the Suctorea.

Family 1. Ancistrocomidae. These ciliates are ovoid, pyriform, or some-
what cylindrical, typically with a more or less pointed anterior pole. The
closely set thigmotactic cilia, not as long as the somatic cilia, are limited
to a few short anterior fields. The body is extensively ciliated in certain
genera; in others, cilia may be limited to the thigmotactic fields. An an-
terior suctorial tentacle (Fig. 7. 23, B, G), continuous with an internal
canal, is characteristic. This tentacle enables the organism to become
attached to epithelial cells and ingest their contents.

The following genera have been referred to the family (31): Ancislrocoiua Chatton
and Lwoff (— Parachaenia Kofoid and Bush) (128; Fig. 7. 23, H). Anisocomides Chatton
and Lwoff (31), Cepedella Poyarkoff (31), Crebicoma Kozloff (126; Fig. 7. 23, A),
Enerthecoma Jarocki (129; Fig. 7. 23, J), Goniocoma Chatton and Lwoff (31), Hetero-
cineta Mawrodiadi (125, 129; Fig. 7. 23, G), Heterocinetopsis Jarocki (31), Holocoma
Chatton and Lwoff (31), Hxpoconiagalma Jarocki and Raabe (128), Hypoconiatidium
Jarocki and Raabe (31; Fig. 7. 23, E), Hypocoinella Chatton and Lwoff (31),
Hypocomoides Chatton and Lwoff (127; Fig. 7. 23, B), Hypocomidium Raabe
(31), Hypocomina Chatton and Lwoff (126; Fig. 7. 23, D), Insignicotna Kozloff (127;
Fig. 7. 23, C), Isocomides Chatton and Lwoff (31), Raabella Chatton and Lwoff (31),
Syri7igupliary)ix Collin (31, 106).

Family 2. Conchophthiriidae. There is a functional cytostome in the
posterior half of the body. The thigmotactic cilia are represented by the
anterior portions of somatic rows instead of forming separate fields.
The body is laterally compressed and the cytostome lies on the narrow
ventral surface (102).

The following genera have been referred to this family: Atidreula Kahl (106),
Cochliophilns Kozloff (124; Fig. 7. 23, I), Conchophthirius Stein (106, 111; Fig. 7. 23,
F), Kidderia Raabe (46, 108).

Faynily 3. Hemispeiridae. These are rather heavily ciliated types with a
posterior or subterminal cytostome. The adoral ciliature shows two typical
components (Fig. 7. 24, B). On the right a single row, row 1, curves
slightly to the left throughout most of its length and then sharply to the

372 Ciliophora

Fig. 7. 23. A. Crebicoma carinata (Raabe) Kozloff, ventral surface, x565
(after K.). B. Hypocomoides mytili Chatton and Lwoff, suctorial tentacle
slightly protruded at anterior end; x935 (after Kozloff). C. Insignicoma
venusta Kozloff. xl495 (after K.). D. Hypncomina tegnlarum Kozlolt, xl400
(after K.). E. Hypocomatidium sphaerii Jarocki and Raabe; three fields of
basal granules in the ventral thigmotactic area; silver impregnation, x900
(after J. & R.). F. Conchoplitliirius anudontae Stein; upper surface, showing
peristomial area and .slender pharynx; x300 (after Kidder). G. Heterocinela
phoroiiopsidis Kozloff, view of right side showing macronucleus, contractile
vacuole, and internal canal continuous with the suctorial tentacle; xl455
(after K.). H. Ancistrocoma dissimilis Kozloff, xl345 (after K.). I. Cochlio-
philus depressus Kozloff, dorsal view, x450 (after K.). J. Enerthecoma pro-
perans Jarocki, xl290 (after Kozloff).

Ciliophora 373

Fig. 7. 24. A, B. Hemispeira asteriasi Fabre-Domergue, view of right side
(A), x600; adoral ciliature and adjacent ciliary rows (/, 2, n and ii-l, first,
second, last, and next to last somatic rows of cilia; A, B, adoral cilia),
sciiematic (after Ciiatton and Lwoff). C. Sphenophrya dosiniae Chatton and
Lwoff, showing long narrow sucker on upper surface, nuclei, rows of basal
granules; length reaches 120^ (after C. & L.). D, E. C.argarius gnrgarius
Chatton and Lwoff: ciliary pattern (D); view showing papillae and nuclei
(E); x900 (after C. & L.). F. Cheissinia (Tiarella) haicalensis (Cheissen)
Chatton and Lwoff, adoral membrane projecting beyond posterior rows of
cilia; length, 50-72/^ (after Cheissin). G. Ancistrum pcriiix (MacLennan and
Connell) Chatton and Lwoff, longitudinal optical section showing nuclei and
pharynx; x490 (after MacL. & C). H. Hypocoma parasitica Gruber, dorso-
lateral view, xI350 (after Chatton and Lwoff). I, J. Boveria teredini Nelson,
dorsal side (I), x465; posterior end (J), showing basal granules of adoral
zone, x620 approx. (after Pickard).

374 Ciliophora

left at the cytostome. At the left of row 1 a double row of cilia (row B)
follows much the same path but does not extend so far posteriorly. In
addition, a short row of cilia lies near the anterior end of row B in some
species and there are also several rows of cilia in the pharynx (30). This
pattern varies in details in different species. The thigmotactic cilia of
some genera are merely the anterior cilia in normal rows. In others, the
posterior somatic cilia have disappeared, leaving only the thigmotactic
cilia.

The family includes the following genera: Ancistrella Cheissen (37), Ancistrospira
Chatton and Lwoff (30), Ancistrum Maupas (30, 109; Fig. 7. 24, G), Boveria Stevens
(142, 169; Fig. 7. 24, I, J), Cheissinia Chatton and Lwoff (30, 37; Fig. 7. 24, F),
Hemispeira Fabre-Domeigue (30; Fig. 7. 24, A, B), Plagiospira Issel (30, 102), Pro-
boveria Chatton and Lwoff (30), Protophrya Kofoid (30).

Family 4. Hypocornidae. These are ovoid to somewhat flattened ciliates
in which cilia are limited to the dorsal surface. An anterior or antero-
dorsal suctorial tentacle is present and there is no cytostome. However,
there is an antero-lateral field of supposedly vestigial adoral cilia (31).

Three genera have been characterized by Chatton and Lwoff (31): Heterocoma
Chatton and Lwoff, Hypocotna Gruber (Fig. 7. 24, H) and Parahypocoma Chatton and
Lwoff.

Family 5. Sphenophryidae. The adult stage is not ciliated although in
reproduction the "embryo" develops cilia and resembles the more spe-
cialized Ancistrocomidae (31). The suctorial tentacle is generally shorter
than that of the Ancistrocomidae and Hypocomidae and tends to be
funnel-shaped.

Three genera have been recognized: Gargarius Chatton and Lwoff (27, 31; Fig. 7.
24, D, E), Pelecyophrya Chatton and Lwoff (31) and Sphenophrya Chatton and Lwoff
(31; Fig. 7. 24, C).

Family 6. Thigynophryidae. The ciliation is essentially uniform. The
thigmotactic field is represented by short, closely set cilia in various
somatic rows (31). Conchophyllum caryoclada (Kidder) Raabe shows an
unusual branched macronucleus (HO).

Three genera have been referred to the family: Conchophyllum Raabe (110),
Myxophyllum Raabe (106), and Thigmophrya Chatton and Lwoff (22).

Suborder 5. Apostomina

This group was established (24) for a number of ciliates with a
ventral cytostome so small that ingestion is probably limited to liquids
or minute particles. A peculiar "rosette" is characteristic of the ventral
surface (26, 28, 45). In Foettingeria (Fig. 7. 25, A-D, F) the cytostome lies

Ciliophora 375

Fipf. 7. 25. AD. The rosette in Foettingaria actinarium; superficial and
progressively deeper optical cross-sections (A-C), showing septa and cilia;
vertical optical "dissection" (D), showing septa and cilia and portion of the
oral pouch below; schematic (after Chatton and Lwoff). E. Young ciliated
stage ("tomite") of Spirophrya subparasitica Chatton and LwofE, ventral
surface showing rosette and cilia; x600 (after C. & L.). F, G. Foettingaria
actinarium (Clapartde) Caullery and Mesnil: ventral side showing rosette
and basal granules (F), xl70; young ciliate (G) ready to leave cyst, x570
(after De Morgan). H, I. Chromidina elegans (Foettinger) Gonder, outline
of "tropho-tomonte," x250; dorsal surface of "tomite," xlOOO (after Chatton
and LwofF). J. Synophrya hypertrophica Chatton and Lwoff, ventral surface
of young "trophonte," x800 (after C. & L.). K. Sessile stage ("phoronte")
of Foettingaria actinarium, x990 (after Chatton and Lwoff). L. Gymno-
dinioides inkystans Chatton and Lwoff, ventral surface of "trophonte,"
xl400 (after C. & L.). M. Reproductive cyst of Spirophrya subparasitica,
x530 (after Chatton and Lwoff).

376 Ciliophora

at the end of a groove, in a small depression next to the rosette, and opens
into a canal which leads inward, along the concave wall ("typhlosole")
of the rosette, to an "oral pouch." The rosette contains 8-10 vertical septa
which join the outer wall to the "typhlosole," and the base of the typhlo-
sole is equipped with a ring of cilia (Fig. 7. 25, C). De Morgan (45)
never observed movement of the rosette in living F. octiyioriiim and ob-
tained no clvies in regard to its functions. There are fewer than 22 com-
plete rows of somatic cilia but their exact number and the organization
of the adoral ciliature vary in different genera.

A complex life-cycle is characteristic (28, 1,H5). In the growth-stage, or
"trophonte" (Fig. 7. 25, F, J, L), food is ingested and accumulated during
growth but there is no reproduction. At maturity, the "trophonte" stops
feeding, and with or without encystment in different species, becomes
transformed into a "protomonte." If the "protomonte" is an encysted
stage, the cilia are discarded. The "protomonte" develops into a "to-
monte" in which the accumulated food is transformed into stored reserves.
Then repeated fission occurs to produce many small "protomites" (Fig.
7. 25, M), each of which becomes an actively swimming "tomite" (Fig. 7.
25, E, G, I). This migratory stage becomes attached to the body of a host,
usually a crustacean. However, Chromidina elegans has been reported
from the kidney of a cephalopod (222). After attachment, the "tomite"
develops into a resting cyst, or "phoronte" (Fig. 7. 25, K). When the host
is ingested by a coelenterate or a ctenophore, or when the host molts, a
young "trophonte" emerges from the cyst. The vegetative stage ("tropho-
tomonte") of Chromidina elegans is unusual in that it may undergo fission
to form chains (222) similar to those produced by Astomina.

A detailed survey of the Apostomina has been published by Chatton and Lwoff (28)
who have characterized the following genera: Calospira Chatton and Lwoff, Chromidma
Gonder (222; Fig. 7. 25, H, I), Foettingaria Caullery and Mesnil (45; Fig. 7. 25, F, G,
K), Gymnodinioides Minkiewicz (135; Fig. 7. 25, L), Ophiuraespira Chatton and Lwoff,
Pericaryon Chatton, Plioretophrya Chatton and A. and M. Lwoff, Phtorophrya Chatton
and A. and M. Lwoff, Polyspira Minkiewicz, Spirophrya Chatton and Lwoff (Fig. 7.
25, E, M), Synophrya Chatton and Lwoff (Fig. 7. 25, J), Traumatiophora Chatton and
Lwoff, Vampyrophrya Chatton and Lwoff. The genus Chromidina also has been assigned
to the Astomina by some workers.

Suborder 6. Astomina

These are parasites without a cytostome. The body is often uniformly
ciliated but there is sometimes a small naked area at the anterior pole.
The average length, for the majority, probably falls within the range,
200-500[jL, but such species as Haptophrya gigantea, H. michiganensis
(225), and Mesnilella radiata (36) may reach lengths of 1.5-2.0 mm. The
cortex shows little specialization, although it ranges from a very thin
zone in some coelozoic species to a layer 1.0-2.0[i, thick in certain intestinal
parasites.

Ciliophora 377

Fig. 7. 26. A-F. Formation of chains in Astomina: Haptophrya-type (A-
C), Radiophrya-type (D-F), schematic (after Cheissin). G, H. Biitschliella
nosuta RossoHnio; specimen showing nuclei and three contractile vacuoles
(G); anterior end showing ciliation (H); length reaches 200fi (after R.). I.
Anoplophrya garmnari Cheissin, x490 approx. (after C.). J, K. Haprophrya
wichiganensis Woodhead: ventral view showing macronucleus, contractile
canal and sucker (J), x265; section (K) showing sucker attached to intestinal
epithelium of salamander, x360 (after Bush). L. Perseia dogieli Rossolimo;
length, 114-205/^ (after R.). M, N. Buclineriella criodrili Heidenreich; length,
110-22(V; stained specimen (M); skeletal apparatus (N), schematic (after
H.). O, P. Metaradiophrya asymmetrica Beers: ventral view (O) showing
nuclei, eight contractile vacuoles, antero-ventral fibrils and hook; lateral
view of anterior end (P); x400 (after B.).

378 Ciliophora

A number of the Astomina have developed holdfast organelles. Such
structures usually lie at or near the anterior pole which is often in con-
tact with an epithelium of the host. An antero-ventral sucker (Fig. 7.
26, J, K) is characteristic of certain intestinal species. In various other

'MM

Fig. 7. 27. A, B. Holdfast apparatus of Radiophrya lumbrici Cheissin,
ventral view (A), sagittal section through anterior end of body (B), x650
(after C). C. Radiophrya hoplites Rossolimo, primite with three satellites;
xlOO (after Cheissin). D, E. ProtoradiopJirya fissispiciilata Cheissin, primite
and satellite (D), x375; tangential section of anterior end (E), showing
spicules, x975 (after C). F, G. Mrazekiella intermedia Cheissin: entire spec-
imen (F), x240; holdfast apparatus (G), x975 (after C). H-J. Mestiilella
miiltispiculata Cheissin: ventral view (H) showing nuclei, skeletal spicides
and row of contractile vacuoles, x210; transverse sections near anterior ends
(I) and near posterior ends (J) of the spicules, xl650 (after C). K. Anterior
end of HopUtophrya secans Stein, showing skeletal apparatus; x900 (after
Heidenreich).

Astomina the anterior end is equipped with an apparatus composed of
barbs or spicules (Figs. 7. 26, N, P; 27, B, K; 28, C, G). In addition, skeletal
fibrils of unknown function may extend for some distance near the sur-
face of the body (Fig. 7. 27, H-J) and sometimes pass from the cortex
into the endoplasm (183).

Ciliophora 379

Contractile vacuoles — or sometimes a contractile canal (15, 147) as in
Haptophrya (Fig. 7. 26, J) — are generally present. There may be one
contractile vacuole or, at the other extreme, many vacuoles arranged in
one or more longitudinal rows (Fig. 7. 26, L, O).

Little is known about the life-cycles of Astomina. Most species are
known from oligochaetes; a few, from amphipod Crustacea and from the
digestive tracts of Turbellaria and Amphibia. Reproduction may involve
typical binary fission, fission in which one daughter organism is a little
smaller than the other, or consecutive fissions leading to the production
of chains (Fig. 7. 26, A-F). In some chains both the anterior (primite)
and the posterior (satellite) organisms undergo repeated fission; in others,
the primite produces several satellites without undergoing a reduction in
size.

The families described below represent five of the six recognized by
Cheissin (36). Other workers have subdivided the Astomina in somewhat
different fashion.

Family 1. Anoplophryidae. The body may be ovoid or distinctly elon-
gated and the cilia are arranged in longitudinal rows. A poorly developed
sucker is commonly present but skeletal elements are typically absent.
There may be one, two, or more contractile vacuoles, or sometimes none.

Cheissin (36) has assigned the following genera to the family: Anoplophrya Stein
(41, 84, 201; Fig. 7. 26, I), BiltschUeUa Awerinzew (84; Fig. 7. 26, G, H), Dogielella
Poljansky, Herpetophrya Siedlecki, Kofoidclla Cep^de, Metaphrya Ikeda, Orchitophrya
CepMe, Perezella Cepcde, Perseia Rossolimo (181; Fig. 7. 26, L), Protoanoplophrya
Mijaschita, Rhizocarium Caullery and Mesnil.

Family 2. Haptophryidae. A long contractile canal, instead of separate
contractile vacuoles, is characteristic. An antero- ventral sucker is present
in some species. Spicules or hooks may or may not be present at the
anterior end.

The following genera have been referred to the family: Haptophrya Cep^-de (15,
147, 225; Fig. 7. 26, J, R), Laclnimnnelln CepMe and SteineUa Cepcde.

Family 3. Hoplitophryidae. These Astomina are ec[uipped with a hold-
fast apparatus, longitudinal supporting spicules, or both types of struc-
tures. There may be several to many contractile vacuoles.

The family includes the following genera: Buchneriella Heidenreich (84; Fig. 7. 26,
M, N), Hoplitophrya Cepcde (85; Fig. 7. 27, K), Mesnilella CepMe (36, 84, 183; Fig. 7.
27, H-J), Metaradlophrya Heidenreich (5, 84; Fig. 7. 26, O, P), Mrazekiella Kijenskij
(36, 84; Fig. 7. 27, F, G), Protoradiophrya Rossolimo (36, 183; Fig. 7. 27, D, E), Radio-
phrya Rossolimo (36, 183; Fig. 7. 27, A-C).

Family 4. Intoshellinidae. These are elongated ciliates with longitudinal
or spiral rows of cilia. There is a holdfast apparatus in the form of a

380 Ciliophora

spiny collar or a toothed disc, the area anterior to which is non-ciliated.
A number of contractile vacuoles are arranged in one or two longitudinal
rows or distributed irregularly. Chains are usually formed.

Two genera have been referred to the family: Intoshellina Cep^de (36, 84; Fig. 7.
28, A-C) and Monodontophrya Vejdowsky (36; Fig. 7. 28, D, E).

Fig. 7. 28. A-C. Intoshellina poljansky Cheissin: primite and satellite
(A), x225; holdfast apparatus (B, C), polar and ventral views, x975 (after
C). D, E. Monodn?itoplirya kijenskiji Cheissin: anterior end (D), showing
thick ectoplasmic cap and holdfast organ, x650; lateral view (E), x75 (after
C). F, G. Maupasella criodrili Heidenreich, 50-1 50/i long; entire specimen
(F), skeletal apparatus (G), x3300 (after H.).

Family 5. Manpasellidae. Little is known about this group which con-
tains only two genera, Maupasella Cepede (84; Fig. 7. 28, F, G) and
Schulzellina Cepede. These ciliates are similar to the Hoplitophryidae
and Cheissin (36) has suggested that further investigation might justify
combining the two families.

In addition to the five families described above, Cheissin (36) included
the Chromidinidae, containing the genus Chromidina Gonder (= Opali-
nopsis Foettinger). Chatton and Lwoff (28), on the other hand, concluded
that Chromidina belongs in the Apostomina.

Order 2, Spirotrichida. The most characteristic feature is the adoral
zone of membranelles, the narrow bases of which usually lie at right or
oblique angles to the long axis of the adoral zone. This series of mem-
branelles extends anteriorly from the left margin of the cytostome, and
in certain genera, may turn dorsally at the anterior pole and extend to
the right for some distance along the antero-dorsal surface. The basal

Ciliophora 381

plate of each membranelle usually contains two rows of basal granules,
although three rows (rarely, four) may be present (104). The group may
be divided into six suborders.

Suborders of the Spirotrichida

Suborder I. Heterotrichina. Somatic ciliation is usually complete. How-
ever, the dorsal surface may be sparsely ciliated in some families and
shows more extensive reduction of ciliation in exceptional cases. The
peristome, usually elongated and fairly narrow, bears the adoral zone
of membranelles along the left wall. In addition, an undulating mem-
brane often extends for some distance along the right margin.

Suborder 2. Oligotrichina. Although the adoral membranelles are well
developed, there is a marked reduction in somatic ciliation and the peri-
stomial field, around which the adoral zone extends, is free from cilia.
An undulating membrane lies at the right margin of the adoral area in
certain genera.

Suborder 3. Tintinnina. These ciliates, sometimes grouped with the
Oligotrichina, are typically conical forms with a lorica. The adoral zone
of membranelles follows a spiral course on the flattened oral pole.

Suborder 4. Entodiniomorphina. This group, sometimes placed in the
Oligotrichina, includes parasites of the rumen and intestine of herbivores.
The ciliation may be limited to the adoral zone or there may be one or
more additional bands or gioups of membranelles.

Suborder 5. Hypotrichina. The somatic cilia are replaced by cirri which
are generally distributed in particular fields and limited essentially to the
ventral surface.

Suborder 6. Ctenostomina. These are laterally compressed, wedge-
shaped ciliates with a rigid pellicle decorated with longitudinal ribs. The
body is sparsely ciliated, and the peristome is a pouch containing an
adoral zone of eight membranelles.

Suborder 1. Heterotrichina

Since the somatic ciliation is practically complete in the majority, these
ciliates are usually considered the most primitive Spirotrichida. However,
there is a trend toward reduction of the dorsal ciliature in some genera,
and the Peritromidae are ciliated only on the ventral surface. In addition
to the adoral zone of membranelles on the left, there is often an undulat-
ing membrane at the right of the peristome. This membrane is sometimes
replaced by a double row of heavy cilia. Thirteen families have been
recognized.

Family 1. Balantidiidae. This family includes Balantidiiun Claparede
and Lachmann (130, 139, 174, 214; Fig. 7. 29, A-E), represented by para-
sites of the digestive tract in both vertebrates and invertebrates. Somatic
ciliation is complete and the cilia are arranged in approximately longi-

382 Ciliophora

Fig. 7. 29. A. Balantidium praenucleatum Kudo and Meglitch, longitu-
dinal optical section; Feulgen preparation; x475 (after K. & M.). B, C.
Transverse sections of Balantidium coli: anterior region showing peristome
(B); more posterior section through the pharynx (C), x550 (after Rees). D,
E. Balantidium sushilii Ray: longitudinal section through peristome (D),
x750; transverse section (E) through peristome near anterior end, x540 (after
R.). F-H. Transverse sections of Bursaria truncatella O. F. M.: anterior por-
tion of deep peristome (F), near posterior end of peristome (G), and at
level of cytostome (H); x96 (after Wetzel). I. Bursaria truncatella, ventral
view of specimen 600yu long (after Kahl). J. Bursaridium pseudobursaria
(Faure-Fremiet) Kahl, ventral view; part of the dorsal peristomial wall is
striated but not ciliated; x260 (after Wang and Nie). K. Chattonidium
setense Villeneuve, membranelles and undulating membrane, macronucleus,
postero-axial cavity (cytoproct?); xl88 (after Villeneuve-Brachon). L. C.
setense, polar view, membrane, bases of membranelles; x250 (after V-B.).

Ciliophora 383

tudinal rows. The peristome is a pouch with a triangular opening,
through which the short adoral band of membranelles is not easily
recognizable from the outside (214). Numerous long fibrils extend into
the endoplasm from the basal granules of cilia and membranelles.

Family 2. Bursariidae. The most characteristic feature is a large funnel-
shaped peristome, closed ventrally throughout part or most of its length
(Fig. 7. 29, F-I). This ventral closure is perhaps the result of overgrowth
of the "oral lip," a plate-like extension of the body wall which extends
mediad from the right margin of the peristome in various Heterotrichina.
In the Bursariidae this extension presimnably has fused with the right
margin of the peristome. The undulating membrane has disappeared in
most species.

Three genera are referred to the family: Bursaria Miiller (134; Fig. 7. 29, F-I),
Bursaridium Lauterborn (104; Fig. 7. 29, J), and Thylacidium Schewiakoff (104).

Family 3. Chattonidiidae. These ciliates show a superficial resemblance
to the Oligotrichina in that the peristome is shifted to the anterior pole.
The membranelles form an almost complete spiral around the margin of
the peristomial funnel, at the base of which lies the cytostome (Fig. 7.
29, K). Within the ring of membranelles, an undulating membrane ex-
tends for some distance around the peristome. The somatic ciliation is
uniform.

The genus Chattonidium Villeneuve (214; Fig. 7. 29, K, L) is the only one assigned to
the family.

Family 4. Clevelandellidae. These are completely ciliated heterotrichs
which taper toward the anterior (aboral) pole. A zone of membranelles
extends into the funnel-shaped peristome to the pharynx (Fig. 7. 30,

G, N).

The family includes two genera, both represented by species in the digestive tract
of wood roaches (Panesthia): Clevelandella Kidder (112; Fig. 7. 30, E, F, N) and
Paradevelandia Kidder (112; Fig. 7. 30, G).

Family 5. Condylostomidae. The large broad peristome is bordered on
the left by the adoral zone of membranelles (Fig. 7. 30, D). On the right,
a long undulating membrane arises from a groove hidden by a ventral
ledge ("oral lip"). This ledge is extended to the left in several species to
form a floor for part of the peristomial cavity. On the antero-ventral
surface, there is sometimes a progressive replacement of simple cilia by
fused groups of cilia, culminating in a zone of cirri at the right margin of
the peristome (Fig. 7. 30, C).

384 Ciliophora

Fig. 7. 30. A. Transverse section of Condylostonia vorax through the un-
dulating membrane and a membranelle; schematic, x800 (after Villeneuve-
Brachon). B. Lorica of Pamfolliculina liinindo (Kent) Kahl, 125^ long
(after K.). C. Antero-ventral region of Condylostoma arenarium showing
undulating membrane and the transition from somatic cilia to cirri; x250
(after Villeneuve-Brachon). D. Condylostoma vorax Villeneuve-Brachon,
ventral view, x250 (after V-B.). E, F. Transverse sections of Clevelandella
elongata: through pharynx (E), x525; through peristome (F), x640 (after
Kidder). G. Paraclevelandia hrevis Kidder, ventral view; karyophore attached
to macronucleus anteriorly; xl230 approx. (after K.). H. Lorica of Micro-
folliculma Umnoriae (Giard) Kahl (after K.). I. Migratory larva of Follic-
ulina aculeata, xl50 (after Dewey). J. Folliculinopsis producta (Wright)
Villeneuve-Brachon, extended specimen showing macronuclear chain; x300
(after V-B.). K. Lorica of FollicuUna viridis (AVright) Kahl, 150^ long (after
K..). L. Polar view of peristome in Folliculinopsis producta, showing bases
of adoral membranelles and rows of somatic cilia; x300 (after Villeneuve-
Brachon). M. Lorica of Pseudofolliculina arctica (Dons) Kahl, about 430ju
long (after K.). N. Clevelandella elongata Kidder, ventral view, x302
(after K.).

Ciliophora 385

The genus Condylostoma Bory (13, 104, 214; Fig. 7. 30, A, C, D) is the only one
assigned to the family.

Family 6. Folliculinidae. These are widely distributed marine ciliates
which live attached to various plants and animals. The body is enclosed
in a relatively thin "pseudochitinous" lorica. At the oral pole, the body
is extended into two mobile lobes traversed by a spiral zone of mem-
branelles extending down to the cytostome (Fig. 7. 30, L). The rest of
the body is rather uniformly ciliated. Upon completion of fission in
Folliculina, the anterior daughter leaves the lorica as a free-swimming
larva (Fig. 7. 30, I) which, after a short migratory period, becomes
attached and secretes a lorica (47).

The following genera, which are distinguished mainly by differences in general form
of the lorica, have been recognized: Folliculina Lamarck (1, 47, 56, 104; Fig. 7. 30, K),
Folliculinopsis Faure-Fremiet (57. 214; Fig. 7. 30, J, L), Metajolliculina Dons (104),
Microfnlliculina Dons (104; Fig. 7. 30, H), Pnrafolliculina Dons (104; Fig. 7. 30, B),
Pehrilla Giard (104), and Pseudojolliculina Dons (104; Fig. 7. 30, M).

Family 7. Liclmophoridae. Both ends of the elongated body are dis-
coidal, while the mid-region is somewhat constricted. The zone of mem-
branelles surroimds most of the antero-ventral, or oral, disc and extends
into a depression of the peristome. The posterior disc (basal disc) is sur-
rounded by several concentric undidating membranes, and just anterior
to these, by a flexible flange, or velum. The basal disc is somewhat ctip-
shaped and serves for attachment lo the host. With one or two exceptions,
the Liclmophoridae are marine ectocommensals.

There is only one known genus, Lichnophora Clapar^de (3; Fig. 7. 31, J).

Family 8. Metopidae. These are often uniformly ciliated heterotrichs in
which the peristome tends to curve to the right posteriorly. The zone of
membranelles is rather straight in primitive Metopidae but spiral torsion
may be marked, as in Caeiiomorpha (Fig. 7. 31, G). A relatively short
undulating membrane often extends along the right margin of the
peristome. Trichocysts may be present, sometimes underlying pellicular
ridges or bands separating the rows of cilia.

The Metopidae, represented in fresh and salt water, include the following genera:
Bryoinetopus Kahl (104), Caenomorpha Perty (104, 214; Fig. 7. 31, G), Copemetopus
Viheneuve-Brachon (214), Ludio Penard (104, 165), Metopus Claparede and Lachmann
(104, 214; Fig. 7. 31, A-C, I), Palmarium Gajevskaia (104), Spirorhynchus da Cunha
(104; Fig. 7. 31, H), Trochella Penard (104, 165), and Tropidoatractus Levander (104).

Family 9. Peritromidae. These are marine ciliates which superficially
resemble hypotrichs in their dorso-ventrally flattened bodies and the
reduction of ciliation to the ventral surface. The dorsal surface may bear

386 Ciliophora

Fig. 7. 31. A-C. Cross-section of Metopus sigmoides through the anterior
part of the peristome and more posterior levels, showing some of the
membranelles (A, B) and the undulating membrane; x815 (after Wetzel).
D, E. Peritromus kahli Villeneuve-Brachon: ventral view, x300; sagittal sec-
tion, schematic, x450 (after V-B.). F. Plagiotoma lumbrici Dujardin, showing
adoral ciliation, somatic cilia (schematic), and a contractile vacuole; x240
approx. (from Kent, after Stein). G. Caenoniorpha medusula Perty, showing
ciliary pattern and bases of membranelles; x450 (after Villeneuve-Brachon).
H. Spirorhynchus verrucosa da Cimha, x425 (after Kirby). I. Metopus
mathiasi Villeneuve-Brachon, ventral view, x375 (after V-B.). J. Lichnophora
macfarlandi Stevens, showing basal and peristomial discs, adoral ciliation,
lateral membrane extending toward cytostome; x565 (after Balamuth).

scattered bristles, and in some cases, so-called mucilaginous trichocysts.
The band of membranelles extends across the anterior end or antero-
ventral surface and then posteriorly along the left ventral margin to the
cytostome, usually near the middle of the body.

Ciliophora 387

Fig. 7. 32. A-C. Nyctotherus kypliodes Geiman and Wichterman: A.
Specimen showing nuclei, karyophore, contractile vacuole, cytoproct; x375.
B. Transverse section through peristome and nuclei, x375. C. Ciliated cyto-
proct into which the contractile vacuole empties, xl025 approx. (after G.
& W.). D. Balantidioides muscicola Penard, about 80/i long (after Kahl).
E. Reichenoiuelln nigricans Kahl, specimen 250^ long, showing adoral mem-
branelles but no undulating membrane or distinct pharynx (after K.). F-
H. Transverse sections of Nyctotherus macropharyyigeus through anterior,
middle and posterior regions of the peristome; x240 approx. (after Wetzel).
I-K. Paranyctotherus kirbyi (Rodriguez) Sandon: view of left side (I), show-
ing endoplasmic fibrils, macronucleus and peristome, xl63; section through
peristome (J), schematic; antero-ventral region (K) showing peristomial
ciliatiue and endoplasmic fibrils (after S.). L, M. Nyctotherus cordiformis
Stein: longitudinal section (L) showing membranelles, undulating membrane,
contractile vacuole and macronucleus, x300 (after Villeneuve-Brachon);
.schematic sagittal section (M), showing nuclei, karyophore, peristome, and
cytostome (after ten Kate).

388 Ciliophora

Only two genera have been assigned to the family: Pediostomum Kahl (104) and
Peritromus Stein (104, 214; Fig. 7. 31, D, E).

Family 10. Plagiotomidae. This group includes parasites of oligochaetes
and other invertebrates and various vertebrates. The body is densely
ciliated, the band of membranelles (Fig. 7. 32, A, B, L, M) is well de-
veloped, and an undulating membrane lies at the right margin of the
peristome (Fig. 7. 32, L). A ciliated cytoproct has been described in
Nyctotherus (Fig. 7. 32, C).

Three genera have been referred to the family. Nyctotherus Leidy (75, 81, 179, 214;
Fig. 7. 32, A-C, F-H, L, M) is represented by intestinal parasites of vertebrates and
invertebrates. Paranyctotherus Sandon (187; Fig. 7. 32, I-K), erected for a ciliate from
a South African clawed toad, is similar to Nyctotherus but shows a row of mem-
branelles along the right margin of the peristome. Plagiotoma Dujardin (84, 166; Fig.
7. 31, F) includes parasites of coelomic cavities in earthworms.

Family 11. Reichenowellidae. This family was erected by Kahl (104) for
Reichenowella Kahl (Fig. 7. 32, E) and Balantidioides Penard (Fig. 7. 32,
D). These ciliates are said to differ from other Heterotrichina in the pres-
ence of a slit-like mouth, usually closed and not easily detected, and in the
lack of a definite oral pit. A band of membranelles is present, but no
undulating membrane.

Family 12. Spirostomidae. Some of these are elongated, with more or
less contractile bodies; certain others are dorso-ventrally flattened to
some extent. A long band of membranelles (Fig. 7. 33, L, M), or a homolo-
gous double row of cilia, extends to the cytostome. An undulating mem-
brane, sometimes fairly short, or a corresponding row of cilia extends
along the right margin of the peristome. The peristome may be rather
straight or may show some degree of spiral torsion. In at least certain
species, bands of trichocysts alternate with rows of cilia (214).

The following genera have been assigned to the family: BlepJiarisma Perty (104,
192; Fig. 7. 33, G, I), Gruberia Kahl (13, 104; Fig. 7. 33, F), Parablephnrisma Kahl
(104, 214; Fig. 7. 33, L), Phacodinium Prowazek (104), Protocrucia da Cunha (214;
Fig. 7. 33, H), PseudobJepharisina Kahl (104), Spirostomina Gruber (104), and Spiro-
stomum Ehrbg. (9, 104, 214; Fig. 7. 33, A-C, M).

Family 13. Stentoridae. The zone of membranelles tends to extend
around the anterior pole of the body, and in some cases the peristome
itself has shifted to the pole (Fig. 7. 33, D, J, K). The undulating mem-
brane has disappeared. Somatic ciliation is relatively uniform, with the
cilia arranged in longitudinal or slightly spiral rows.

The Stentoridae, represented in both fresh and salt water, include the following
genera: CUmacostomum Stein (104; Fig. 7. 33, K), Fabrea Henneguy (104, 113, 214;
Fig. 7. 33, E), and Stentor Oken (104, 214; Fig. 7. 33, D, J).

Ciliophora 389

Fig. 7. 33. A-C. Transverse sections of Spirostoniuni anibiguum Ehrbg.
through anterior and middle regions of the peristome and near the level of
the cytostome, x510 (after Wetzel). D. Stent or Relict Villeneuve-Brachon,
showing macronuclear chain, membranelles, somatic ciliary pattern; x375
(after V-B.). E. Fabrea salina Henneguv; bases of membranelles, peristomial
striations, somatic ciliary pattern; xl25 (after Villeneuve-Brachon). F. Gni-
beria calkinsi Beltran, 200-800/i long (after B.). G. Blepharisina hyalinum
Perty, x510 (after Wang and Nie). H. Protocrucia tiizeti Villeneuve-Brachon,
xl320 (after V-B.). I. Blepharisma lateritium (Ehrbg.) Kahl, x3I0 (after
K.). J. Stentor auriculatus (Gruber) Kahl, x200 (after Bullington. K. Clima-
costomum virens (Ehrbg.) Kahl, specimen 200fj. long, with a broad peristome
(after K.). L. Parablepharisma bacteriophora Villeneuve-Brachon, x300 (after
V-B.). M. Spirostomiim teres Claparede and Lachmann, showing membra-
nelles and somatic ciliary pattern; x250 (after Villeneuve-Brachon).

390 Ciliophora

Suborder 2. Oligotrichina

The somatic ciliation is either markedly reduced or has disappeared.
Persisting somatic cilia are often fused into tufts. The zone of mem-
branelles is commonly differentiated into a short oral band and an an-
terior spiral band of more powerful membranelles (Fig. 7. 34, A) which
are the most important or else the only organelles of locomotion. The
suborder is divided into two families.

Fig. 7. 34. A, B. Halteria geleiana Szabo, antero-ventral view (A) showing
oral membranelles and membrane, anterior locomotor membranelles, and
lateral cilia ("Springborsten"), x400; polar view (B) of adoral organelles,
x800 (after S.). C, D. Strobilidium gyrans (Stokes) Kahl, about 60^ long:
polar view of peristome (C), schematic; lateral view (after K.). E. Lohmani-
ella elegans (Wiilff) Kahl, specimen 25/^ long (after K.). F. Tontonia gracil-
lima Faure-Fremiet, specimen 50/^ long; contractile protoplasmic fibre ex-
tends from the posterior part of the body (from Kahl, after F-F.).

Family 1. Halteriidae. The peristome and the band of membranelles
extend posteriorly on the ventral surface. The group is represented in
fresh, salt, and brackish water.

The following genera have been referred to the family: Halteria Dujardin (104, 209;
Fig. 7. 34, A, B), Meseres Schewiakoft (104), Metastrombidium Faure-Fremiet (104),
Strombidium ClaparMe and Lachmann (55, 60, 104), Tontonia Faure-Fremiet (104;
Fig. 7. 34, F).

Family 2. Strohilidiidae. The zone of membranelles forms a spiral
crown at the anterior pole (Fig. 7. 34, C, D). The majority are marine,
biit some are known from brackish water and a few from fresh water.

Ciliophora 391

Six genera have been referred to the family: Cephalotrichium Meunier (104),
Ciliospina Leegaard (104), Lohmaniella Leegaard (104; Fig. 7. 34, E), Strobilidium
Schewiakoff (104; Fig. 7. 34, C, D), Parastrombidium Faure-Fremiet (104), and
Spliaerotrichium Wulff (104).

Suborder 3. Tintinnina

A typical member of this group is a conical or trumpet-shaped ciliate
contained in a lorica to which it is attached by the adhesive aboral tip of
the body (Fig. 7. 35, M). The aboral end may or may not be drawn out
into a slender contractile stalk. The peristomial field (Fig. 7. 35, A)
covers most of the oral pole and is a more or less funnel-shaped area
leading to the cytostome (Fig. 7. 35, B). The zone of 12-24 membranelles
forms a spiral around the peristome. In some species, a protoplasmic
flange lies just outside the membranelles. The adoral zone commonly
bears a series of "tentaculoids," one between each pair of membranelles
(Fig. 7. 35, A). Each tentaculoid is a ball- or club-shaped structure borne
on a stalk, from the base of which a conical "accessory comb" extends into
the peristomial area (17). Nothing is known about the functions of the
tentaculoids or their appendanges. Somatic ciliation is usually sparse,
sometimes limited to the anterior third of the body, sometimes extending
almost to the posterior end. In certain species (18), a paroral zone of
long somatic cilia lies near the oral pole. In addition, a large ciliary
membrane, extending along the ventral surface from the rim of the
peristome (Fig. 7. 35, M), occurs in several families. This membrane helps
to mold the new lorica in fission (17, 18).

The form of the lorica (Fig. 7. 35, C-L) varies considerably. The aboral
end is usually closed but both ends are open in certain species. The
capacity is generally several times the volume of the enclosed ciliate.
Foreign particles are sometimes incorporated in the wall of the lorica,
which is composed basically of secreted organic material, including
xanthoproteins (116). As fission is completed, this material is discharged
through the gullet and worked into shape by the membranelles, perioral
cilia and the ciliary membrane. These organelles seem to function some-
what like trowels in fashioning the new lorica, the anterior part being
shaped by the anterior daughter organism and the posterior part by the
posterior one.

A few Tintinnina have been described from fresh and brackish water
but most of them are marine pelagic ciliates. Campbell (19) and Kofoid
and Campbell (115, 116) have published systematic studies of the group,
the taxonomy of which is based largely upon structure of the lorica.

The following families and genera have been characterized (19, 115, 116): (1)
Codonellidae: Codonaria Kofoid and Campbell, Codonella Haeckel (Fig. 7. 35, G),
Codonopsis K. & C, Tintinnopsis Stein (Fig. 7. 35, M); (2) Codonellopsidae: Codo-
nellopsis Jorgensen (Fig. 7. 35, I), Laackmanniella K. & C, Stenosemella J.; (3) Cox-
liellidae: Climacocyclis J., Coxliella Brandt (Fig. 7. 35, H), Helicostomella J.,

392 Ciliophora

Fig. 7. 35. A. Polar view of peristome in Tintinuopsis miciila; bases of
the membranelles alternate with tentaculoids and their accessory combs;
xI075 (after Campbell). B. Longitudinal section of Tintinnopsis campanula
(Ehrbg.), showing adoral and somatic ciliation; semidiagrammatic (after
Entz). C-L. Variations in form of the lorica in lintinnina: C. Petalotricha
ampulla; D. Salpingella acuminata; E. Dictyocysta mira; F. Craterella urceo-
lata; G. Codonella rapa; H. Coxliella fasciata; I. Codonellopsis longa; J.
Eutintinnus brandti; K. Cyttarocyclis aciitiformis; L. Rhabdonella henseni;
F, x300; others xl88 (after Campbell). M. Tintinnopsis nucula, lorica in
optical section; ciliary membrane extends from the peristome posteriorly
past the middle of the body; nuclei, cytoproct, and somatic cilia are shown;
schematic, x425 (after Campbell). KEY: ac^ accessory comb; m, membranelle;
t, tentaculoid.

Metacyclis J.; (4) Cyttarocyclidae: Cyttarocyclis Fol (Fig. 7. 35, K); (5) Dictyocystidae:
Dictyocysta Ehrbg. (Fig. 7. 35, E), Luminella K. & C, Wailesia R. & C, Wangiella Nie;
(6) Epiplocylidae: Epicanella K. & C, Epiorella K. & C, Epiplocylis J.; (7) Favellidae:
Cymatocyclis Laackmann, Favella J. (18), Poroecus Cleve, Protocymatocyclis K. & C;
(8) Pelatotrichidae: Aranthostomella J., Craterella K. & C. (Fig. 7. 35, F), Pelatotricha

Ciliophora 393

Kent (Fig. 7. 35, C); (9) Ptychocyclidae: Plychocyclis Brandt; (10) Rhabdonellidae:
Epirhabdonella K. & C, Protorhnbdonella J., Rhabdonella B., Rhabdonellopsis K. &
C; (11) Tintinnidae: AlbatrossicUa K. &: C, Amphorella Daday, AmphorcUopsis K.
& C, Brandt iella K. & C, Bursaopsis K. & C, Canthariella K. & C, Dadayiella K. & C,
Daturella K. & C, Epicranella K. & C, Epirhabdosella Campbell, Eutintinnus K. & C.
(Fig. 7. 35, J), Odontophorella K. & C, Ormosella K. & C, Proamphorella K. & C, Pro-
stelidiella K. & C, Rhabdosella K. & C, SaJpingacantha K. fe C. Salpiiigella J. (Fig. 7.
35, D), SalpingcUoides Campbell, Stelidiella K. & C, Steenstrupiella K. & C, Tintinnns
Schrank; (12) Undellidae: Amplcctella K. & C. AmplecteUopsis K. & C, CricundeJla
K. & C, Proplectella K. & C, Undella Daday, Undellopsis K. & C; (13) Xystonellidae:
Parafavella K. & C, Parundella J., Xystonella Brandt, Xystonellopsis J.

Suborder 4. Entodiniojnorphina

These ciliates occur in the rumen of cattle, sheep and other ruminants
and in the intestine of certain other herbivores. The ciliature is much
reduced and in the simplest Ophryoscolecidae, as represented by Ento-
dinium (Fig, 7. 36, P), is limited to the membranelles of the adoral zone.
In most Ophryoscolecidae, however, there is also a dorsal zone of mem-
branelles, ranging from a short anterior band to a longer and more or
less equatorial row (Fig. 7. 36, A-E). The adoral membranelles arise in a
furrow formed by an ectoplasmic fold and extend spirally to the cytostome
which lies in an elevated oral disc at the anterior pole. At least the adoral
zone, and in some genera the dorsal zone also, can be retracted. Between
the adoral and dorsal zones there is often an elevation, the operculum
(Fig. 7. 36, M, R). The Cycloposthiidae have added one or more posterior
or caudal rows or tufts of membranelles (Fig. 7. 38, A).

Beneath the firm pellicle there is a distinct cortical layer (Fig. 7. 36,
P) with a clear matrix containing many granules. The macronucleus,
micronucleus, contractile vacuoles (143), and skeletal plates also lie in
this zone, which is separated from the endoplasm by a membrane con-
tinuous anteriorly with the pharynx and posteriorly with the rectum.
The cytostome (Fig. 7. 38, B) opens into the short pharynx (Fig. 7. 36,
R). The endoplasmic sac, consisting of the bovuidary membrane and the
contained endoplasm, fills most of the body posterior to the pharynx. The
rectum, at the posterior end of the sac, is a thin-walled tube extending
through the ectoplasm to the anus. In certain Ophryoscolecidae, the wall
of the rectum apparently contains myonemes which presumably have a
sphincter-like action.

Skeletal plates are present except in a few of the Ophryoscolecidae
(Diplodinhim, Entodinium, Eodinium). These structures vary in nimiber,
size, form, and arrangement in different genera (Fig. 7. 36, F-L). Each
plate is a flattened structure extending posteriorly in the ectoplasm from
a level shortly behind the adoral zone (Fig. 7. 36, Q). The plates of
Polyplnstron multivesiculatum consist of a protein matrix containing ir-
regular platelets of "paraglycogen" (144).

The Entodiniomorphina are divided into two families: tiie Ophryo-

394 Ciliophora

Fig. 7. 36. A-E. Arrangement of membranelles in Ophryoscolecidae, polar
views: A. Entodinium; B. Opisthotrichum; C. Diplodinium and Epidiniiim;
D. Opiiryoscolex; E. Caloscolex; diagrammatic (after Dogiel). F-L. Variations
in the number and arrangement of skeletal plates in Ophryoscolecidae; dia-
grammatic cross-sections near the anterior pole (after Dogiel). M. Eudiplo-
dinium maggii (Fiorentini) Dogiel, x250 (after Kofoid and Christenson). N.
Eodiniuni polygonale Kofoid and MacLennan, x600 (after M. & MacL.). O.
Ophryoscolex caudalus Eberlein, x250 (after MacLennan). P. Entodinium
biconcavum Kofoid and MacLennan; a, anus; b, boundary layer between
ectoplasm and endoplasm; c, contractile vacuole; e, endoplasm; o, oesoph-
agus; r, rectum; v, ventral lobe; schematic, xlOOO (after K. & MacL.). Q.
Epidiniuni caudatum (Fiorentini) Crawley, xlOO (after Kofoid and Mac-
Lennan). R. Longitudinal section of Diplodinium medium showing adoral
ciliation and cilia extending into the oesophagus; x225 (after Rees).

Ciliophora 395

E F

Fig. 7. 37. A. Diplodinium moy^ocanthum Dogiel, x480 (after Kofoid and
Chiistenson). B. Ostracodinium cUpeolum , x250 (after Kofoid and MacLen-
nan). C. Tetratoxum unifasciculatum (Fiorcntlni) Gassovsky, x222 (after
Hsiung). D. Eremoplastron bovis (Dogiel) Kofoid and MacLennan, x375
(after K. & MacL.). E. Polyplastron multh'csicnlatum, skeletal plates indi-
cated in solid black, x250 (after MacLennan). F. Metadiniiiiu, medium
Awerinzew and Mutafowa, x200 (after MacLennan). G. Ditoxunt funinu-
cleum Gassovsky, x202 (after Hsiung). H. Elytroplastron bubali (Dogiel)
Kofoid and MacLennan, x250 (after K. & MacL.).

scolecidae with not more than one "dorsal" band of membranelles in
addition to the adoral zone; and the Cycloposthiidae, which have added
one or more posterior or caudal groups to the maximum for Ophryo-
scolecidae. The Ophryoscolecidae are rumen-dwelling ciliates charac-
teristic of cattle, sheep, and related hosts. Cycloposthiidae have been
reported mostly from horses, but a few species are known from the
chimpanzee, gorilla, rhinoceros, and elephant. Genera assigned to the two
families are listed below.

Family 1. Ophryoscolecidae. Amphacanthus Dogiel, Caloscolex Dogiel (50), Cunhaia
Hasselmann (50), Diplodinium Schuberg (50, 120, 176; Fig. 7. 36, R), Diploplastron
Kofoid and MacLennan (120), Elytroplastron Kofoid and MacLennan (120), Enopla-
stron Kofoid and MacLennan (120), Entodinium Stein (50, 117, 119; Fig. 7. 36, P), Eodi-
nium Kofoid and MacLennan (120; Fig. 7. 36, N), Epidinium Crawley (50, 117, 121;
Fig. 7. 36, Q), Epiplastron Kofoid and MacLennan (121), Eremoplastron Kofoid and
MacLennan (120; Fig. 7. 37, D), Eudiploditiiutn Dogiel (120; Fig. 7. 36, M), Metadinium
Awerinzew and Mutafowa (120, 143; Fig. 7. 37, F), Ophryoscolex Stein (50, 121, 143;
Fig. 7. 36, O), Opisthotrichum Buisson (50), Ostracodinium Dogiel (117, 120; Fig. 7.
37, B), Polyplastron Dogiel (120, 143; Fig. 7. 37, E).

396 Ciliophora

Fig. 7. 38. A. Trifasciciilaiia parvum Hsiung, x322 (after H.). B. Polar
view of peristome in Tripalmaria dogieli showing outer membranelles and
basal plates of the adoral zone leading to the cytostome; x260 (after Strel-
kow). C. Longitudinal section through antero-dorsal group of membranelles
in Tripahtiaria dogieli; x550 (after Strelkow). D. Triadiniiim caudatum
Fiorentini, x322 (after Hsiung). E. Spirodinium equi Fiorentini, x322 (after
Hsiung). F. Polydinium mysorenm Kofoid, from Indian elephant; x275 (after
K.). G. Tripalmaria dogieli Gassovsky, x.'522 (after Hsiung). H. Cyclopes-
thium bipalmatum (Fiorentini) Bundle, x363 (after Hsitmg).

Family 2. Cycloposthiidae. Cochliatoxum Gassovsky (91), Cycloposthium Bundle (91,
193, 194, 195; Fig. 7. 38, H), Ditoxum Gassovsky (91; Fig.?. 37, G), Polydinium Kofoid
(114; Fig. 7. 38, F), Spirodiniiun Fiorentini (91; Fig. 7. 38, E), Tetratoxum Gassovsky
(44, 91; Fig. 7. 37. C), Triadinium Fiorentini (91; Fig. 7. 38, D), Trifnsciciilaria
Strelkow (197; Fig. 7. 38, A), Tripalmaria Gassovsky (91, 196; Fig. 7. 38, B, G),
Triplumaria Hoare, Troglodytella Brumpt and Joyeux (208).

Suborder 5. Hypotrichina

Somatic cilia are replaced by cirri which are nearly always limited to
the ventral surface. The dorsal surface often bears rows of so-called sen-
sory bristles, which are sometimes present also on the ventral surface
adjacent to cirri. The ventral cirri are typically arranged in groups (Fig.
7. 40, E, F, J): frontal cirri, located between the peristome and the right
side of the body; ventral cirri, posterior to the frontal cirri; marginal
cirri, arising from the right and left margins of the ventral surface; caudal
cirri, arising from the posterior margin; and anal cirri, arising in a trans-
verse or diagonal row a short distance from the posterior end of the body.
Certain cirri, particularly the lateral ones, may be lacking in some species.

Ciliophora 397

The peristomial area is large and more or less triangular in many
species, but is reduced in size and fairly narrow in others. In some cases
the right margin of the peristome is extended toward the left as a flange
("oral lip") which partially encloses the peristome ventrally. Such a de-
velopment increases the efficiency of the peristome, especially in species
which feed on bacteria.

Fig. 7. 39. A, B. Aspidisca turrita (Ehrbg.) Clapar^de and Lachmann,
lateral and ventral views, x765 (after Wang and Nie). C. Certesia quadri-
nudeata Fabre-Domergue, specimen 90/i long (after Kahl). D. Aspidisca
polystyla Stein, x840 (after Wang and Nie). E, F. Uronychia heinrothi
Buddenbrock, ventral view showing the peristomial membranes, and dorsal
view of the three postero-dorsal cirri; x200 (after Bullington). G. Euplo-
taspis cionaecola Chatton and Seguela, 60-70^ long; from branchial cavity
of ascidians (after C. & S.). H, I. Euplotidium agitatum Noland: ventral
view, x600; anterior end showing bases of membranelles, x450 (after N.).

398 Ciliophora

The zone of membranelles often extends from the cytostome anteriorly
and then transversely across the antero-dorsal or antero-ventral surface of
the body (Fig. 7. 39, H). In the Aspidiscidae, however, the membranelles
are reduced to a short band (Fig. 7. 39, B). An undulating membrane
often extends at least part way along the right margin of the peristome
(Fig. 7. 40, A).

Family 1. Aspidiscidae. These are flattened ciliates (Fig. 7. 39, A) with
an armor-like pellicle. The adoral membranelles are reduced to a short
band, while the cirri are limited to a small group of frontals and a group
of anal cirri (Fig. 7. 39, B). Near the anterior end there is sometimes a
small depression containing a few delicate membranelles which represent
the anterior remnant of the primitive adoral zone.

The type genus, Aspidisca Ehrenberg (104; Fig. 7. 39, A, B, D), seems to be the only
one which belongs to the family. Chatton and Seguela (34) have referred their genus
Euplotaspis (Fig. 7. 39, G) to the Aspidiscidae but such an assignment cannot be justi-
fied without major revisions in characterization of the family. Perhaps a better location
for Euplotaspis would be the family Paraeuplotidae.

Family 2. Euplotidae. The number of ventral cirri is reduced, with a
loss of the right marginal series (Fig. 7. 39, C) or both marginal rows
(Fig. 7. 39, H) and usually of all except a few of the primitive ventral
group. The persisting cirri are relatively stout and there is generally a
group of well-developed anal cirri (usually five). A few caudal cirri also
persist, either as rather slender structures or as large "rudders" in
Uronychia and Diophrys. The peristome and the adoral membranelles
are well developed.

The following genera are included in the family: Certesia Fabre-Domergue (104,
188; Fig. 7. 39, C), Diophrys Dujardin (104, 188; Fig. 7. 40, H), Euplotes Ehrenberg
(104, 170; Fig. 7. 40, G), Euplotidium Noland (158; Fig. 7. 39, H, I), and Uronychia
Stein (13, 104, 227; Fig. 7. 39, E, F).

Family 3. Oxytrichidae. The arrangement of the cirri follows the gen-
eralized pattern, although there is some reduction of the ventral cirri in
certain species. Right and left marginal cirri are always present and the
adoral membranelles are well developed.

The following genera have been referred to the Oxytrichidae: Ancystropodium
Faure-Fremiet (104), Balladyna Kowaleski (104; Fig. 7. 40, B), Balladynopsis Ghosh
(104), Caryotricha Kahl (104), Chaetospira Lachmann (104), Cladotricha Gajevskaja
(104), Epiclintes Stein (13, 104), Eschaneustyla Stokes (104), Gastrocirrhus Lepsi (13;
Fig. 7. 41, K), Gastrostyla Engelmann (88, 104, 223; Fig. 7. 40, F), Gonostomum Sterki
(104), Hemicycliostyla Stokes (104). Holosticha Wrzesniowski (88, 104; Fig. 7. 40, I;
41, A), Hypotrichidium Ilowaisky (182; Fig. 7. 41, G), Kahlia Horvath (89, 104; Fig. 7.
41, H), Keroria Ehrbg. (104; Fig. 7. 41, E), Onychodromopsis Stokes (104), Onychodromus
Stein (104; Fig. 7. 40, J), Oxytricha Ehrbg. (10. 88, 104; Fig. 7. 40, E), Paraholosticha
Kahl (88, 104; Fig. 7. 41, A), Pleurotricha Stein (104; Fig. 7. 41, C), Pseudostrombidium

Ciliophora 399

Fig. 7. 40. A. Urostyla limboonkengi Wang and Nie, x320 (after W. &
N.). B. Balladytia parvula Kowalewsky, x780 (after W. & N.). C, D. Sticho-
tricha nankingensis Wang and Nie, ventral view, x250; specimen in gelat-
inous lorica, xl25 approx. (after W. & N.). E. Oxytricha platystoma Ehrbg.,
x300 (after Horvath). F. Gastrostyla steinii Engelmann, x300 (after Hor-
vath). G. Euplotes harpa Stein, x375 (after Wang and Nie). H. Diophrys
appendiculatus (Ehrbg.), x600 (after Wang and Nie). I. Holosticha kessleri
(Wrzesniowski), x290 (after Wang and Nie). J. Onychodromus grandis Stein,
specimen 250/x long (after Kahl).

Horvath (88), Psilotrkha Stein (104), Stichotricha Perty (104; Fig. 7. 40, C, D),
Strojigylidium Sterki (104; Fig. 7. 41, J), Stylocoma Gruber (104), Stylonethes Sterki
(71, 72), Trachelostyla Kahl (104; Fig. 7. 41, I), Uncinata Bullington (13), Uroleptus
Ehrbg. (16, 104; Fig. 7. 41, F), Uroleptopsis Kahl (104), and Urostyla Ehrbg. (104; Fig.
7. 40, A).

In addition, Histrio Sterki, OpistJiotricha Kent, Steinia Diesing, Stylonychia Ehrbg.,
Tachysoma Stokes and Urosoma Kowalewski are listed by Kahl (104) as sub-genera of
Oxytricha; Amphisiella Gourret and Roeser, Keronopsis Penard, Paruroleptus Kahl
and Trichotaxis Stokes, as sub-genera of Holosticha,

400 Ciliophora

Fig. 7. 41. A. Paraholosticha ovata Horvath, x300 (after H.). B. Holo-
sticha (Paruroleptus) novitas Horvath, xl50 (after H.). C. Pleurotricha
grandis Stein, specimen 300/x long (after Kahl). D. Paraeuplotes tortugensis
Wichterman, ventral view, zoochlorellae not shown; x375 (after W.). E.
Kerona polyporum Ehrbg., specimen 160^ lotig (after Kahl). F. Uroleptus
mobilis Engelmann, specimen 150^ long (after Kahl). G. Hypotrichidium
conicum Ilowaisky, x250 (from Rossolimo, after I.). H. Kahlia costata Kahl,
x580 (after Wang and Nie). I. Trachelostyla pediculiformis (Cohn) Kahl,
x350 (after Wang and Nie). J. Strongylidium maritimum Wang and Nie,
x594 (after W. & N.). K. Gastrocirrhus stentoreus Bullington, x375 (after
B.).

Family 4. Paraeuplotidae. This family contains the unusual genus,
Paraeuplotes Wichterman (224; Fig. 7. 41, D), in which the adoral zone
of membranelles is well developed but the only cirri are a group of five
or six extending from the posterior end of the body. Instead of the usual

Ciliophora 401

ventral cirri, there are bands and tufts of free cilia. The genus is rep-
resented by a single marine species.

Euplotaspis Chatton and Seguela (34; Fig. 7. 39, G) resembles Paraeuplotes in several
respects and possibly belongs in the same family. There seem to be two ventral rows of
free cilia, and the frayed compoinid organelles in the frontal field may be homologous
with the tufts of cilia in Paraeuplotes.

Suborder 6. Ctenostomina

These are small laterally compressed, wedge-shaped ciliates in
which the base of the wedge (Fig. 7. 42, B) represents the ventral (oral)
surface. In lateral view (Fig. 7. 42, C, H), the dorsal margin usually
describes about a third of a circle but may approach a semicircle or almost
a circle. Anteriorly, the dorsal keel ends in a brow-like prominence or in
a spur or spine (Fig. 7. 42, E, H, J). Except for the ventral surface, and
the posterior end in certain species, the body is covered with a firm
pellicle differentiated into longitudinal plates (105).

The somatic ciliation is much reduced. On the left surface there are
typically four rows, extending forward for varying distances from the
posterior end (Fig. 7. 42, F, J), and also a fifth frontal row (exception-
ally, two frontal rows) extending posteriorly for some distance from an
origin near the anterior pole. In primitive species, a frontal band of five
rows arises anteriorly on the lower left surface, extends across the narrow
ventral surface, and then upward and posteriorly for some distance on
the right side. A tvift of preoral cilia anterior to the peristome, and two
adoral rows running from the frontal band almost to the peristome, may
also be found on the ventral surface. Modifications of the general pattern
occur in the more specialized types.

The peristome is a ventral pouch covered on the left by a thin wall
(Fig. 7. 42, A, B) apparently analogous to the "oral lip" of various
Spirotrichida. In contrast to most Spirotrichida, the adoral membranelles
are reduced to eight in the peristome proper and a small ninth one in
the pharynx (105).

All except two species are known from fresh water and all are sapro-
pelic (or polysaprobic) types, growing well in the presence of putrefying
materials. Kahl (105) recognized three families.

Family 1. Epalcidae. The posterior end of the body is unarmored, al-
though surrounded by the spurred or spiny ends of the armor plates.
Somatic ciliation is relatively complete. On the left, a frontal band and
the four primitive posterior rows are always present. On the right side
of the body, at least the ventral and dorsal rows are present.

Three genera are listed for this family (105): Epalxis Roux (Fig. 7. 42, A, D),
Pelodinium Lauterbom (Fig. 7. 42, F), and Saprodinium Lauterborn (Fig. 7. 42, J).

402 Ciliophora

Fig. 7. 42. A. Peristome of Epalxis showing peiistoniial wall and bases
of adoral membranellcs; diagrammatic (after Kahl). B. Transverse section
of Epalxis bidens through the peristome; diagrammatic (after Kahl). C.
Atopodinium fibulatum Kahl, 35-45/i (after K.). D, E. Epalxis striata Kahl,
25-35^, right and left sides (after K.). F. Pelod'niiuni reuifonne Lautcrborn,
40-50^ (after Kahl). G, H. DiscoiuorpJia pectinata Levander, about 80^ long;
ventral and lateral views (after Kahl). I. Mylestoina anat'nium (Penard)
Kahl, 20-28;u (after K.). J. Saprodinium integrum Kahl, 40-55;Lt (after K.).

Family 2. Mylestomidae. The right and left dorsal rows of cilia have
disappeared and the ventral cilia are absent or reduced in number (Fig.
7. 42, C). The reinaining posterior cilia may remain free or may be fused
into one or tAvo long "rudder-cirri" (Fig. 7. 42, I). The frontal band is
limited to the ventral surface. The posterior end of the body is almost or
completely covered with armor.

Ciliophora 403

Two genera have been referred to the family (105): Mylestoma Kahl (Fig. 7. 42, I)
and Atopodiniiim Kahl (Fig. 7. 42, C).

Family 3. Discomorphidae. The dorsal keel, which ends anteriorly in a
spine, sweeps back over the posterior end to the ventral surface (Fig. 7.
42, H). Somatic ciliation is limited to the two ventral rows, two posterior
tufts of cilia on the left, and a well-developed frontal band.

Only one genus is known (105): Discomorpha Levander (Fig. 7. 42, G, H).

Order 3. Peritrichida. The adult is usually attached either directly by
its aboral end or by means of a secreted stalk, or else lies within a lorica
which is attached to some solid surface. A number of the stalked types are
colonial. A few species are free-swimming and apparently have no sessile
stage.

In this order, the peristome (or "epistome") is a polar disc which, seen
from the oral end of the body, is approximately circular (Fig. 7. 47, H).
Encircling the peristome counterclockwise are two or more rows of cilia
which usually complete a full spiral before passing through the cytostome
into the vestibule. Two rows of adoral cilia have been described in
Telotrochidhim (138) and Cyclochaeta (146); three, in Vorticella (159).
In all three cases, the cilia of each row are free distally but are fused
basally into a continuous membrane. Each row of cilia is continued into
the vestibule in CyclocJweta and I'orticrlla: only the inner row, in Telo-
trocliidiiim. Within the vestibule, a membrane may be formed by com-
plete fusion of cilia (the outer row of Vorticella). The outer margin of
the peristomial surface, which ranges from a narrow border to a broad
projecting shelf, often forms a contractile rim which can be constricted
to enclose the peristome and its ciliature.

The vestibule receives the contents of the contractile vacuole, some-
times through an intermediate "reservoir" (Fig. 7. 47, H), and also the
luidigested materials from old food vacuoles. As in the ordinary pharynx,
incoming food particles also pass down the vestibule into the developing
food vacuole at its base. Noland and Finley (159) have noted an apparent
separation of incurrent and excurrent channels within the vestibule.

The scopula (54), a differentiated area at the aboral pole, is often evi-
dent as a small invagination, the wall of which sometimes shows fibrils
or rod-like elements. In many of the sessile species withovu stalks, the
scopula apparently secretes some material which insures adhesion to the
substratvmi. In stalked species, the scopula secretes the inert matrix of
the stalk. The non-contractile stalk of the Epistylidae consists entirely
of secreted material. The stalk of the Vorticellidae contains, in addition,
a loosely spiral myoneme, or "stalk-muscle," which appears to be an out-

404 Ciliophora

Fig. 7. 43. A. Horizontally elongated telotroch characteristic of Epistylis
horizontalis Chatton, x500 (after C). B. Pyxidium cotliurnoides Kent, spec-
imen 50;a long; stalk typically unbranched; peristomial disc similar to that
in Opcrcularia (after Kahl). C. Rhabdostyla ovum Kent, x850 (after Wang
and Nie). D. Asfylozoon pirifonne Schewiakoff, with rudimentary stalk; x560
(after Wang and Nie). E. Telotrochidiuyn (Opisthonecta) henneguyi (Faure-
Fremiet) Kahl, x450 (after Kofoid and Rosenberg). F. Geleiella vagans
Stiller, body enclosed in gelatinous mantle (after S.). G. Hastatella radians
Ehrbg., two rings of cytoplasmic "spines"; x740 approx. (after Wang and
Nie). H. Epistylis chrysemidis Bishop and Jahn, two zooids; xll5 (after B.
S: J.). I. Telotrochidium johanninae Faur^-Fremiet, xl025 (after F-F.).

growth from the body. The stalk-muscle of colonial Vorticellidae may be
continuous throughout the colony (Fig. 7. 48, D), except perhaps in the
basal section of the main stalk (200), or the myonemes of individual
stalks may be independent (Fig. 7. 48, E). In the former, the colony as a

Ciliophora 405

Fig. 7. 44. A. Glossatella fnUhmabuhun (Kent) Kahl, 30-43^: large adoral
membrane (after K.). B. ParavorticeUa clyrnencUae (Shumway) Kahl, about
100^ long (after S.). C. Ophrydium lemnae Kahl, specimen 70^ long (after
K.). D, E. Lagenophrys labiata Wallengren, ventro-lateral and lateral views;
x360 (after Wang and Nie). F. Sryphidia physarum Lachmann, about QOyn
long (after Kahl). G, H. Scyphidia ameirui Thompson, Kirkegaard and
Jahn, 34-45/i; telotroch and sessile stages (after T., K. & J.). I. Ophrydium
(Gerda) glaus (Clapar^de and Lachmann) Kahl, specimen 200;^ long; long
canal extends from reservoir of contractile vacuole to the vestibule (after
K.). J. Opercularia ramosa Stokes, x210 approx. (after Bishop and Jahn).

whole may be retracted toward the point of attachment; in Carchesium,
contractions of the stalks affect individual zooids separately.

The life-cycles of Peritrichida are commonly dimorphic and sometimes
polymorphic. Reproduction apparently should be considered fission
rather than budding, although one daughter organism is often smaller

406 Ciliophora

than the other. The plane of fission in Vorticella passes from near the
center of the peristome to a point just to one side of the stalk. One
daughter organism thus retains the parental stalk. The other develops
an aboral band of locomotor cilia and becomes a telotroch (Fig. 7. 47,
C) which swims actively for a short period. Metamorphosis into the adult
follows adhesion of the scopula to a suitable surface. In this attached
stage, the young peritrich resembles Scyphidia which lacks a stalk. How-
ever, the Scyphidi a-st3.ge of Vorticella lasts for only a short time. Soon
after attachment, secretion of a stalk begins and the aboral cilia disap-
pear within a few minutes (186). In fission during development of a
colonial type, such as Zoothamnium (200), the daughter organism not
retaining the old stalk secretes a new stalk which becomes a branch of
the original one.

The telotroch is a common stage in the life-cycle and is not limited to
stalked species, since it occurs in such forms as Scyphidia (Fig. 7. 44, H).
Direct transformation of a stalked form into a telotroch sometimes occurs
in Vorticella (Fig. 7. 47, D-F) and is a normal means of asexual propaga-
tion in such genera as Zoothamnium (200). After liberation from the
parental colony, the migratory stage swims away and then settles down
on some object to develop into a new colony. The Urceolariidae are
sometimes considered highly specialized permanent telotrochs. In this
connection, the telotroch of Epistylis liorizontalis (Fig. 7. 43, A) is in-
teresting in its superficial resemblance to the Urceolariidae and in its
comparable ability to glide over surfaces (20). Telotrochidiiim (Fig. 7.
43, E) also seems to represent a permanent telotroch, although it is not
impossible that this genus contains telotrochs whose sessile stages have
not been recognized.

The life-cycle of the commensal Ellohiophrya donacis (25) is more com-
plicated than that of most Peritrichida. The adult lives in a lamelli-
branch, Donax vittatns, attached to a gill-filament (Fig. 7. 45, A). At the
completion of fission one daughter organism (the future telotroch) re-
mains attached to the larger by a narrow isthmus of cytoplasm (Fig. 7.
45, B). A scopula soon develops in the embryonic telotroch and secretes
a stalk wdiich extends for some distance into the attached sister organism
(Fig. 7. 45, D). Elongation of the developing telotroch and differentiation
of the aboral cilia occur next (Fig. 7. 45, C). The telotroch then becomes
free-swimming (Fig. 7. 45, E), usually breaking away at the junction of
stalk and scopula, but sometimes carrying its stalk along to be discarded
later. After reaching a gill-filament, this migratory stage becomes attached
by its aboral end and develops the protoplasmic arms which anchor the
organism to its host (Fig. 7. 45, F).

Axial homologies between the Peritrichida and other ciliates are some-
what uncertain. Although the peristome is usually considered anterior,

Ciliophora 407

V ^ . v\\\VSi\l!ii//7//; ' ' '''^''' ''-^

"■''''^'-<,^i:i!.:::!;:,l]l:B.;^,i.^.:S---'

H G

Fig. 7. 45. A-F. Ellobiophrya doiiacis Chatton and Lwoff: mature stage
(A) attached to gill-filament of Donax vittatiis, x400; completion of fission
(B), x575; development of migratory larva (C), x575; stage in formation of
embryonic stalk (D), x675; swimming larva (E), x675; young organism re-
cently attached to gill-filament (F), aboral cilia still present, x675 (after C.
& L.). G-I. Cyclochaeta domerguei Wallengren: view of aboral surface (G),
x-500; lateral view (H); section through vestibule and contractile vacuole (I),
x750 (after MacLennan).

Telotrochidium henenguyi (138) and Cyclochaeta domerguei (146) both
swim with the aboral pole directed forward.

Family 1. Astylozoonidae. These are actively swimming peritrichs which
travel with the peristome directed forward. Instead of a stalk, one or two
apparently thigmotactic bristles are developed at the aboral pole. The
family has been considered a highly specialized group (106).

408 Ciliophora

Fig. 7. 46. A. Vaginicola annulata Stokes, lorica reaches length of 120/i
(after Kahl). B. Vaginicola amphora Kahl, lorica lOO/j. (after K.). C. Cothur-
nia canthocampti Stokes, lorica about 80jn (after Stokes). D. Pyxicola entzi
(Stiller) Kahl, 70-75/i (from K., after S.). E. Caulicola I'alvata Stokes, lorica
about 50/A (after Kahl). F, G. Urceolaria patellae (Cuenot) Kahl, diameter
50-60/x; lateral view; portion of aboral disc (after K.). H. Platycola longi-
collis Kent, about 125yii (after Penard). I. Trichodina spheroidesi Padnos and
Nigrelli, x712 (after P. & N.). J. Thuricola obconica Kahl, about 210/i (after
K.). K. Cothurnia acuta Wang and Nle, x334 approx. (after W. & N.).

Three genera have been assigned to the Astylozoonidae: Astylozoon Engelmann (106;
Fig. 7. 43, D), Geliella Stiller (106; Fig. 7. 43, F), and Hastatella Stiller (95, 106; Fig.
7. 43, G).

Family 2. Epistylidae. In sessile stages, the scopula produces a stalk
which contains no myoneme (stalk-muscle). Some species are solitary and
others colonial.

Ciliophora 409

Six genera have been referred to the family: Ballodora Dogiel and Furssenko (106),
Epistylis Ehrenberg (106; Fig. 7. 43, A, H), Opercularia Stein (106; Fig. 7. 44, J),
Pyxidium Kent (106; Fig. 7. 43, B), Rhabdostyla Kent (106; Fig. 7. 43, C) and Telo-
trochidiiim Kent (63, 122, 180; Fig. 7. 43, E. I). Since no stalked stage is known for
Telotrochidium, the status of this genus as a member of the family is uncertain.

Family 3. Lagenophryidae. These are loricate ciliates in which the
peristomial disc lies at the tip of a stout neck which is usually the only
part of the body to be extended through the mouth of the lorica.

The family contains only the genus Lagenophrys Stein (2, 106; Fig. 7. 44, D, E).

Fig. 7. 47. A-F. Vorticella microstoma Ehrbg.: extended form (A), con-
tracted specimen (B), and telotroch (C), x540; development of a telotroch
from a stalked stage (D-F), x360 (after Noland and Finley). G. Vorticella
mayeri Faure-Fremiet, x575 approx. (after Wang and Nie). H. The peri-
stomial area in Vorticella: ac, basal granules of adoral ciliature; cv, con-
tractile vacuole with its adjacent reservoir; r', vestibule; vm, adoral membrane
in vestibule; diagrammatic (after Noland and Finley). I. Vorticella picta
Ehrbg.. x5I0 (after Noland and Finley).

410 Ciliophora

Family 4. Ophrydiidae. The oral end of the body is prolonged into a
long contractile neck. The aboral end tapers to a point in some species
but is broadly rounded in others. The scopula may or may not produce
a short stalk.

Only two genera have been recognized: Ophrydium Ehrbg. (106; Fig. 7. 44, C, I) and
Ophridiopsis Penard (106, 165).

Family 5. Scyphidiidae. These are sessile peritrichs in which the scopula
functions as a holdfast organ. The body is sometimes broadly flattened
at the aboral pole; in other cases it tapers to a stalk-like basal region.

Four genera have been assigned to the family: Ellobiophrya Chatton and Lwoff (25;
Fig. 7. 45, A-F), Glossatella Biitschli (106; Fig. 7. 44, A), Paravorticella Kahl (106;
Fig. 7. 44, B), and Scyphidia Dujardin (87, 106, 212; Fig. 7. 44, F-H).

Family 6. Urceolariidae. This family includes specialized ectoparasites
and endoparasites in which the oral-aboral axis is often much shortened.
The aboral end is a flattened disc equipped with rings of cuticular ele-
ments (Fig. 7. 45, G). These skeletal elements o£ the basal disc seem to be
composed of scleroproteins and they have no continuity in fission, each
daughter organism forming a new set (68). Although the ribs and den-
ticles (or plates) of the basal disc have sometimes been considered impor-
tant in attachment to the host, their functional significance is somewhat
vmcertain. Cyclochaeta domerguei (146), for instance, is not really at-
tached to its host. The organism is equipped with a series of aboral loco-
motor structures (Fig. 7. 45, H, I) — a posterior row of membranelles, a
row of slender cirri just above the membranelles, and an undulating
velum, a delicate membrane lying above the cirri. The cirri are important
in swimming, whereas the membranelles are responsible for gliding move-
ments, spinning the ciliate counterclockwise, and at the same time holding
it in contact with the host.

Kahl (106) included three genera in the family: Cyclochaeta Jackson (35, 146; Fig.
7. 45, G-I), Trichodina Ehrbg. (49, 68, 87, 161; Fig. 7. 46, I), and Urceolaria Stein (87,
228; Fig. 7. 46, F, G). Hirschfield (87) has discussed the suggestion of Faurc-Fremiet
that Cyclochaeta Jackson should be reduced to a sub-genus of Trichodina Ehrenberg.
This simplification of the family would recognize only two genera: Trichodina, in
which the denticles show projections (Fig. 7. 45, G); and Urceolaria, in which the
denticles (or plates) lack such projections (Fig. 7. 46, G).

Family 7. Vaginicolidae. These are loricate peritrichs which differ from
the Lagenophryidae in that the entire oral end of the body is extended
beyond the mouth of the lorica.

Seven genera have been assigned to the family: Caulicola Stokes (106; Fig. 7. 46, E),
Cothiirnia Ehrenberg (106; Fig. 7. 46, C), Platycola Kent (106; Fig. 7. 46, H), Pyxicola

Ciliophora 411

Kent (106; Fig. 7. 46, D), Thuricola Kent (106; Fig. 7. 46, J), Thuricolopsis Stokes
(106), and Vaginicola Ehrenberg (106; Fig. 7. 46, A, B).

Family 8. Vorticellidae. This family includes four genera of typically
sessile forms which develop contractile stalks.

Fig. 7. 48. A, B. Vorticella cousonia Stokes, x700 approx.: extended and
contracted specimens; contraction involves the attenuated aboral portion of
the body but not the stalk proper (after Faure-Fremiet). C. Carchesium
polypiuiuu (Linn.) Kahl, l^ranching pattern of large colony, zooids not
shown; diagrammatic (after Kahl). D. Zoothamnium adamsi Stokes, con-
tinuous stalk-muscle; zooids about QQfi long (after S.). E. Carchesium lim-
neticitni Svec, portion of colony showing separate stalk-muscles in individual
stalks; x200 approx. (after Faure-Fremiet). F. Stalked cyst of Zoothanuiiuni
arbuscula, x215 approx. (after Furssenko).

Intrastyhiin Faure-Fremiet (106) includes ectocommensals, either solitary or forming
small colonies. In Carchesium Ehrenberg (61, 106; Fig. 7. 48, C, E), the stalk of each
zooid in the colony is independently contractile. Vorticella Ehrenberg (11, 106, 159;
Fig. 7. 47, A-I; 48, A, B) contains solitary types. In the colonial Zoothamnium Ehren-
berg (70, 106, 200; Fig. 7. 48, D, F), there is a continuous stalk-muscle.

Order 4. Chonotrichida.^ These are ectocommensals, mostly on actively
swimming Crustacea. Except for Trichocluma lecythoides (154) from the

* The life-cycles of several species have been traced in a recent paper by Y. Guilcher
(1951. Ann. Sci. Nat., ZooL, Ser. 11, T. 13: 33).

412 Ciliophora

California coast, these ciliates have been reported from European waters.
Species of Spirochona occur on fresh-water gammarids but the other
genera all seem to be marine.

The body is usually more or less vase-shaped, and is attached to the
host by either a basal disc or a fairly short stalk. The peristome lies at
the upper end of the body and is usually surrounded by a thin-walled
funnel, near the base of which several rows of cilia extend to the cyto-
stome. The body is generally constricted at the base of the funnel. The
funnel in Kentrochona is a simple structure with a continuous wall (Fig.
7. 49, F). More often, the wall is incomplete and one end is rolled up to
form a secondary spiral funnel (Fig. 7. 49, A, H). Stylochona is unusual
in that a wide funnel of the Kentrochona-type surrounds an inner and
apparently separate funnel (Fig. 7. 49, B). The general surface of the body
is usually not ciliated. The contractile vacuole opens into the pharynx
(vestibule), as in the Peritrichida.

Budding and conjugation (with oral ends in contact) have been re-
ported. In Trichochona lecythoides there is a lateral pouch ("marsu-
pium") into which the developing bud extends. As the bud grows, it
protrudes from the marsupium and finally becomes separated from the
parent (154). The bud in Cliilodochotia quennerstedti (82) is set free as
a migratory larva with a ciliated ventral surface, an apparently undif-
ferentiated cytostome, and a scopula-like organelle which will produce
the stalk of the adult (Fig. 7. 49, J). Similar migratory stages have been
reported for species of Heliochona and Spirochona (82).

The relationships of the Chonotrichida remain somewhat uncertain
and further work is needed on the morphology and life-cycles. However,
the present scanty information is believed by some workers to indicate
that this group is more closely related to the Holotrichida than to any
other ciliates (62). Three families have been recognized by Mohr (154).

Family 1. Chilodochonidae. This family includes Chilodochona Wal-
lengren (82, 106; Fig. 7. 49, I, J) in which there is no very marked con-
striction ("neck") at the base of the funnel .A well-developed stalk is
characteristic. The "funnel" is rudimentary and the peristome is better
described as a groove bordered by two lips. This type of organization
would require a rather simple metamorphosis of the migratory stage.

Family 2. Stylochonidae. Funnels are well developed but are not spi-
rally twisted. A stalk may or may not be present.

Four genera have been assigned to the family: Heliochona Plate (106; Fig. 7. 49, E),
Kentrochona Rompel (106; Fig. 7. 49, F), Stylochona Kent (106; Fig. 7. 49, B), and
Trichochona Mohr (154; Fig. 7. 49, G).

Fam,ily 3. Spirochonidae. The funnel is folded into spirals and there is
no stalk. The single genus, Spirochona Stein (106, 202, 207; Fig. 7. 49, A,
C, D, H), includes species from fresh-water gammarids.

Ciliophora 413

Fig. 7. 49. A. Spirochona patella Swarczewsky, x248 (after S.). B. Stylo-
chona coionata Kent, about 60/^ long (after K.). C, D. A stage in conjugation,
and the formation of two buds in Spiroclio7ja elegans, schematic (after
Swarczewsky). E. Heliochona sessilis Plate, about 60/i long (from Kahl, after
Wallengren). F. Keiitrochona nebaliae Rompel, a loricate type about 40^^
long; pharynx and macronucleus shown in outline (from Kahl, after R.). G.
Trichochona lecythoides Mohr, x83 (after M.). H. Spirochona elegans Swar-
czewsky, x248 (after S.). I, J. Chilodochona quennerstedti Wallengren: stalked
form (I), showing basal granules of peristome and the macronucleus, x750;
migratory larva (J), showing cytostome, ventral ciliature which will become
the peristomial cilia of the adidt, and the postero-lateral scopula-like organ
which will secrete a stalk during metamorphosis; x875 (after Guilcher).

CLASS 2. SUCTOREA

The form of the body, in different species, may be approximately
spherical, conical, club-shaped, cylindrical, vermiform, or irregularly
branching. The most obvious features of the group are the presence of

414 Ciliophora

tentacles and the absence of cilia in the mature stage. Even the tentacles
are lacking in Endosphaera, which includes endoparasites of certain
ciliates.

Tentacles may be distributed over the surface or they may arise in

Fig. 7. 50. A, B. Tentacles of Echinophrya horrida, extended and partly
retracted; x750 (after Swarczewsky). C, D. Tentacles of Tokophrya lemnarum,
extended, and during feeding; x2000 approx. (after Noble). E. Tentacle of
Discoplirya piscijormis: c, internal canal; le, lateral expansions of the pel-
licle; p, pellicle; sr, spiral ridge in pellicle; t, tip of tentacle containing pores
(?); uc, undulations in wall of the internal canal; diagrammatic (after
Dragesco and Guilcher). F. Changes in form observed in tentacles of Sphae-
rophrya magna; diagrammatic (after Wang and Nie). G. Attachment of
stalk to body in Acineta conimensalis; x495 (after Swarczewsky). H. Dendro-
cometes paradoxus feeding on several ciliates; x295 approx. (after Pestel). I.
Capture of a hypotrich by Tokophrya lemnarum, x268 approx. (after Noble).

clusters or from lobes or extensible arms. Two varieties are known. One
type is capitate (Fig. 7. 50, CT), ending distally in a flattened or rounded
expansion. The other type tapers more or less to a point (Fig. 7. 50, A,
B). In at least some species, the tentacles contain an inner tube (Fig, 7.

Ciliophora 415

50, C, E) which extends into the endoplasm for a short distance. The
tentacles adhere to a suitable ciliate which comes in contact with them
and are powerful enough to hold prey much larger than the captor (Fig.
7. 50, H, I). Prompt paralysis of the captured organism has often been
reported. Shortly after contact, protoplasm of the prey starts flowing down
the tentacle to the base of the tube, where food vacuoles are formed.
Whether the pellicle of the prey is ruptmed by suction or undergoes lysis
upon contact with the tentacle is uncertain. Ingestion is rapid. Tokophrya
le.jnnoriim, for instance, ingests Eiiplotes patella in about fifteen minutes
(156).

The flow of material through the tentacle during feeding suggests the
exertion of suction, the source of which has remained an intriguing prob-
lem. Perhaps it is significant that activity of the contractile vacuole is
increased about five-fold in Tokoplirya infusionum as the organism begins
to feed (184). Dragesco and Guilcher (53) have noted, by means of phase-
contrast microscopy, that the wall of the inner canal may undergo con-
tractions suggesting a sort of peristaltic activity. Whether such activity
plays a major part in ingestion is not yet certain.

The suctorian stalk, present in many species, is always non-contractile
although not necessarily homogeneous in structure (156). The upper end
of the stalk may be expanded as a small cup in which the base of the
body rests, or in other cases, the distal end of the stalk fits into a depres-
sion in the body (Fig. 7. 50, G). In the metamorphosis of ciliated larvae,
the stalk apparently arises from an organelle analogous to the scopula
of Peritrichida (Fig. 7. 51, L).

Some of the Suctorea are equipped with a secreted lorica which is often
open distally, leaving the apical end of the body free (Fig. 7. 56, A), or
may be a fairly heavy wall enclosing the body as in Squalophrya macro-
styla (Fig. 7. 53, H, I).

The relationships of the Suctorea to ciliates are indicated in the life-
cycles of most species. Reproduction typically involves budding, either
internal (Fig. 7. 51, N-P) or external. Although it appears to be unusual,
both internal and external budding may occur within a single species, as
reported for Anarma inultiriiga (80). The bud usually develops into a
ciliated larva (Fig. 7. 51, A-K), which after a short period of swimming,
undergoes metamorphosis. After the larval stage of Tokophrya lemnarum
becomes attached (Fig. 7. 51, L, M), a stalk is secreted within a few min-
utes, the tentacles have grown to normal length about fifteen minutes
after they are first detectable, and the adult form is fully developed within
an hour (156). Even more rapid metamorphosis has been noted in
Tokophrya infusionum (185).

The disappearance of cilia during metamorphosis apparently does not
include their basal granules, which persist in the adult stage of Podophrya
fixa (32). In reproduction, the bud receives some of the parental basal

416 Ciliophora

Fig, 7. 51. A-K. Ciliated larvae of various Suctorea: A. Podophrya soli-
formis; B. Tokophrya qiiadripartata; C. Podophrya sandi; D. Podophrya
globulifera; E. Podophrya fixa; F. Parapodophrya denticulata (A-F, sche-
matic, after Kahl); G. Cyclophrya magna, xl70 (after Gonnert); H.
Ephelota geniinipara, silver impregnation of basal granules, schematic
(after Giiilcher); I. Deiidrocouietes paradoxus, x380 (after Pestel); J. Toko-
phrya lenitiaruni, x482 (after Noble); K. Tokophrya infusionum, silver
impregnation of basal granules, x750 approx. (after Guilcher). L, M. Toko-
phrya lemnarum, larva shortly after attachment (L), and early metamor-
phosis (M), x482 (after Noble). N. Endogenous buds in Gorgonosoina
arbuscula, xI65 (after Swarczewsky). O. Endogenous buds in Acineta
corniita, x248 (after Swarczewsky). P. Emergence of larva in Dendrocometes
paradoxus, x600 (after Pestel).

Ciliophora 417

Fig. 7. 52. A. Acineta livadiana Mereschkowski, x548 (after Wang and
Nie). B. Mtiltifascictilatuin elegans Goodrich and Jahn; body, 50-90x20-50/*
(after G. & J.). C. Tnkophrya lemnarum Stein. x268 (after Noble). D.
Acinetopsis elegn7is Swarczewsky. xl65 (after S.). E. Acinetides xmriaris Swar-
czewsky, xl65 (after S.). F. Tokopliryopsis gigantea Swarczewsky, xl65 (after
S.). G. Thecacineta baikalica Swarczewsky, x248 (after S.). H. Acineta
corniita Swarczewsky, x248 (after S.).

granules, which then multiply and give rise to the larval cilia. If this case
may be considered representative, there is thus a genetic continuity of
basal granules throughout the life-cycle.

In the life-cycle of Podophrya fixn (59), an intermediate stage inter-
venes between the larva and the adult, parasitic on Nassula ornata (Fig.
7. 56, F). The result of metamorphosis is a Sp}werophrya-st3.ge which floats
until it makes contact with its ciliate host. Endospltaera also includes un-
usual types with an endoparasitic adult, embedded in the cytoplasm of a

418 Ciliophora

..\vJf^ l.fm

Fig. 7. 53. AD. Penetration of a ciliate host by Endosphaera engel-
manni; stained preparations; x600 (after Noble). E. Anarma brei'is Good-
rich and Jahn; body about 125x75^ (after G. & J.). F. Cometodendron
digitatuin Swarczewsky, xl65 (after S.). G. AUantosoma intestinalis Gas-
sovsky, from large intestine of horse; x854 (after Hsiung). H, I. Squalo-
phrya macrostyla Goodrich and Jahn, a loricate type, lateral view and
cross-section; body about 90x40/i (after G. & J.). J. Dendrocometes para-
doxus Stein, x285 (after Pestel).

ciliate host (138). In reproduction, the parasite produces a typical ciliated
larva which is set free as a migratory stage and later invades a new host
(Fig. 7. 53, A-D).

In addition to the common occurrence of ciliated larvae, transforma-
tion of the adult into a migratory stage also may occur, as in Podophrya
parasitica (Fig. 7. 56, D). This migratory stage (59), in tinn, becomes

Ciliophora 419

Fig. 7. 54. A. Lernaeoplirya cnpitata Perez, with branched macronucleus;
x83 (after Gonnert). B. Baikalodeudron augustatuin Svvarczewsky, xl40
(after S.). C. Dendrosoma radians Ehrbg., x68 (after Gonnert). D. Gorgono-
soma arbusmla Swarczewsky, portion of a yoinig specimen, x31 (after S.).
E. Baikalophrya acaiithogammari Swarczewsky, x248 (after S.). F. Dendro-
somides truncafa Dons, x200 (after D.).

attached by development of a stalk, loses its cilia, and develops tentacles
on its upper surface to become a Paracineta-sta.ge (Fig. 7. 56, G). This
stage does not feed and apparently is a temporary stage preceding encyst-
ment (Fig. 7. 56, H).

Conjugation, comparable to that in typical ciliates, has been described
in several genera, including Acineta (148), Dendrocometes (86), and
Tokophrya (156). In contrast to conjugation in Tokophrya lemnarum,

420 Ciliophora

in which the two conjugants eventually separate much as in ciliates (156),
complete fusion and the production of a single synkaryon have been
reported in Lernaeophrya capitata (79).

Encystment is known in a number of species. In Tokophrya leinnarum
(156), encystment involves the deposition of a transparent secretion, at
first basally, and finally over the apical end of the body. Specimens in
early encystment resemble certain of the loricate Suctorea. Precystic with-
drawal of the tentacles has not been observed. Instead, these structures
remain matted over the apical surface and are engulfed by the material
secreted to form the cyst membrane.

Taxonomy

The Suctorea, which are probably more closely related to the gymno-
stomatous Holotrichida than to other ciliates (62, 103), seem to have
undergone little basic diversification in the course of their evolution. Since
there seems to be no logical basis for differentiating orders, the group has
usually been divided into a number of families. Even this simple arrange-
ment might be more satisfactory if some of the families v.ere more sharply
defined so as to take care of genera Vvhich show apparently intermediate
combinations of characteristics. Perhaps a more intensive study of life-
cycles, with detailed comparisons of the migratory larvae, might yield
useful information. For example, it is interesting that larvae of Podophrya
soliformis and Parapodophrya denticulata (Family Podophryidae) have
been described with a polar circlet of cilia similar to that reported for
Discophryn cybistri (Family Discophryidae); the larva of Podophrya sandi,
with an equatorial belt of cilia resembling that noted in species of Toko-
phrya (Family Acinetidae); larvae of Podoplirya globidifera, P. fixa, and
P. parasitica, with ciliary rows w'hich parallel the long axis of the body
instead of encircling it transversely.

Family 1. Acinetidae. This family is characterized by endogenous bud-
ding and by the possession of capitate tentacles, usually arranged in
groups. A lorica is often present, and a stalk may be present or absent.

The following genera have been included in the family: Acineta Ehrbg. (148, 206;
Fig. 7. 52, A, H), Acinetides Swarczewsky (206; Fig. 7. 52, E), Acinetopsis Robin (206;
Fig. 7. 52. D), AUantosoma Gassovsky (90; Fig. 7. 53, G), Anarma Goodrich and Jahn
(80; Fig. 7. 53, E), Dactylophrya Collin (40), Endosphaera Engelmann (138; Fig. 7.
53, .\-D), Multijasciculatum Goodrich and Jahn (80; Fig. 7. 52, B), Paracineta Collin
(40), Poitsia Chatton and Lwoff (23), Pseudogemma Collin (40), Soleiwphrya ClaparMe
and Lachmann, Squalophrya Goodrich and Jahn (80; Fig. 7. 53, H, I), Tachyblaston
Martin (148), Thecacineta Collin (206; Fig. 7. 52, G), Tokophrya Biitschli (156, 206;
Fig. 7. 52, C), Tokophryopsis Swarczewsky (206; Fig. 7. 52, F).

Family 2. Dendrocometidae. These stalkless forms undergo endogenous
budding. Capitate tentacles may be distributed over the surface or local-
ized on slender extensions of the body.

Ciliophora 421

The following genera have been referred to this family: Cometodendroji Swarczewsky
(204; Fig. 7. 53, F), Dendrocometes Stein (168, 204; Fig. 7. 53. J), Discosoma Swarczewsky
(204), and Stylocometes Stein.

Farnily 3. Dendrosomidae. The stalkless body is irregular in form and
often branched, and the basal surface is usually attached to the sub-

Fie 7 55 A. Discophrya longa Swarczewsky. xl65 (after S.). B. Echi-
rwplfrya' hor'rida Swarczewsky. x248 (after S.). C. Sphaerophrya magna
Maupas, xl65 (after Wang and Nie). D. Ephelota gemmipara (Hertwig)
Biitschli, x345 (after Wang). E. Cydophrya magna Gonnert, showing
macronucleus and several raicronuclei; x255 (after G.). F. Tnchophrya
epistylides Claparede and Lachmann. x210 (after Gonnert). G. Platophrya
rotunda (Hentschel) Gonnert. containing a bud; stained preparation; xiHb
(after G ) H. Stylophrya polymorpha Swarczewsky, x248 (after S.). I. Ex-
ogenous formation of a vermiform bud. characteristic of Ophryodendron;
diagrammatic (after Collin).

422 Ciliophora

Fig, 7. 56, A. Paracineta pleuromammae Steuer, lorica 57-114;* (after S.).
B. Parapodophrya atypica Gonnert, attached to a ciliate; x900 (after G.).
C-H. Podophrya parasitica Faure-Fremiet: C. Several specimens attached to
Nassula ornata, xll6 approx. D. Migratory stage formed by direct transforma-
tion of the adult. E. Ciliated larva produced by budding. F. Specimen at-
tached to Nassula ornata, tentacles apparently penetrating the pellicle of
the host. G. The Paracineta-sia^e, which does not feed. H. Encysted form,
derived from the Paracineta-stn^e (after Faure-Fremiet).

Stratum. The tentacles are arranged in clusters. Reproduction typically
involves endogenous budding, either single or multiple.

The family includes the following genera: Astrophrya Awerinzew, Baikalodendroji
Swarczewsky (203; Fig. 7. 54, B), Baikalophrya Swarczewsky (203; Fig. 7. 54, E), Den-
drosoma Ehrenberg (79, 86a; Fig. 7. 54, C), Dendrosomides Collin (40; Fig. 7. 54, F),
Gorgonosnma Swarczewsky (203; Fig. 7. 54, D), Lernaeophrya P^rez (79; Fig. 7. 54, A),
Platophrya Gonnert (79; Fig. 7. 55, G), Rliabdophrya Chatton and Collin, Staiirophrya

Ciliophora 423

Zachaiias. Stylophrya Swarczewsky (203; Fig. 7. 55, H), Trichophrya Claparede and
Lachmann (79; Fig. 7. 55, F).

Family 4. Discophryidae. Endogenous budding and capitate tentacles
are characteristic, although some species with pointed tentacles have been
assigned to the family (205). A lorica is lacking and a stalk may or may
not be present.

The following genera have been referred to the family: Choanophrya Hartog (40),
Cyclophrya Gonnert (79; Fig. 7. 55, E), Discophrya Lachmann (205; Fig. 7. 55, A),
Echmophrya Swarczewsky (205; Fig. 7. 55, B), Rhyncheta Zenker, Rhynchophrya
Collin (40), and Thaumatophrya Collin (40).

Family 5. Ephelotidae. These are stalked marine Suctorea with capitate
or pointed tentacles. Budding is typically exogenous and may be multiple.
A lorica may or may not be present.

Two genera, Ephelota Wright (148; Fig. 7. 55, D) and Podocyathus Kent, have been
referred to the family.

Family 6. Ophryodendridae. In these marine forms the tentacles are
concentrated on one or more mobile proboscis-like extensions. In addi-
tion to the usual type of larva, so-called vermiform buds, with no tentacles
or proboscis, may be produced by exogenous budding (149).

The type genus is Ophryodendron Claparede and Lachmann (40, 149; Fig. 7. 55, I).

Family 7. Podophryidae. Reproduction involves external budding, or
in some cases "fission" in which the two daughter organisms are almost
equal in size. A stalk and a lorica may be present or absent.

The following genera have been referred to the Podophryidae: Lecanophrya Kahl,
Metacmcta Biitschli, Ophryocephalus Wailes, Paracineta Collin (40, 191; Fig. 7. 56, A),
Parapodophrya Kahl (79. 103; Fig. 7. 56, B), Podophrya Ehrljg. (59, 178; Fig. 7. 56,
C-H), Sphaerophrya Claparede and Lachmann (Fig. 7. 55, C), Spelaeophrya Stammer
(190), and Urnula ClaparMe and Lachmann.

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424 Ciliophora

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VIII

Physiology

Nutritional requirements of Protozoa
Pine cultures as material for research
General types of nutrition
The determination of food requirements
Autotrophic nutrition
Mineral requirements
\'itamin requirements

Thiamine

Riboflavin

The pyridoxine complex

Pantothenic acid

Nicotinic acid

Biotin

Pteroylglutamic acid and /)-aminohen-
zoic acid

Nucleic acid derivatives

Ascorbic acid

Sterols

Hematin

Vitamin Bij (cyano-cobalamin)

Protogen

Biosynthesis of vitamins
The requnements of various groups

Cryptomonadida

Phytomonadida

Euglenida

Protomastigida

Trichomonadida

Sarcodina

Ciliates

Oxygen relationships and oxidations
Ecological distribution
Oxidation-reduction potentials
Oxygen consinnption
Respiratory quotients
Oxidations

The cytochrome system

Pyridine nucleotide enzymes

Diphosphothiamine enzymes

Flavoprotein enzymes

Pyridoxine enzymes

Peroxidase and catalase

Glutathione

Pantothenic acid enzymes
Adenosine phosphate system
Tricarboxylic acid cycle

Digestion

Food vacuoles
Digestion of proteins
Digestion of carbohydrates
Digestion of lipids

Nitrogen metabolism

Carbohydrate metabolism

Synthesis of carbohydrates and lipids

Contractile vacuoles in hydrostatic regula-
tion
The vacuolar cycle

Growth of Piotozoa

Individuals and populations

Initial stationary phase

1 he lag phase

Phase of logarithmic growth

Phase of negative growth acceleration

Phase of maximal density

Phases of death

Size of the inoculum in relation to
growth
Initial pH of the culture medium
Temperaluie
Light and darkness
Effects of certain toxins and venoms
Effects of certain therapeutic drugs
Effects of carcinogenic hydrocarbons

Effects of irradiation

Locomotion

Amoeboid movement
Flagellar locomotion
Swimming in ciliates

Responses to stimuli
Responses to light
Responses to electric current
Responses to temperature

Literature cited

428

Physiology 429

NUTRITIONAL REQUIREMENTS
OF PROTOZOA

Pure cultures as material for research

HE ESTABLISHMENT OF various spccics in cultures free from
other inicroorganisms opened a new era in the study oi protozoan nutri-
tion, making possible for the first time a realistic approach to the deter-
mination of basal food requirements. As a result, the investigation of
metabolic activities in Protozoa is steadily expanding, and is leading to
increasingly precise interpretations.

One of the important factors in this rapidly developing field has been
the availability of many phytofiagellates in pine culture. For this, much
credit is due E. G. Pringsheim^ whose unfailing determination was
resj^onsible for maintaining an invaluable collection intact through a
number of trying years. The phytofiagellates are particularly favorable
material for investigation because their absolute requirements are much
less extensive than those of higher Protozoa. Consequently, they afford
the most direct routes to the determination of mineral requirements, and
also the need for certain vitamins and organic foods. The relatively simple
requirements of certain phytofiagellates (Table 8. 1), in contrast to the
complex brews needed by higher I^rotozoa, also should expedite the study
of metabolic pathways. Furthermore, the diversity of phytofiagellates, with
respect to the j^ossession or lack of chlorophyll and the presence or absence
of holozoic habits, encourages consideration of the evolutionary and taxo-
nomic aspects of protozoan nutrition.

Until recently, ciliates and Zoomastigophorea had been grown only in
broths of unknown chemical composition which endangered the validity
of conclusions concerning basal food requirements. The development of
almost completely defined media (Table 8. 1) for at least a few of these
higher Protozoa (121, 282, 349, 540, 568) insures much the same results
as those now obtainable with many phytofiagellates.

Aside from the intrinsic interest to protozoologists, the study of pro-
tozoan nutrition promises significant contributions to the general fields
of biochemistry and physiology. In the study of plant nutrition, the rap-
idly growing chlorophyll-bearing flagellates are readily adaptable to the
investigation of various fundamental problems. The study of animal nu-
trition might be served in the same way by the typical animals among
the Protozoa. As microscopic animals, which approach bacteria in rates
of growth and ease of handling. Protozoa in pure cultures also offer ma-

^ Pringsheim, E. G. 1946. Pure Cultures of Algae (Cambridge University Press).

430 Physiology

TABLE 8. 1. CONSTITUENTS OF CULTURE MEDIA FOR THE
COLORLESS PHYTOFLAGELLATE, CHILOMONAS PARAMECIUM,
AND THE CILIATE, TETRAHYMENA PYRIFORMIS, STRAIN E (121)

Chilomonas Paramecium

Tetrahymena

NH4CI

Arginine

riboflavin

(NH4)2S04

histidine

pteroylglutamic acid

KH2P04

isoleucine

biotin

MgCla • 6H2O

leucine

thiamine

CaCl2 • 2H2O

lysine

choline

FeCls • 6H2O

methionine

yeast nucleic acid

Acetate (or ethyl alcohol)

phenylalanine

protogen

Thiamine

serine

MgS04 • 7HoO

threonine

K2HPO4

tryptophane

CaCl2 • 2H2O

valine

FeCls • 6H2O

dextrose

Fe(NH4)2(S04)2 • 6H2O

sodium acetate

CuCb • 2H2O

pantothenate

MnCl. • 4H2O

nicotinamide

ZnCl2

pyridoxine

(In addition to the listed components, various trace elements are present as impurities.)

terial for studying animal metabolism under conditions controllable to a
degree not attained with tissues of higher animals. In addition, precise
control of the food supply favors use of these organisms in the search for
new vitamins, as in the discovery of protogen (540), as well as in micro-
biological assays of known growth-factors (230, 579) and amino acids
(494). To the parasitologist and the pathologist, parasitic Protozoa in
pure cultures offer unique opportunities for correlating metabolic activ-
ities of parasites with susceptibilities to chemotherapeutic agents and with
reactions of the host's tissues to infections. To the explorer, these organ-
isms extend a challenge to trace nutritional evolution from the possibly
complete synthesis of needed vitamins to an essentially complete depend-
ence upon external sources. Did plant-like Protozoa suddenly become
"animals," with wholesale loss of synthetic abilities, or did they lose their
original abilities one by one as evolution tempted them toward the animal
kingdom? Or have the phytoflagellates arisen from more animal-like Pro-
tozoa, acquiring in their evolution various synthetic powers unknown to
their ancestors?

General types of nutrition

For many years, general types of nutrition were classified merely as
autotrophic (or holophytic), saprozoic, and holozoic, the last two repre-
senting varieties of heterotrophic nutrition. By definition, autotrophs
could grow in inorganic media while heterotrophs required organic foods.
With the exception of saprozoic types. Protozoa probably are not limited

Physiology 431

to one method in a natural environment. Chlorophyll-bearing species are
often saprozoic and some can grow in darkness. A number of the green
flagellates also ingest solid food, while typically holozoic organisms also
may carry on saprozoic nutrition. The relative importance of one method
or another depends largely upon environinental conditions.

The ecological classification of Kolkwitz and Marsson (301) divides
Protozoa into katharobes, living in water containing almost no organic
matter; and saprobes, found in water containing appreciable quantities of
organic matter. Saprobes are divided into oligosaprobes, mesosaprobes,
and polysaprobes, living in the presence of small, moderate, and large
amounts of organic matter. This classification involves oxygen relation-
ships as well as food supply, since katharobes are typically aerobic, while
polysaprobes are more probably anaerobes or facultative anaerobes.

The earlier results with pure cultures soon demonstrated that these
older concepts were inadequate. For instance, it became clear that the
term, autotroph, could no longer be applied automatically to any green
flagellate that occurs naturally in fairly pure water exposed to light. In
fact, it is not yet certain that the existence of complete autotrophs, as
originally defined, has been demonstrated. Furthermore, some of the
"saprozoic" flagellates proved to be "autotrophic" with respect to nitrogen
sources, although needing organic foods as a source of energy. This situa-
tion furnished the stimulus for several more modern classifications of
protozoan nutrition (99, 176, 343, 358, 459). Although such classifications
were a distinct improvement over the older systems and were a conven-
ience in discussions, it now appears that even the more modern classifica-
tions have a tendency to oversimplify protozoan nutrition. From present
indications, food and vitamin requirements of Protozoa will show many
variations from species to species, so that much more information will be
needed before definitive classification can be attempted.

The determination of food requirements

Two general methods have been followed in the study of proto-
zoan food requirements. In one procedure the experimental media have
been the simplest ones which would support giowth in serial transfers.
In such media, growth is often at a minimum and the organisms do little
more than maintain themselves in successive transfers. In the other gen-
eral procedure, media have been devised to maintain growth at a maxi-
mum and any constituent which cannot be omitted is considered essential
to growth. This method is based upon the generalization that growth of
any species will reach a maximum when all conditions are optimal —
qualitative and quantitative aspects of the substrate, concentrations of
stimulatory and essential growth-factors, concentrations of essential min-
erals, and non-dietary environmental factors.

On a theoretical basis, the first procedure might seem to offer the more

432 Physiology

direct analysis. In a medium reduced to bare essentials, it might be pos-
sible to recognize species capable of synthesizing required materials at a
rate so slow that growth could never reach the maximum attainable in a
rich medium. In the second procedure, with maximal growth as the goal,
slow synthesis of a particular factor might conceivably be overlooked. In
practice, however, the first method has certain limitations. Validity of the
results obviously depends upon purity of the reagents and cleanliness of
the culture vessels. In addition, contamination of the medium with dust
or with volatile materials from the atmosphere of a laboratory could be a
possibly serious source of error. Even minute contaminations might turn

density of

population

^ » * *

-/<

^ » — — *

-■i^^l'i'-'' -'■■"?

^ ^ ^

" /

y/^

^ /

y /

^y J

/ f

yjs

/
/

concentration
— 1 1 1

^supp

ement

■ 1 1 —

Fig. 8. 1. Hypothetical growth responses of a test organism to essential
and stimulatory growth-factors.

the balance in favor of slight growth, with resulting faulty interpreta-
tions of experimental data. Hence, it is essential, in following the first
procedure, to take all possible precautions. In the use of media which
support maximal growth, the influence of minute contaminations would
be less likely to account for positive instead of negative results. Further-
more, the response to graded increments of a given growth-factor can be
traced over a wide range of growth. An approximately linear growth-
response to a vitamin or a mineral in concentrations ranging from zero
to an optimum (Fig. 8. 1) would indicate that the factor is essential.
Omission of a stimulatory substance, on the other hand, would decrease
growth but not prevent it completely.

Physiology 433

Autotrophic nutrition

This general variety of nutrition,- in which inorganic nitrogen is
adequate for growth, is sometimes considered a primitive type which was
gradually lost during the "regressive" evolution of heterotrophs. Another

TABLE 8. 2. REPORTED CASES OF "AUTOTROPHIC" NUTRITION

IN FLAGELLATES

"Chemoauto-

"Photoauto-

Heteroauto-

Species

trophic"

trophic

CRYPTOMONADIDA

Chilomonas paramechtrn

NG(390)

NC(390)

Chilomonas Paramecium

CP(220)

Chilomonas Paramecium

*NC(354)

Chilomonas Paramecium

NC(73)

PHYTOMONADIDA

Chlamydnmonas agloeformis

CP(340)

Chlorogonium el on gat um

CP(321)

C. euchlorum

CP(321)

Eudorina elegans

NC(108)

Haematococcus pluvialis

CP(340)

Haematococcus pluvialis

*CP(420)

Lobomonas piriformis

CP(422)

Polytoma caudatum

*CP(352)

P. obtusum

NC(355)

P. ocellatum

*NC(353)

P. uvella

CP(340, 452)

P. uvella

NC(355)

Polytomella caeca

*NC(353)

EUGLENIDA

Astasia longa (Jalin strain)

NC(505)

NC(503)

Euglena anabaena

CP(105, 175,

Euglena anabaena

NC(109)

E. gracilis

CP(104, 189)

NC(504)

E. klebsii

CP(105)

E. stellata

CP(105)

E. viridis

CP(177), NC(508)

* Supplementary growth-factors said to be required; CP, cotton plugs used in culture
tubes or flasks; NC, glass-covered culture vessels.

view (210, 411) is that the evolution of autotrophic organisms has in-
volved the acquisition of synthetic abilities lacking in more primitive
ancestral types which were dependent upon the environment for critical

- In this chapter, the following terms will be applied to species which can obtain
their required nitrogen from inorganic sources: (1) photoautotroph, or photosynthetic
autotroph, utilizing the energy of light; (2) chemoautotroph, or chemosynthetic auto-
troph, obtaining energy from inorganic substrates; (3) heteroaiitotroph, requiring an
organic source of energy (e.g., acetate, lactate, ethanol).

434 Physiology

organic materials. Whether autotrophic nutrition should be considered
primitive or not, the food requirements of the supposedly autotrophic
phytoflagellates differ considerably from those of other Protozoa.

Chemoautotrophic, photoautotrophic, and heteroautotrophic nutrition
have been reported in various phytoflagellates (Table 8. 2). Three species
have been giown under conditions suggesting chemoautotrophy — two of
the three in glass-covered culture vessels. The investigation of chemo-
autotrophic nutrition illustrates, to an extreme degree, the difficulties in-
herent in the use of media supplying a bare minimum for growth. After
an adequate number of transfers, it may be concluded that the food sup-
ply of the organism is limited to constituents of the test medium — plus
any contaminants absorbed from glassware, introduced by way of dis-
tilled water, stock solutions and dust or absorbed from the air. The first
obvious refinement in technique is the elimination of cotton plugs from
culture tubes or flasks. Although bleached cotton may be inactive as a
source of thiamine or its components (509), plugs of this material con-
tribute significantly to the growth of Chilojnonas Paramecium in an in-
organic medium and the effect is not eliminated by the addition of
thiamine in excess (73). Organic pollution of distilled water and stock
solutions by bacterial growth can be avoided by aseptic techniques or by
the use of a volatile preservative (227). However, other difficulties remain.
Certain inorganic salts used for culture media contain organic contami-
nants, which in terms of carbon balance could account for the observed
growth in an experimental medium (73). Furthermore, there must be a
demonstrable inorganic soiuce of energy. In the case of Chilomonas para-
7necium, there is no significant oxidation of ammonia (73), although
tests have not been reported for several other "chemoautotrophs." Ac-
cordingly, it must be concluded that the evidence for chemoautotrophy
in Protozoa is still inadequate, and that under experimental conditions
now attainable it is seemingly impossible to limit a flagellate to chemo-
autotrophic nutrition.

So far as photoautotrophic nutrition is concerned, it should be much
less difficult to prove that organic contaminants are an unimportant factor
in growth. In this connection, the failure of Chlamydomonas moexvusii
to grow without both light and carbon dioxide — even in inorganic media
supplemented with a variety of nitrogen sources, vitamins and oxidizable
carbon sources — seems very significant (315). Investigations are still
handicapped, however, by inadequate knowledge of qualitative and quan-
titative mineral requirements. "Photoautotrophic" nutrition has been re-
ported in several Phytomonadida and Euglenida (Table 8. 2). In most
cases, culture vessels have been plugged with cotton, but Eudorina elegans
(108) has been maintained in all-glass vessels. Such results (109) also have
supported earlier observations on Euglena anahaena. Similar confirma-
tion is needed for other reports of photoautotrophy. All-glass vessels do

Physiology 435

not eliminate all potential organic contaminants, but it is possible that
such impurities could not account for the observed growth without cotton
plvigs.

In contrast to Euglena anahaena, E. gracilis is said to require the py-
rimidine component (353), and E. pisciformis, both the pyrimidine and
thiazole components of thiamine (107) for growth as "photoautotrophs."
Whether or not any or all of the currently reported photoautotrophs will
eventually prove to be such, it appears at present that certain green flag-
ellates may not be photoautotrophs. For example, failures to demonstrate
"photoautotrophy" have been reported for Euglena deses (105, 188). Such
failures could have been caused by inadequate culture media. On the
other hand, E. deses may represent a more advanced stage in the type of
regressive evolution suggested for E. gracilis (353) and E. pisciformis
(107).

Heteroautotrophic nvitrition was first noted by Pringsheim (452) in
Polytoma uvella. These observations have been repeated and comparable
findings have been reported for P. obtusum (348, 353, 355). The related
flagellates, Polytoma caudatum, P. ocellatum, and Polytomella caeca, have
been grown in inorganic salt and acetate media with supplementary
growth-factors (352, 353, 354). More recently. Astasia longa (503), Euglena
gracilis (504), and Lobomonas piriformis (422) have been maintained as
heteroautotrophs, the green species being incubated in darkness. Chilo-
monas Paramecium, also is a facultative heteroautotroph (73, 220, 390),
although there is one report that supplementary growth-factors are re-
quired under such conditions (354). At present, it appears that certain
flagellates are facultative heteroautotrophs for which thiamine and pos-
sibly other vitamins are stimulatory but not essential, while other species
require one or more supplementary growth-factors under such conditions.
This possibility needs further investigation with careful attention to
mineral requirements and with a variety of substrates.

Mineral requirements

In contrast to older beliefs that some ten or eleven elements are
essential to life, modern investigations have detected about fifty elements
in the tissues of different animals and plants. It remains to be determined
just how many are essential and how many are chance accumulations con-
ditioned by a particular chemical environment. So far as the Protozoa
are concerned, little is known about qualitative and quantitative mineral
requirements. For microorganisms in general, certain metal requirements
are related to particular enzyme systems. Such metals may be integral
parts of enzymes or may serve as "activators" whose exact functions are
not yet understood. Consequently, it is at least conceivable that certain
mineral requirements may vary quantitatively, and possibly even qualita-
tively, in the presence of different substrates.

436 Physiology

Growth in organic media cannot be expected to yield many clues be-
cause such materials contain quite a variety of trace elements. In yeast
extract, for example, barium, bismuth, boron, calcium, chromium, copper,
gold, iron, magnesium, manganese, phosphorus, potassium, silicon, silver,
and strontium have been demonstrated by spectroscopic analysis, and a
preparation of asparagine has contained as many as 21 trace metals (483).
Such difficulties have been illustrated in the investigation of mineral re-
quirements in Tetrahymena pyriformis (292).

For various phytoflagellates, culture media have been prepared from
analyzed reagents and used in all-glass culture vessels. Within such limits
of accuracy, the composition of the medium is known except for possibly
significant contaminations from glassware or other sources. Some of these
media contain, as impurities in the salts, barium, cobalt, chromium, cop-
per, gold, iron, manganese, sodium, zinc, and other metals, in addition to
the intentionally supplied calcium, magnesium, phosphorus, potassium,
sulfur, chlorine, ammonium salts, and carbon dioxide. It has been pos-
sible, from such starting points, to detect a lew trace-mineral require-
ments. The determination of others, merely by selecting different salts so
as to exclude particular elements, has been impossible.

The purification of reagents permits some further progress. The re-
moval of certain trace metals has been accomplished with chelating
agents, such as 8-hydroxyquinoline, which form metal complexes soluble
in chloroform or ethyl acetate (130). In addition, several other methods
for purifying important components of culture media are available (229),
and a number of purified elements and reagents can be obtained com-
mercially.^

Additional advantages are promised by the use of non-toxic chelating
agents in culture media (226, 229). Such substances as citrate and ethyl-
enediamine-tetraacetic acid, which form soluble and fairly stable metal
complexes, minimize and tend to eliminate certain technical difficulties.
A metal-chelate complex forms a sort of metal "buffer" with an action
somewhat analogous to that of pH-buffers. Since the precipitation of
metals as hydroxides, phosphates, or sulfates is prevented, it is possible
to add quantities sufficient for heavy growth. Metals which are toxic
above certain concentrations also can be supplied at levels favoring heavy
growth without danger of toxic effects. Aside from such general improve-
ment of the simpler culture media, chelating agents can be of assistance
in analyzing mineral requirements, rhe addition of a chelating com-
pound to a medium containing essential trace metals in minimal amounts
may induce a metal deficiency which will prevent growth. Incidentally,
materials often supplied as substrates — a-amino acids, glycerol, malate,
annnoniiun salts — also may be active enough as chelating agents to induce

^Johnson, Matthey & Co., Ltd. 1947. Catalogue of standardized substances for spec-
trography, chemical analysis and research (London).

Physiology 437

metal deficiencies. Therefore, they might be considered of little value to
the organism unless allowance is made for chelating activity. Intentional
induction of an inhibitory metal deficiency makes it possible, by trial and
error, to identify various trace elements which seem to be essential (229).
After preliminary qualitative observations, individual requirements can
be analyzed quantitatively by adding an excess of all needed elements ex-
cept one and then determining the amounts of this element which will
compensate for graded increases in the chelating agent. A curve plotted
from such data and then extrajiolated to zero chelate should give a fairly
good approximation of the basal requirement for a particular metal.
There remains to be considered the situation in which a trace element
minutely contaminating a supposedly required metal may actually be the
essential factor (229). 1 his conqjlication can be eliminated, if elimination
is possible, only by the use of highly purified metal sources. However,
the failure of progressive purifications to alter the apparent requirements
quantitatively would suggest that the original indications were valid.

The older techniques were adequate only to the extent of indicating
qualitative requirements for certain metals in a few species. Calcium —
apparently needed by Eiiglena annbaena (109), E. stellata (105), Hyalo-
gonium klebsii (457), Chilomonas Paramecium (395), Oikomonas termo
(192), and Tetrahymena pyriformis (184) — may prove to be a general
requirement. Magnesium, necessary for Chilomonas Paramecium (395)
and Tetrahymena pyriformis (292), is a component of carboxylase and
should be a general requirement. As a constituent of chlorophyll, mag-
nesium also is obviously essential to green flagellates. Iron was foimd to
he a requirement of Chilomonas Paramecium (220), Eudorina elegans
(108), Euglena anabaena (109), Polytoma obUisum and P. uvella (355),
and Tetrahymena pyrijormis (184, 287). Since this metal is a constituent
of cytochromes, cytochrome oxidase, catalase, and peroxidase, it may be
impossible to find Protozoa which do not need iron unless it can be
shown that obligate anaerobes have absolutely no iron requirements.
Phosphorus, essential for Chilomonas Paramecium (423) and Tetrahy-
mena pyriformis (292), is obviously a general requirement for phosphory-
lation of metabolites and vitamins. Observations on the phosphate cycle
in T. pyriformis (123, 497) indicate that during the lag phase of growth
there is a rapid liberation of inorganic phosphate from organic sources,
whereas uptake of inorganic phosphate is more characteristic of logarith-
mic growth. Manganese, which favors growth of Euglena anabaena in
inorganic media (109, 174), and apparently participates in oxidation of
pyruvate and other metabolites by Plasmodium gallinaceum (535), may
be generally needed as an activator of phosphorylases and peptidases and
possibly other enzymes. Potassium, required by Chilomonas Paramecium
(423) and T. pyriformis (292), seems to be needed in certain phosphoryla-
tions and probably is a general requirement. The possible significance of

438 Physiology

sodium, apparently needed by C. paramechnn (423), is uncertain. Sulfur,
as a constituent of several vitamins and amino acids, is presumably essen-
tial. A variety of inorganic salts, cystine, glutathione, and cysteine are
satisfactory sources for C. Paramecium (392). Silicon stimulates growth of
C. Paramecium, (398) and prolongs life in a phosphate-deficient medium
(396). Whether this effect is attributable to silicon or to impurities (73)
in the silicate used, remains to be determined. Vanadium also seems to
accelerate growth of C. Paramecium (25). A need for copper, apparently
a component of ascorbic and phenol oxidases, became apparent in Tetra-
hymena pyriformis when natural products were replaced by purified con-
stituents of culture media (287). Zinc, apparently involved in aldolase,
carbonic anhydrase, and uricase activity, has often been included in cul-
ture media on the assumption that it is essential to protozoan growth.
Cobalt, as a constituent of vitamin Bjo (cyano-cobalamin), is required by
Euglena gracilis (230) and probably various other Protozoa. Molybdenum,
which accelerates nitrogen-fixation by bacteria and seems to be essential
for certain molds, needs investigation as a protozoan requirement.

In summary, fragmentary evidence now indicates that, in addition to
carbon, hydrogen, oxygen, and nitrogen, at least twelve other elements —
calcium, cobalt, copper, iron, magnesiimi, manganese, phosphorus, potas-
sium, silicon, sodium, sulfur, and vanadium — are either stimulatory or
essential to growth of certain Protozoa. In addition, there are reasons for
believing that others, such as molybdenum and zinc, may be important.
Highly purified chemicals and the newer techniques of investigation may
expand the list of required trace elements, and should clarify the status
of some of them in protozoan metabolism. Even so, a complete list of the
basal requirements apparently remains unobtainable with the inorganic
materials and culture vessels now available.

Vitamin requirements^

The requirements of many Protozoa, although incompletely known,
are probably comparable to those of Metazoa. Colpoda steinii (duo-
denaria) needs more than five vitamins, TetraJiyfnena pyriformis needs
at least nine or ten, and not less than six are important in the metabolism
of malarial parasites. At the other extreme, a few phytoflagellates have
been grown in media apparently free from vitamins. There is every reason
to believe that such differences depend upon the ability or inability to
synthesize particular vitamins.

Among the phytoflagellates, Chilomonas Paramecium, in an acetate,
inorganic salt and thiamine medium, synthesizes nicotinic acid and the
diphosphopyridine nucleotide (DPN, or coenzyme I) which contains nico-
tinamide and adenine (223). Microbiological assays of comparable cul-
tures have confirmed the synthesis of nicotinic acid and demonstrated that

* Several reviews of the earlier literature arc available (99, 180, 347, 348).

Physiology 439

of pyridoxal and riboflavin (207). In addition, growth of Tetrahymena
pyriformis on C. Paramecium and on Polytoma ocellatum in similar cul-
ture media (182) suggests that these flagellates are able to synthesize a
variety of vitamins needed by the ciliate. Therefore, it may be assumed,
in the absence of evidence to the contrary, that metabolic activities of the
phytoflagellates involve essentially the same vitamins as do those of the
higher Protozoa. Under favorable conditions, which must include a
medium satisfying essential mineral requirements, some species may be
able to synthesize all of their needed vitamins. Present indications, that
certain other phytoflagellates cannot synthesize at least one or two vita-
mins from simple materials, raise interesting possibilities. Perhaps it will
be feasible, in this group, to trace a series of stages in the development of
multiple vitamin requirements (or multiple losses in synthetic powers) as
represented by ciliates, for example. As the scope of the pure-culture tech-
niques is broadened, the ability to visualize vitamin requirements on a
taxonomic framework may prove very interesting — possibly to the extent
of furnishing clues to the phylogeny of the higher Protozoa. From the
practical standpoint, the determination of vitamin requirements for many
different Protozoa may reveal unsuspected new vitamins and may also
furnish additional tools for luicrobiological assays. Both possibilities have
already been realized to a limited extent.

Some information on vitamin requirements is now at hand for a num-
ber of species (Table 8. 3). Although the present data may be definitive
for a few phytoflagellates, this is far from true for nearly all of the other
Protozoa which have been investigated.

Thiamine. This vitamin is an absolute requirement for certain strains
of ciliates and parasitic flagellates and probably for malarial parasites.
The case of Chilomonas Paramecium is still puzzling. Certain strains
apparently require either thiamine or its thiazole and pyrimidine com-
ponents, while others have been grown in all-glass vessels without added
thiamine on acetate as a substrate (73, 390). Under such conditions, sup-
plementary thiamine markedly increases growth on acetate and becomes
essential instead of stimulatory when pyruvate is substituted for acetate
(73). Thiamine is stimulatory for Polytoma obtusuyn and P. uvella in
simple media, although both will grow without the added vitamin (348).
In the same types of media, certain other colorless phytomonads need
thiazole or both the pyrimidine and thiazole components of thiamine
(Table 8. 3). Several substituted thiazoles and pyrimidine also are active
for Polytomella caeca and Chilomonas Paramecium (348). In addition,
heavy growth of Euglena gracilis var. bacillaris, in an amino acid and
inorganic salt medium containing vitamin Bjo, depends upon an adequate
concentration of thiamine (230).

Riboflavin. Earlier reports of growth-acceleration in ciliates (120, 181,
183, 283) were soon followed by evidence that this vitamin is essential for

440 Physiology

TABLE 8. 3. VITAMIN REQUIREMENTS OF VARIOUS PROTOZOA

Species

Vitamins

PHYTOMASTIGOPHOREA

Chilomonas Paramecium
Euglena gracilis
E. piscijorrnis
Haematococcus pluvialis
Poly torn a caudatum
P. obtusum
P. oc ell a turn
P. uvella
Polytomella caeca

ZOOMASTIGOPHOREA

Eutrichomastix colubrorum

Leishmania agamae

L. ceramodactyli

L. donovani

L. tropica

Leptomonas ctenocephali

L. pyrrhocoris

Strigomonas culicidarum

S. fasciculata

S. muscidarum

S. oncopelti

Trichomonas columbae

T. foetus

T. gallinarum

T. vaginalis

Trypanosoma cruzi

T. lewisi

T. rabinnwitchi

SARCODINA

Acanthamoeba castellanii

SPOROZOA

Plasmodium gallinaceum
P. knowlesi
P. lophurae

CILIATEA

Colpidium campylum
Colpoda duodenaria
Pleurotricha lanceolata
Stylonychia pustulata
Tetrahymena pyrijormis

A, B (353); none (73)

B (353); N (230)

A, B (107)

C, K (420); none (340)

A (352, 353)

none (355)

A (353)

none (355)

A, B (351)

K, L, Q (49)

M, Q (366)

K, M, Q (366)

M (366)

C, M (366); G, D, E, M (566)

C, M (366)

M (366)

C (366)

K (50), L (47)

K, L, Q (48)

K, Q (52)

F (303), L (537)

K, M, Q (366)

M (432)

M (410)

A, B (345)

c, g, j (535)
d,j, p(ll)
f, h (556)

Q (437)

C, E, G (545); D, F, Q (139)

D, E, Q (317)
D, E, Q (317)

C (120, 122, 179, 183, 357);

D (179, 289, 290); E, F, G (121, 289, 290);

I (122, 275); J (284); O (121, 540)

KEY: A, thiazole component of thiamine; B, pyrimidine component of thiamine;
C, thiamine; D, riboflavin; E, pyridoxine; F, pantothenic acid; G, nicotinic acid or
nicotinamide; H, biotin; I, pteroylglutamic acid; J, nucleic acid components (purines,
pyrimidines) ; K, ascorbic acid; L, sterols; M, hematin; N, cyano-cobalamin (vitamin
B12); O, protogen; P, /)-aminobenzoic acid; Q, unidentified growth-factors. Small letters
indicate probable requirements.

Physiology 441

Colpoda steinii (diiodenaria) (139) and Tetrahymena pyriformis (179,
289, 290) and probably for malarial parasites (11). The synthesis of ribo-
flavin by Chilomonas Paramecium (207) suggests the probable importance
of this vitamin in phytoflagellate metabolism.

The pyridoxine complex. In the earlier investigations, a stimulation of
growth by pyridoxine was noted in several ciliates (120, 283, 317). A
ciliate, Colpoda steinii (545), also was the first protozoon shown to need
pyridoxine. Tetrahymena pyriformis has since been found to require pyri-
doxine, pyridoxal, or pyridoxamine, the two derivatives being 100-500
times as active as pyridoxine (289), a relationship similar to that pre-
viously reported for certain bacteria. Pyridoxine proved to be a com-
ponent of "Factor 11" (287), a concentrate of natural origin previously
found essential to growth of T. pyriformis (92). Since pyridoxine inhibits
the action of quinine and atebrin against Plasmodium cathemerium and
P. lophurne in ducklings (519), the vitamin probably is a requirement of
malarial parasites. Among the phytoflagellates, Chilomonas Paramecium
synthesizes pyridoxal (207).

Pantothenic acid. In the first tests on Protozoa, Elliott (1 17) found that
growth of T. pyriformis was accelerated, within the pH range 5.5-6.5, by
a concentrate of pantothenic acid. Garnjobst, Tatum, and Taylor (139)
next found pantothenate essential for Colpoda steinii, and it now appears
that Tetrahymerm pyriformis has the same requirement (290, 121). Sup-
plementary evidence involves inhibition of growth of T. pyriformis by
a-methyl-pantothenic acid and reversal of the efl^ect by pantothenic acid
(502). This vitamin also favors survival of P. lophurae (556) in vitro.
Furthermore, a pantothenate deficiency in chickens decreases the severity
of infections with P. gallinaceum, and dosage with certain analogues is
more effective than quinine therapy (28). Pantothenate analogues are
active likewise against Trichomonas foetus, T. gallinae, and T. vaginalis
in pure cultures (256). Supplementary pantothenate in the diet of rats
also increases the populations of Eimeria nieschulzi (15).

Nicotinic acid. Colpoda steinii (545) was the first protozoan species
shown to require nicotinic acid. Later on, Tetrahymena pyriformis, at
first believed to grow without nicotinic acid (283), was found to need the
vitamin (290). Diphosphopyridine nucleotide (DPN), which contains
nicotinamide, also has been demonstrated in T. pyriformis (512). As a
component of DPN and TPN, nicotinamide also is involved in oxidative
metabolism of Plasmodium gallinaceum (535) and Trypanosoma hippi-
cian (194). Among the phytoflagellates, growth of Eugena viridis in an
asparagine medium (110) and that of Chilomonas Paramecium as a
heteroautotroph (425) are stimulated by nicotinic acid. The latter also
synthesizes this vitamine (207, 223).

Biotin. Although a biotin deficiency has decreased division-rate and

442 Physiology

reduced the density of populations (283), evidence that this vitamin is
essential for Tetrahymena pyriformis is still lacking (290). However, it
seems to be important in the metabolism of malarial parasites (535, 556).

Pteroylglutamic acid and p-aminobenzoic acid. In the first report on
Protozoa, Kidder (275) found this vitamin essential to growth of T.
pyriformis. Calculated on the basis of free pteroylglutamic acid, the
vitamin has about the same activity as it conjugates, pteroylglutamylglu-
tamic acid and pteroylhexaglutamic acid (289). Apparently, p-amino-
benzoic acid cannot be substituted for folic acid. This ability to use
conjugates and the holozoic nature of T. pyriformis suggest the probable
value of this ciliate in assays of natural products.

The action of sulfadiazine against P. gaUinaceum in chickens is re-
versed by pteroylglutamic acid (166); p-aminobenzoic acid has the same
effect on sulfonamides used against P. lopliurae (520) and P. gaUinaceum
(376). Some of these sulfonamides, such as sulfanilamide (68, 562) and
sulfathiazole (562), inhibit oxygen consumption of malarial parasites. In
addition to the evidence obtained with analogues, growth of P. knozolesi
is stimidated in vitro by p-aminobenzoic acid (8, 11). For the phytoflagel-
lates, a reversal of sulfanilamide action by p-aminobenzoic acid has been
reported in Polytojuella caeca (359).

Nucleic acid derivatives. Ribonucleic acid contains certain purines
(adenine, guanine), pyrimidines (cytosine, uracil) and D-ribose; in de-
soxyribonucleic acid, uracil is replaced by thymine and D-ribose by d-2-
desoxyribose. Several nucleic acid derivatives have been tested on Tetra-
hymena pyrijorynis (284). Together with folic acid, the purines (guanine
apparently being essential) form the active components of 'Tactor I," an
undefined concentrate previously found essential to growth of T. pyri-
formis (92). Although adenine and hypoxan thine show a guanine-sparing
action (286), neither can replace guanine. However, the inhibitory action
of an adenine analogue (adenazolo) on growth of T. pyriformis is re-
versed specifically by adenine (293). Among various substituted purines,
1-methyl-guanine is about 75 per cent as active as guanine, several are
inert, and others are inhibitory (291).

'Tactor III," another concentrate which appeared necessary to growth
of T. pyriformis, has been resolved into the pyrimidine derivatives, uracil
and cytosine, or their ribosides or ribonucleotides (281, 291, 293). T.
pyriforjnis is believed to synthesize thymine from non-pyrimidine precur-
sors in reactions involving pteroylglutamic acid (293), as reported previ-
ously for bacteria (539).

Ascorbic acid. Although a need for this vitamin has been attributed to
Haematococcus pluvialis and several parasitic flagellates (Table 8. 3),
there is at present no conclusive evidence that ascorbic acid is essential
to growth of Protozoa.

Sterols. Several flagellates (Table 8. 3) require sterols, a requirement

Physiology 443

which may be satisfied by cholesterol or certain other sterols. Among 66
different sterols tested on Trichomonas gallinae (T. cohimhae), com-
parable activity was shown by cholesterol, cholestanol, sitosterol, and
several others. Ergosterol was moderately active if not heated, whereas
irradiated ergosterol ("vitamin D") was inactive (47). Cholesterol also
seems to be required by Entamoeba histolytica (530) and is a possible
requirement of Trichomonas vaginalis (537). Growth of Colpidium cam-
pylum is slower with certain concentrations of cholesterol but reaches a
greater density than in the control medium (541).

Hematin. That species of Trypanosojua and related flagellates need
blood in culture media was first noted many years ago. Later on Salle
and Schmidt (498) found that, for Leis/imania tropica, blood could be
replaced by hemoglobin, which they suggested as a probable growth-
factor. This question has been investigated extensively by M. Lwoff (349,
366), who has shown that certain Trypanosomidae can grow in ordinary
peptone media while others require supplementary blood or a more active
substitute, hematin (Table 8. 3). The latter are unable to synthesize
porphyrin groups in the production of cytochrome, cytochrome oxidase,
and related enzymes (347). Strigomonas fasciculata apparently can com-
bine iron and exogenous protoporphyrin to produce heme (341), On
the other hand, certain Trypanosomidae and free-living Protozoa con-
taining the cytochrome system apparently can synthesize porphyrins from
simpler materials.

Vitamin B^n (cyano-cobalamin). It is interesting that the first evidence
for protozoan requirements has been obtained with a phytoflagellate.
Vitamin Bjo, or "cyanocobalamin" (265), tremendously stimulates growth
of Englena gracilis var. baciUaris in the presence of adequate thiamine
and is believed to be an absolute requirement (230). These findings have
extended earlier observations (225) that heavy growth of E. gracilis de-
pends upon certain factors present in crude casein. This growth-response
of E. gracilis has been applied to microbiological assay of cyano-cobalamin
(230, 579).

Protogen. A previously undefined "Factor II," a concentrate of natural
origin essential for Tetrahymena pyriformis (92), has been resolved into
fractions IIA and IIB (540). The name, protogen, was proposed for
Factor IIA, which is not identical with any known vitamin or with the
"animal protein factor." Protogen, which may prove to be a fundamental
requirement of animals, is unique as the first vitamin to be discovered
throvigh the study of protozoan growth requirements. The search for
natural sources of protogen will be facilitated by the ability of T. pyri-
formis to digest complex foods as well as by its growth in media suitable
for assays.

BiosyntJiesis of vitamins. The synthesis of vitamins by Protozoa has
been suggested occasionally, but specific evidence has been presented in

444 Physiology

only a few cases. Among the phytoflagellates, experimental evidence is
available for Chilomonas paromec'nim (182, 207, 224) and Polytoma
oceJlatitm (182). There is also some presumptive evidence in the case of
heteroautotrophs which have been grown without exogenous thiamine.
Thiazole can replace thiamine in stimulating growth of Polytoma cauda-
tum (352) and P. ocellatum (353), while the thiazole and pyrimidine
components together replace the vitamin for PoJytomeUa caeca (351).
Several substituted thiazoles and pyrimidines also can be utilized instead
of the natural components of thiamine (348). Although actual synthesis
by a phytoflagellate has not been demonstrated, it is assumed that some
species can produce thiamine from simple raw materials while others need
thiazole or both components. In investigating such problems, composition
of the medium must be considered carefully since the supply of trace
elements, such as iron (348), and the nature of the substrate may be
important factors in a potential synthesis. The significance of the sub-
strate is suggested by failure of Chilornonas Paramecium to grow on
pyruvate without added thiamine, although the flagellate grows slowly on
acetate in a thiamine-free medium (73). This is an interesting parallel
to Prototheca zopfi which can oxidize acetate in a thiamine-deficient
medivnn but apparently requires thiamine for utilization of pyruvate (4).
In general, the burden of proof seems to rest upon those who would deny
that phytoflagellates can synthesize a variety of vitamins. For the higher
Protozoa, such assumptions are not justified because these organisms have
been grown almost exclusively in chemically undefined media. So long
as materials of natural origin are included, it is unsafe to assume that a
particular vitamin has been eliminated from a culture medium.

Synthesis of thiamine from its thiazole and pyrimidine components has
been reported for Acanthamoeba castellaTiii (345), and from unspecified
intermediates in the case of Tetrahymena pyriformis (277). However,
A. castellanii was grown in peptone media of unknown vitamin content,
and the interpretation of the earlier data for T. pyriformis has been
questioned (183, 184). Although more recent data have been supplied for
the ciliate (278, 282), there is no conclusive evidence that either A. castel-
lanii or T. pyriformis can synthesize thiamine.

The Trypanosomidae which need exogenous hematin presumably are
unable to synthesize the porphyrins necessary to the formation of heme.
Others, which possess the cytochrome system but do not require ready
made porphyrins, obviously synthesize heme from simpler constituents
of culture media. The problem of obtaining suitable raw materials is a
minor one because such a substrate as acetate (450) may serve as a starting
point. Syntheses of this nature may be assumed for Chilomonas Para-
mecium, Polytoma uvella. Astasia klebsii, Euglena gracilis, and Tetra-
hymena pyriformis, for example. As a source of direct evidence, the

Physiology 445

hematin-requiring Trypanosomidae should be useful for microbiological
assays.

The synthesis of nicotinic acid (207, 223), adenine (223), pyridoxal,
and riboflavin (207) has been demonstrated in Chiloynonas Paramecium.
Synthesis of riboflavin, pantothenic acid and probably of biotin has been
reported for Tetrahy?7iena pyriformis on the basis of microbiological
assays (283), but these conclusions were later withdrawn (290). The syn-
thesis of p-aminobenzoic acid and inositol by T. pyriformis has been
reported on the basis of Neurospora assays (276). Synthesis of the former
would seem to be no advantage to the ciliate since p-aminobenzoic acid
apparently cannot replace pteroylglutamic acid as an absolute require-
ment.

The requirements of various groups

At present, little has been published on two major groups of the
phytoflagellates,^ the Chrysomonadida and Dinoflagellida. Earlier work
on Oikomonas termo (192) and dinoflagellates (13) was interrupted, and
although extensive investigations are in progress, the two orders offer quite
a variety of unsolved nutritional problems. Both groups include colorless
and chlorophyll-bearing species and a number of holozoic types, and both
are represented in fresh and salt water. Representatives of two other
orders, Heterochlorida and Chloromonadida, apparently are not yet avail-
able in pure cultures.

Cyyptomonadida. So far, the chlorophyll-bearing cryptomonads have
been neglected in favor of Chilomonas paramedian, several strains of
which have been investigated. As nitrogen sources, ammonium salts are
satisfactory, nitrate is inadequate (73), and utilization of nitrate has not
been demonstrated. Reported chemoautotrophy (390) had not been con-
firmed (73). Although C. Paramecium has been grown in glycine and
acetate medium (186, 390), little is known about amino acids as nitrogen
sources or their possible value as sources of both carbon and nitrogen.

Excellent carbon sources,^ added to a basal inorganic medium supple-
mented with thiamine (or its components), include acetate, ethanol, lac-
tate, and pyruvate (73, 222, 354). In such a medium, about 45 per cent of
the available acetate is oxidized while the rest is assimilated (224). With-

'^ Current data on food requirements and metabolism of the phytoflagellates have
been discussed by Hutner and Provasoli (228).

" Extending the earlier observation of Provasoli (467) with peptone media, B. K.
Swanson (1951. M. S. Thesis, University of Iowa) has tested various alcohols as carbon
sources for Chilomonas Paramecium in a simple medium. Several straight-chain alcohols
— ethyl, n-butyl, and to a lesser degree, hexyl alcohol — were good carbon sources.
Methyl, rz-propyl and n-amyl alcohols were inadequate for growth, and this was true
also for certain alcohols with side-chains (secondary-butyl, tertiary-butyl, wo-amyl, etc.).
Furthermore, these nonutilizable alcohols produced significant inhibition of growth
when mixed with the lUilizable alcohols.

446 Physiology

out added thiamine, acetate has supported growth in all-glass culture
vessels (73, 390) — about one per cent of the growth obtained with thia-
mine. In thiamine-supplemented acetate medium, growth of C. para-
rnecimn has been tripled by raising the carbon dioxide concentration
from that of the atmosphere to 100 mm Hg at atmospheric pressure (428).
Similar stimulation by carbon dioxide had previously been detected in
peptone media (245). Certain alcohols (467), fatty acids (299, 467), and
carbohydrates (321, 327) also accelerate growth in peptone media, al-
though a number of these supplements have not yet been tested as carbon
sources in heteroaiuotrophic nutrition.

Phytomonadida. As inorganic nitrogen somxes in "photoautotorphic"
nutrition, ammonium salts have been more satisfactory than nitrates for
Haematococcus pluvialis (340) and Lobomonas piriformis (422). No
appreciable differences have been reported for Chlamydomonas agloe-
formis (340), Chlorogonium elongatum, and C. euchlorum (321). How-
ever, comparative tests of nitrates and ammonium salts over an adequate
range of salt and hydrogen-ion concentrations have not been reported.

In heteroautotrophic nutrition Polytoma ocellatum has been grown in
a nitrate medium (353), but species of PolytomeUa and other species of
Polytoma apparently are limited to an ammonium-N source. This situa-
tion deserves further investigation in view of Lwoff's (347) characteriza-
tion of an autotroph as an organism which can reduce nitrate in an
inorganic medium.

Organic nitrogen sources have not been investigated extensively but
growth on asparagine or a single amino acid has been reported for species
of Chlamydomonas and Haematococcus (340, 367), Chlorogonium (350,
457), Lobomonas in darkness (422), Polytoma (340, 360, 452, 458), and
PolytomeUa (342, 455, 458).

Acetate, butyrate, and lactate are good carbon sources in heteroauto-
trophic nutrition. A number of other substrates probably would be satis-
factory since salts of additional acids, including propionic, valerianic, and
caproic (323, 456, 463, 464, 465, 466), and also certain alcohols (467)
accelerate growth of various colorless species in peptone media. Certain
carbohydrates also have stimulated growth of Polytoma (360, 361) and
PolytomeUa (342) but may or may not be adequate substrates in hetero-
autotrophic nutrition. The ability to use an amino acid, as the sole source
of nitrogen, carbon, and energy, has not yet been demonstrated.

Several green species — Chlorogonium elongatum (321), C. euchlorum
(321, 350), Lobomonas piriformis (422), Chlamydomonas agloeformis,
and Haematococciis pluvialis (367) — have been grown in darkness, par-
ticularly in peptone media supplemented with acetate, and are obviously
facultative heterotrophs. Under such conditions, acetate is a rather satis-
factory substitute for photosynthesis. On the other hand, Chlamydomonas
moewusii is an obligate phototroph in a wide variety of media (315).

Physiology 447

Eiiglenida. Further study of these flagellates should prove interesting
because the order includes chlorophyll-bearing species, colorless saprozoic
types {Astasia, Menoidium, etc.), and various holozoic genera {Hetero-
nema, Peranema, etc.). Furthermore, such species as Etiglena anahaejia, E.
deses, E. klehsii, E. pisciformis, and E. stellata have failed to grow in dark-
ness (105), whereas Euglena gracilis grows well under such conditions
(377, 460, 549). Although investigations on holozoic types are in progress,
previous reports are limited to Euglena and Astasia.

Inorganic sources of nitrogen have been tested for several species of
Euglena (104, 105, 109, 175, 177, 189, 377, 508). The available data in-
dicate that ammonium salts are generally more satisfactory than nitrates.
However, nitrates apparently are adequate for Euglena anahaena (109).
Among the colorless Euglenida, Astasia longa has been maintained on
ammonium-N (503).

Organic sources of nitrogen have been investigated for several species
of Euglena (104, 105, 549). Asparagine and various amino acids have sup-
ported growth of one species or another, but several species have shown
interesting differences in their apparent abilities to utilize particular
amino acids (105). Possible relations of mineral requirements and pH of
the basal media to the utilization of amino acids have not been investi-
gated adequately.

The available information on carbon sources is based mainly on growth
of Euglenida in peptone media, although acetate has supported slow
growth of Astasia longa (503) and Euglena gracilis (504) in heteroau to-
trophic nutrition. A number of organic acids — including acetic, propi-
onic, butyric, valerianic, caproic, /^o-caproic, octylic, nonilic, lactic, and
pyruvic — have stimulated growth of Astasia (456, 465, 466, 467) and
Euglena (456, 467) in peptone medivmi. Growth of Euglena (332, 456,
467) and Astasia (456, 467) also is stimulated by certain alcohols, includ-
ing ethyl, propyl, butyl and hexanol.

Protomastigida. Pure cultures of parasitic Protomastigida have been
available for many years but most investigators have been interested in
these flagellates as parasites rather than in their nutrition. However, the
investigations of Marguerite Lwoff (349, 363, 366) on several parasites of
insects showed that Strigomonas oncopelti grows well in peptone media
while certain other species have more complex requirements. 5. jasciculata,
from mosquitoes, requires a small amount of blood or hematin as a
supplement to peptone medium. Leptonionas ctenocephali, from the
dogflea, requires such a supplement in higher concentrations (349). Spe-
cific requirements of S. culicidarum include at least nine amino acids,
hematin, thiamine, riboflavin, pyridoxamine, trace minerals, and possibly
two or three additional vitamins (567).

Certain Trypanosomidae of vertebrates require, in addition to hematin,
other growth-factors not supplied by peptone solutions (Table 8. 3).

448 Physiology

Trypanosoma cruzi, Leishmania brasiliensis, L. donovani, and L. tropica
need factors in serum other than thiamine, /?-aminobenzoic acid, pyri-
doxine, or nicotinic acid (521). Hence, it appears that one aspect of
physiological specialization among the trypanosomes and their relatives
involves an increasing dependence upon the host for essential growth-
factors.

Trichomonadida. Relatively little work has been reported on these
flagellates (349). Peptone solutions enriched Avith whole blood, serum,
and fragments of liver have supported growth of Eutrichomastix colu-
brorum (59), Trichomonas gallinae (T. columbae) (20), T. foetus (7,
163, 575), and T. vaginalis (255, 557, 558). Liver-infusion agar slants,
overlaid with serum-enriched Ringer's solution, also have been satisfactory
for T. vaginalis. The investigations of Cailleau have shown that the first
three species need certain growth-factors apparently not required by free-
living flagellates (Table 8. 3). More recently, T. vaginalis has been grown
in a peptone solution supplemented with acetate, maltose, about 15
growth-factors, and after sterilization, diluted serum and ascorbic acid
(536). In such a medium, two fractions of human serum — an ether-soluble
and an ether-insoluble aqueous fraction — are essential. Linoleic acid
seems to be the most active component of the ether-solvible fraction and
serum albumin of the ether-insoluble one. The first fraction could be
replaced by a mixture of linoleic and oleic acids, cholesterol, ergosterol,
lecithin, a-estradiol, a-tocopherol, vitamin A, and ^-carotene (537). Tri-
chomonas foetus has been grown in a mixture of thirteen amino acids,
various vitamins, and minerals (568).

Sarcodiiia. Published reports on the Sarcodina include little more than
the development of suitable media for pure cultures, although detailed
investigations are in progress. Acanthamoeba castellanii has been grown
in a medium containing serum and liver fragments (44) and in a simpler
peptone and inorganic salt medium (45). A medium containing peptone,
dextrose, and inorganic salts also is satisfactory for Mayorella palestiiien-
sis (472, 473). Progress is also being made toward pure-culture techniques
for parasitic amoebae. Entamoeba invadens has been maintained bacteria-
free for several transfers after elimination of a single bacterial contami-
nant by treatment with penicillin (307). In addition, E. histolytica has
been grown on non-viable bacteria for more than 200 transfers (500),
and also in a non-particulate medium without bacteiial growth (501).

Ciliates. Under natural conditions free-living ciliates feed mainly upon
ingested microorganisms. In the investigation of such natural foods,
strains of ciliates have been grown in cultures with other living or killed
microorganisms (36, 417, 418). Such "species-pure" cultures with living
bacteria involve complex relationships, and the ciliates tend to be
swamped unless the initial proportions between ciliates and bacteria are
satisfactory (259, 260, 261). Such relationships, particularly important

Physiology 449

when the medium supports growth of the bacteria, may be controlled by
using non-nutrient basal media (14, 42, 253, 258, 315).

A wide variety of bacteria may serve as food for particular ciliates.
About 20 species, as individual suspensions in salt solutions, were each
adequate for growth of Tetrahymena pyriformis (253). Killed yeasts and
washed and killed suspensions of green flagellates also were satisfactory,
although living flagellates failed to support growth in serial transfers. On
the other hand, Perispira ovum thrives on living Eiiglena gracilis (93),
and Tetrahymejia pyriformis grows on either Chilomonas parainecium or
Polytoma oceUatum (182). For Colpidium colpoda, species of Entero-
bacteriaceae are more satisfactory than Bacillaceae (42). Killed bacteria
seem to be an inadequate diet for Colpoda duodenaria (548). Likewise,
Pleurotricha lanceolata and Stylonychia piistiilata can be grown on living
Tetrahymena geleii but not on killed ciliates (317). Just what heat-labile
factors, supplied by living organisms, are significant in such cases is still
unknown. However, Didinium nasutum is said to have lost the ability to
synthesize peptidases and must obtain these enzymes from the living
Paramecium which it ingests (101).

Definitive observations on food requirements of ciliates awaited, first
of all, the establishment of pure cultures. This step was taken some
thirty years ago when A. Lwoff isolated Tetrahyrnerm (Glauco7na) pyri-
formis'' in a peptone medium. Comparative data on various culture media
were published later (340). These observations furnished a timely stimu-
lus, and within a relatively few years, additional bacteria-free strains —
referred to the genera Colpidium, Colpoda, Glaucoma, Tetrahymena,
Loxocephalus, and Paramecium — were isolated by other workers (274).
With a few exceptions, culture media included solutions of commercial
peptones, yeast-extract or yeast autolysates, usually supplemented with
inorganic salts. Such media as those of Glaser and Coria (160, 161, 162)
Avere more complex. Although Parameciinn bursaria has been maintained
in peptone media (322), cultivation of other species of Paramecium has
proven more difficult. However, P. aurelia (564) and P. multi-micronu-
cleatum (262) are now in pure culture and investigations on their food
requirements are in progress.

The next progressive step led toward the development of chemically
defined culture media (349). The first apparently successful results were
those of Kline (298) with Colpidium striatum. Unfortunately, Kline's
strain of C. striatum seems to have been lost and several other strains
have failed to grow in his medium.

Much better results have been obtained with somewhat similar media

' This ciliate is a strain of Tetrahyinena gelii, a genus and species erected by Furga-
son (138) to include strains of "Colpidium campyhim," "C. striatutn," "Glaucoma pyri-
formis." Letter designations for various strains, such as T. geleii H and T. geleii W,
have since been proposed (72, 279). The name of the species apparently should be
Tetrahymena pyriformis (see Chapter VII).

450 Physiology

developed for Tetrahymena geleii W (280, 282). The simpler of these
media contained eleven amino acids (arginine, histidine, isoleucine, leu-
cine, lysine, methionine,^ phenylalanine, serine, threonine, tryptophane,
valine), glucose, eleven known vitamins, several inorganic salts, and in
the proportion of 1:10, a plant or animal tissue extract apparently con-
taining a nimiber of amino acids, certain unidentified growth factors and
known vitamins and several minerals. Further progress has made possible
the gradual substitution of known growth-factors (Table 8. 3) for supple-
ments of natural origin. This general type of medium, which has proven
satisfactory for several strains of Tetrahymena (121, 122, 494), is illus-
trated in Table 8, 1, although nucleic acid may be replaced by known
purines and pyrimidines. Such media are now almost completely defined
in a chemical sense and are potentially useful in the assay of certain vita-
mins and also such amino acids as histidine, isoleucine, lysine, and trypto-
phane (494).

OXYGEN RELATIONSHIPS AND
OXIDATIONS

Ecological distribution

The distribution of Protozoa suggests that some species are obli-
gate aerobes, that others are microaerophiles (requiring only a little
oxygen), and that many intestinal parasites and some free-living types may
be obligate or facultative anaerobes. Natural waters containing much
putrefying material are anaerobic at their lower levels, and such artificial
environments as Imhoff sewage tanks also insure anaerobiosis beneath the
surface (304, 305). Species characteristic of such a fauna, sometimes
termed polysaprobic or sapropelic, are at least facultative anaerobes. They
include Ctenostomina and scattered species of other ciliates, as well as a
few flagellates and Sarcodina. Another practically anaerobic environment
is found near the bottom of deep fresh-water lakes, but very little is
known about this fauna. Other Protozoa, typical of clean waters with a
high oxygen content, are aerobes and some may be obligate aerobes. How-
ever, such ciliates as Coleps Jiirtus and Frontonia leucas, which are not
sapropelic, may survive anaerobically for several weeks (318).

Oxygen relationships of parasites doubtless vary with the usual site of
infection. Species which invade the blood and other tissues probably
have access to about as much oxygen as the surrounding tissue cells and
may be predominantly aerobic. Oocysts of Eimeria stiedae and E. magna,
for instance, apparently cannot sporulate under strictly anaerobic con-

^The reported ability of T. geleii to use homocystine, with supplementary "liver-
fraction," as a replacement for methionine (285) has been refuted (150) on the basis
that the observed growth in the presence of homocystine can be attributed to the
methionine content of the liver-fraction used by Kidder and Dewey.

Physiology 451

ditions (61). On the other hand, conditions in the vertebrate intestine
suggest that intestinal parasites are anaerobes. Experimental evidence in-
dicates that the rumen ciliate, Eudiplodinium neglectum, is an obligate
anaerobe (218). Such is true also for flagellates of termites (217, 555).
Entamoeba histolytica, in contrast to many other intestinal parasites,
normally invades the wall of the colon. Yet this species grows as an
anaerobe in cultures (56).

Relationships between oxygen tension and growth of laboratory popu-
lations have been investigated in a few cases. Aeration of flask cultures
increases giowth of Tetrahymena pyriformis (245, 440), reduction of the
oxygen supply (pyrogallol technique) decreases populations to about half
the normal density (171), and complete anaerobiosis prevents growth
(340). Growth of Chilomonas Paramecium, on the other hand, is retarded
by aeration of cultures (245). Quantitative data also have been reported
for Trichomonas vaginalis (255) and for C. Paramecium and T. pyri-
formis (428). Growth of T. vaginalis is heaviest in complete anaerobiosis
and is inhibited progressively by increasing oxygen tensions. Oxygen pres-
sures of 0.5 to 500 nnn Hg permit growth of C. para?necium, with an
optimum at about 75 mm Hg (about half the normal atmospheric con-
centration of oxygen), while pressures of 600 mm Hg and higher are
lethal. Growth of T. pyriformis increases from 10 mm Hg to a maximum
(about twice the growth with atmospheric concentrations of oxygen) in
an atmosphere of pure oxygen (739 mm Hg).

Oxidation-reduction potentials^

The oxidation-reduction potential of the culture medium is an-
other factor related to the giowth of microorganisms. In a general sense,
this potential is a measure of the reducing intensity or oxidizing intensity
of a given system. Examples of such systems — each of which consists of a
more reduced and a less reduced substance — are leuco-methylene blue/
methylene blue, lactate/pyruvate, and reduced cytochrome a/oxidized
cytochrome a. If a platinum electrode and a calomel electrode, in a po-
tentiometer hookup, are immersed in such a system, a potential difference
can be measured. Since the calomel electrode is standardized against the
hydrogen electrode, measurements are expressed in millivolts in terms of
the hydrogen electrode potential. The more negative the potential, the
greater is the reducing power of the system; the more positive, the greater
the oxidizing power (and the lower the reducing power). Each system has
a characteristic Ef/ value at which it is half reduced at a particular tem-
perature and pH. Consideration of pH is necessary because the potential

" Compact discussions of oxidation-reduction potentials have been published by John-
son (257) and Stephenson (538). In addition, there is available a table of potentials for
more than 200 different systems (6). Early literature on Protozoa has been reviewed by
Jahn (246).

452 Physiology

varies with pH. For the methylene blue system, Eq at pH 7 is 11 mv; for
the cytochrome a system, 290 mv at pH 7.4; for lactate/pyruvate, —180 mv
at pH 7. If two systems with different E^' values are mixed together, a
reaction, which may be considered the transfer of electrons from one sys-
tem to the other, continues until equilibrium is reached. The higher
potential is lowered and the lower potential raised to a common level; or
reduction of the first system (gain of electrons) and oxidation of the
second (loss of electrons) take place.

The potential of the culture medium undoubtedly influences the
growth of inicroorganisms. For bacteria, it is possible to lower the po-
tential of a liquid medium with a suitable reducing agent so as to inhibit
growth of aerobes and permit growth of anaerobes. In the case of Chilo-
monas Paramecium, appropriate additions of the sulfhydryl radical (— SH)
both lower the oxidation-reduction potential of the medium and stimu-
late growth (238). Influence of the potential on Entamoeba histolytica
apparently varies with the type of medium. According to one worker,
growth of E. histolytica decreases from a maximum, at a potential below
— 300 mv, to almost none at —200 mv; unencysted amoebae die after an
hour or more at —50 mv (56). Jacobs (234), on the other hand, found that
the potential of the medium was about —25 mv while E. histolytica was
growing most rapidly in cultures containing "organism t." In contrast to
E. histolytica, Trypanosoma criizi and several species of Leishmania grow
best in cultures at a potential of about 330 mv (57). Growth of micro-
organisms themselves also may modify the potential. For instance, a drop
of about 290 mv has been traced in cidtures of Chilomonas Paramecium
(241). It is uncertain just how extensively the potential of the medium
influences internal oxidations and reductions, although the "internal
potential" of Amoeba proteus, as measured by injected indicators, may
be changed from about —70 mv (pH 7) under aerobic conditions to —143
mv in anaerobiosis (55, 70).

Oxygen consumption^**

Measurements of oxygen consumption make it possible to trace
effects of environmental factors on metabolic rates, to investigate the
utilization of particular substrates, and to correlate stages in the life-cycle
with metabolic activity. Such measurements are necessary in the study of
oxidative mechanisms by the use of poisons or stimulants, and may indi-
cate the relative importance of particular systems, such as the cytochrome
system, in the metabolism of a particular species. Manometric techniques
can be used also in the estimation of specific enzyme systems and meta-
bolites. Comparative data on oxygen consumption of different species

" A monograph by Umbreit and his associates (560) supplies a comprehensive survey
of manometric techniques and their various applications. The earlier literature on
Protozoa has been reviewed by Jahn (246).

Physiology 453

should be interpreted cautiously, since extensive variations apparently
occur even in single species. For example, oxygen consumption of Para-
meciiun caudatum has been recorded as 0.00014 (339), 0.0004 (213) and
0.0052 mm-'^/hour/organism (266). In addition to differences attributable
to different manometric techniques, the physiological condition of the
test organisms may be a significant factor. Starvation significantly reduces
oxygen consumption of Paramecium caudatum (339) and Pelomyxa caro-
Unensis (526). Likewise, a marked decrease occurs in old cultures of
Colpidhnyi colpoda (563), Bodo caudatus (310), Chilomonas paramechim
(221), Tetrahymena pyrijormis (9, 431), Trichomonas foetus (484), and
Trypanosoma cruzi (33). In T. pyriformis the change occurs after the
logarithmic phase of growth (421) and is not correlated with any decrease
in cytochrome content (9). Changes in consumption also have been traced
during conjugation of Paramecium caudatum (585).

Environmental conditions also may influence oxygen consumption. For
Tetrahymena pyriformis consumption is at a maximum in media at pH
5.5 and is distinctly lower on each side of the optimum (170). Increasing
temperatures, within physiological limits, stimulate oxygen consumption
of ciliates (267, 563) and Strigomonas fasciculata (341). The oxygen con-
sumption of Spirostomum ambiguum increases with increasing oxygen
concentration of the atmosphere to which cultures are exposed. The maxi-
minn, observed with pure oxygen, was about 50 per cent higher than for
ciliates exposed to air (532). On the other hand, changes in oxygen
tension within fairly wide limits have produced little effect on Para-
mecium caudatum (4).

Respiratory quotients

The ratio of the carbon dioxide produced to the oxygen consumed
— the respiratory quotient (R.Q.) — has interested physiologists as a theo-
retical index to the type of material being consumed. The R.Q. for com-
plete oxidation of carbohydrate is 1.0 and is about the same for acetate;
for butyrate, about 0.8; for fats, approximately 0.7; for proteins, about 0.8
(urea as the nitrogenous waste) or about 0.9 (ammonia as the nitrogenous
waste). Quotients well above I.O may indicate synthesis and storage of fat
produced from carbohydrate. Low values (0.4-0.6) might indicate con-
version of protein to carbohydrate, or incomplete oxidation of carbo-
hydrate.

Most of the R.Q. values reported for Protozoa (Table 8. 4) fall within
the usual range. This is especially true of Trypanosomidae, some of which
have shown a higher R.Q. with glucose than without (415, 531), as would
be expected. Unusually low values for several phytoflagellates have been
attributed to synthesis of carbohydrates from carbon dioxide (398) and
to the conversion of protein into carbohydrate or the incomplete oxida-
tion of carbohydrate (247). Varying quotients for a species may reflect

454 Physiology

TABLE 8. 4. RESPIRATORY QUOTIENTS REPORTED FOR
VARIOUS PROTOZOA

Species

R.Q.

PHYTOMASTIGOPHOREA

Astasia longa (strain J)
Chilornonas Paramecium
Khawkinea halli

ZOOMASTIGOPHOREA

Leishmania tropica

Strigomonas fasciculata

S. oncopelti

Trypanosoma cruzi

T. equiperdum

T. lewisi

T. rhodesiense

SARCODINA

Amoeba proteus
Pelomyxa carolinensis

SPOROZOA

Plasmodium knowlesi

CILIATEA

Balantidium coli
Blepharisma undulans
Paramecium aurelia
P. caudatum
P. multirnicronucleatum
Spirostomum ambiguum
Tetrahymena pyrijormis

0.34 (247)

0.28-0.37 (398); 0.74-0.93 (221)

0.56 (247)

0.84-0.95 (531)
1.0 (341)
1.0 (341)
0.74-1.06 (33)
0.60 (126)
0.74-0.94 (531)
0.2 (64)

1.03 (124)

0.56-0.87 (526); 0.45-0.94 (430)

0.87-0.93 (63)

0.84 (85)

1.12 (124)

0.73-0.90 (429)

0.69 (4); 0.62 (495); 0.70-0.99 (429)

0.72 (398)

0.84 (532)

0.81-1.27 (431)

differences in condition of the organisms. An R.Q. of 0.87 has been re-
ported for well-fed Pelomyxa carolinensis and one of 0.56 for starved speci-
mens (526). Age of the culture also is a factor. The R.Q. of Chilornonas
Paramecium drops from 0.91-0.93 in 24-hour cultures to 0.75 at 72 hours
(221); that of Tetrahymena pyrijormis from 1.21-1.27 at three days to 0.81
after seven days (431); that of Trypanosoma cruzi, from a maximum of
1.06 to a low of 0.74 after the population reaches its peak. In the last case,
the lower quotient is attributed to exhaustion of carbohydrates and subse-
quent utilization of proteins (35). The R.Q. also may vary with tempera-
ture— 0.73 at 20° to 0.90 at 30° for Paramecium aurelia; 0.70 at 15° to
0.99 at 35° for P. caudatum (429); 0.45 at 10° to 0.94 at 30° for Pelomyxa
carolinensis (430).

Oxidations^^

Most biological oxidations consist of series of oxidations and re-
ductions catalyzed by a variety of enzymes, and may be pictured as

" For detailed discussions of oxidative enzyme systems, the reader is referred to
such sources as Baldwin (10), Lardy (308), and Stephenson (538).

Physiology 455

involving the transfer of hydrogen step by step from one acceptor to
another. Each step involves an oxidation-reduction system and each dehy-
drogenation yields energy for anabolism. For cells in general, a number of
enzymes and oxidation-reduction systems are known to be involved in
metabolism. Gradually accumulating evidence indicates that at least some
of these are operative in Protozoa, as woidd be expected.

The cytochrome system. In aerobes the final stages of the oxidative re-
actions— the transfer of hydrogen to oxygen (2H to Oo) — involve the
cytochrome system. This system includes several cytochrome pigments
which, in their reduced forms, show different absorption bands spectro-
scopically. Each cytochrome is an iron-porphyrin-protein which can exist
in either the oxidized or the reduced form. The oxidation-reduction po-
tentials (Eq') of cytochromes a and c are about 290 and 270 mv; that of
cytochrome h, about —40 mv. The oxidation of reduced cytochrome c,
catalyzed by cytochrome oxidase, involves the transfer of 2H to atmos-
pheric oxygen:

cytochrome-H2 -f- cyt. oxidase — > cytochrome -\- cyt. oxidase-H2
cyt. oxidase-H2 -|- 1/2^2 -^ cytochrome oxidase -|- HoO

The reduction of cytochrome c is catalyzed by dehydrogenases which
bring about oxidation of reduced DPN, reduced TPN, and such sub-
strates as succinate. Cytochrome h also may be involved in the reduction
of cytochrome c. Reduction of cytochrome c may be blocked by heat and
by such reagents as alcohol, formalin, and urethanes. The transfer of
hydrogen from cytochrome to cytochrome oxidase is inhibited by cyanide
and azide, thus maintaining cytochrome in the reduced condition. Trans-
fer of hydrogen from cytochrome oxidase to oxygen is inhibited by carbon
monoxide.

As would be expected, aerobic Protozoa resemble other aerobic micro-
organisms in possessing cytochrome pigments. Cytochromes a, h, and c
have been reported in Astasia klebsii (83) and Tetrahymena pyrifor?nis
(9); cytochromes b and c, in Eiiglena gracilis, Tetrahymena (Glaucoma)
pyrijormis, Polytoma uvella, Strigomonas jasciculata, and 5. oncopelti
(341); cytochrome c, in Colpidium campylum (510, 512), and with cyto-
chrome oxidase, in Chilomonas Paramecium (220). Cytochrome oxidase
is said to occur in the pigment granules (mitochondria) of Stentor coeru-
leiis (569). In contrast to the typical aerobes, Trichomonas foetus ap-
parently contains no cytochrome (484).

Poisoning techniques have supplied additional information. The
respiration of Tetrahyynena pyrijormis is decreased by 9-57 per cent in
different concentrations of methyl-, ethyl-, and propylurethanes (364).
Cyanide decreases oxygen consumption about 61-64 per cent in Astasia
longa and Khawkinea halli (247), about 90 per cent in Polytoma uvella
(364), and about 95 per cent in Astasia klebsii, for which azide is almost

456 Physiology

as inhibitory (83). A number of Trypanosomidae (349) also are sus-
ceptible to such poisons. Leptomonas ctenocephali, Strigomonas fascicu-
lata, and S. oncopelti show 83-95 per cent inhibition with cyanide and
are about as sensitive to carbon monoxide (341). Cyanide in certain con-
centrations inhibits respiration about 90 per cent in Trypanosoma cruzi
(33), 11-82 per cent in T. congolense (34), 97-98 per cent in Leishmania
tropica, L. brasiliensis, and L. donovani, 85-88 per cent in T. leiuisi from
cultures, and 66-69 per cent in T. conorhini (32). Certain other Trypano-
somidae are relatively insensitive to cyanide — T. equiperdum (127),
T. brucei, T. hippicitm, T. rhodesiense (32, 64, 126), T. evansi, and T.
equinum (34). Although the oxygen consumption of T. gambiense from
blood is not decreased by cyanide (34), flagellates from cultures are
moderately sensitive (32). Among the Sarcodina, Pelomyxa caroUnensis
is sensitive to cyanide (427), and sensitivity increases with temperature in
the range, 10-35° (430). Respiration of Plasmodium knoxvlesi also is
inhibited by cyanide (64, 371) and carbon monoxide (371). Earlier re-
ports (338, 364, 526) that free-living ciliates are insensitive to cyanide, are
contradicted by later observations. Respiration of Tetrahymena pyri-
formis is sensitive both to carbon monoxide (9) and to cyanide (9, 170),
while that of well-fed, but not of starved specimens, also is cyanide-sensi-
tive in Paramecium aurelia (424) and P. caudatum (66, 424).

The results obtained with poisoning techniques indicate that in gen-
eral, aerobic Protozoa are cyanide-sensitive and presumably oxidize sub-
strates mainly through the cytochrome system. On the other hand, some
questions are unanswered. Why are starved ciliates, in contrast to well-fed
ones, relatively insensitive to cyanide? What converts insensitive T. gayn-
biense from the blood into cyanide-sensitive flagellates in culture media?
Is it necessary for these organisms to oxidize certain substrates only par-
tially ("anaerobically") in the blood but completely, or nearly so, in
culture media? And what are the biochemical differences between the
cyanide-sensitive "lewisi group" of trypanosomes and such insensitive spe-
cies as T. brucei and T. evansi? Such problems are of practical as well as
theoretical interest, since the response of parasites to chemotherapeutic
agents may depend to an important extent upon the oxidative mech-
anisms of particular species.

Pyridine nucleotide enzymes. The pyridine nucleotides are coenzymes
for a number of important oxidative enzymes. Diphosphopyridine nucleo-
tide (DPN), or coenzyme I, contains nicotinamide, D-ribose, adenine, and
two phosphoric acid groups. Triphosphopyridine nucleotide (TPN), or
coenzyme II, contains a third phosphoric acid group. Both coenzymes are
involved in protozoan metabolism. Chilomonas Paramecium (223) and
Tetrahymena pyriformis S (510, 512) contain DPN, while Trypanosoma
hippicum requires DPN for glycolysis in vitro (194). In addition, supple-

Physiology 457

mentary DPN and TPN accelerate oxidation of pyruvate by Plasinodium
gallmaceum in the presence of dicarboxylic acids (535).

Diphosphothiamine enzymes. This phosphoric acid ester of thiamine
is the coenzyme of carboxylase which catalyzes the decarboxylation of
pyruvic acid and probably certain other a-keto monocarboxylic acids.
Supplementary thiamine is necessary for the oxidation of pyruvate by
Tetrahymena pyriformis (552), is essential to growth of Chilomonas Para-
mecium on pyruvate (73), and accelerates oxidation of pyruvate by Plas-
modium gallinaceum (535). The thiamine content of Tetrahymena pyri-
formis is at least 60 per cent that of yeast (574), and the vitamin is
essential to growth of this and certain other Protozoa (Tijble 8. 3). There-
fore, thiaminoprotein enzymes are probably of general importance in
protozoan metabolism.

Flavoprotein ejizymes. In these enzymes the prosthetic groups contain
riboflavin, either as riboflavin-phosphate or as flavin dinucleotide (a
union of riboflavin-phosphate and adenylic acid). Enzymes of the first
type apparently catalyze the oxidation of reduced TPN and oxidation of
L-amino acids (L-amino acid dehydrogenase). Enzymes of the second type
include xanthine oxidase, D-amino acid dehydrogenase, glycine dehydro-
genase, and apparently "diaphorase I" (catalyzing oxidation of reduced
DPN). These flavoprotein enzymes, which are not significantly affected
by cyanide poisoning, are probably present in Protozoa. Riboflavin occurs
in high concentration in Tetrahymena pyriformis (574) and is essential
to growth of several ciliates (Table 8. 3), although synthesized by Chilo-
monas parajnecium (207).

Pyridoxine enzymes. Pyridoxal phosphate appears to be the coenzyme
for transaminases, tryptophanase, and certain amino-acid decarboxylases.
The presence of comparable enzymes in Protozoa may be suspected since
pyridoxine is essential to growth of certain ciliates (Table 8. 3) and
inhibits the antimalarial action of quinine and atebrine against P.
lophurae (519), and also since pyridoxal is synthesized by Chilomonas
Paramecium.

Peroxidase ayid catalase. These are iron-porphyrin-protein enzymes.
Catalase probably catalyzes coupled oxidations by means of the hydrogen-
peroxide formed in some primary reaction (271), the peroxide being de-
composed to water and molecular oxygen. Peroxidase catalyzes the oxida-
tion of such substrates as tyrosine, adrenaline, bilirubin, pyrogallol, and
various other phenols in the presence of hydrogen peroxide. Peroxidase
has been demonstrated in Tetrahymena pyriformis (311) and catalase in
certain related ciliates (43, 204), but the activities of neither enzyme in
protozoan metabolism have been investigated.

Glutathione. In the reduced form, this is a tripeptide of glycine, cys-
teine, and glutamic acid. Although reduced glutathione has been demon-

458 Physiology

strated in Tetrahynnena pyriformis (oil, 512) and may play a part in
respiration of this ciliate (364), its functions in protozoan metabolism are
still unknown.

Pantothenic acid enzymes. Pantothenic acid is a component of coenzyme
A (319) which may be involved in acetylation reactions in general and
perhaps in the utilization of acetylmethylcarbinol by certain bacteria. Al-
though pantothenate is essential to giowth of certain ciliates and malarial
parasites, its possible functions in protozoan metabolism have not been
investigated.

Adenosine phosphate system. The adenosine phosphate system includes
adenylic acid (adenosine monophosphate), adenosine diphosphate
(ADP), and adenosine triphosphate (ATP). Each contains adenosine (a
riboside of adenine) and one, two, or three phosphoric acid groups, re-
spectively. The system functions in phosphorylation of metabolites and
enzymes and especially in the transfer of high-energy phosphate bonds.^^
This system apparently makes available for anabolic activities the energy
derived from oxidation of metabolites. Essentially, TPN serves as a par-
ticipant common to a variety of exergonic and endergonic reactions, mak-
ing it possible for reactions of the first type to drive those of the second
variety. Little is known about the adenosine-phosphate system in Proto-
zoa. However, adenylic acid, ADP, and ATP have all been demonstrated
in Euglena gracilis (1). In addition, Trypanosoma hippicum needs ATP
in the formation of hexose-phosphates (194), and Tetrahymena pyri-
jorynis contains adenosine triphosphatase (510).

Tricarboxylic acid cycle.^^ This so-called cycle (Fig. 8. 2) involves the
oxidation of various metabolites through a common catalytic system to
carbon dioxide and water under aerobic conditions. The cycle is fed by
glycolysis, leading to pyruvate and thence to acetyl; by the breakdown of
fats, yielding acetyl from fatty acids; and by the breakdown of proteins,
through the deamination of certain amino acids to a-keto acids which
enter the cycle. At each turn of the oxidative cycle, COo and HoO are
produced as end-products in certain reactions, and energy is made avail-
able by the generation of high-energy phosphate bonds in several de-
hydrogenations. Aside from its importance in the oxidation of substrates,
the tricarboxylic acid cycle may also be considered a basic reservoir of
important materials which can be drawn upon for the synthesis of amino
acids, carbohydrates, and fatty acids.

The tricarboxylic acid cycle has been studied both by the use of isotopes
(578) and by the control of enzyme systems ^\•ith blocking reagents. In the
presence of cyanide at a suitable concentration, oxalacetate is trapped;

^^Tor a discussion of the energetics of high-energy phosphate bonds, a review by
Ogsdon and Smithies (419) may be consulted.

"Representative discussions (10, 165, 416) may be consulted for details of the tri-
carboxylic acid cycle and its general importance in metabolism.

Physiology 459

in the presence of sufficient malonate, which inhibits succinic dehydro-
genase, the cycle stops with the accumulation of succinate. Arsenite checks
the cycle by inhibiting the oxidation of malate to oxalacetate. In addition,
the ability of a species to use components of the cycle may be tested by

CARBOHYDRATE

FATTY
ACIDS

ASPARTAT

PYRUVATE

NH3 ^ACETYL
OXALACETATE

C^ * C3
(acetate,

PYRU VATE ^

•NH3

ALANINE

FUMARATE

cis-ACONITATE

iso-CITRAT£

OXALO-
SUCCINATE

cx-KETOGLUTARATE

Fig. 8. 2. The tricarboxylic acid cycle.

GLUTAM-
ATE

measuring oxygen consumption with each as a substrate or by detennin-
ing possible stimulation of growth.

Little work has been done on the tricarboxylic acid cycle in protozoan
metabolism. There is no reason a priori, for suspecting that all aerobic
Protozoa must complete the oxidation of metabolites through a typical
tricarboxylic acid cycle; there may be some species which do not. There
apparently are such reactions as Co + Co condensations which skip the Ce
acids of the typical cycle. Rhizopiis nigricans can carry out this condensa-

460 Physiology

tion, producing succinate and fumarate from labeled acetate or ethanol
with essentially quantitative recovery of radioactive carbon, indicating no
decarboxylation of intermediate C^ acids (131). In relation to hetero-
autotrophic nutrition of phytoflagellates, it is interesting that a mutant
form of Azotobacter agilis has lost the ability to use glucose, lactate, pyru-
vate, and various Krebs-cycle acids, but retains the ability to use acetate
and ethanol (269, 270).

Evidence for the occurrence of the tricarboxylic acid cycle in certain
ciliates seems conclusive. Tefrahyynena pyriformis takes up carbon dioxide
with formation of succinate in the anaerobic dissimilation of glucose
(561). Studies on oxygen consumption show that pyruvate, succinate,
a-ketoglutarate, fumarate, malate, and oxalacetate are utilized, and that
malonate produces typical inhibition (513, 528). It is interesting that
malonate in low concentrations (5 [xg/ml) serves as a substrate for T.
pyriformis (528), although it is distinctly inhibitory at high concentra-
tions. With the exception of citrate and r/5-aconitate, the various inter-
mediates of the cycle are readily utilized by Plasmodium gallinaceum
(407, 535), and fumarate, pyruvate, and succinate are known to be oxi-
dized by P. lophurne (21). The evidence for such a cycle in phytoflagel-
lates is not yet conclusive. Added to a peptone medium, malate stimulates
growth of Astasia longa, Euglena gracilis, and Polytoma ocellatinn, while
succinate stimulates growth of these and six other species (565), and also
accelerates growth of E. gracilis in darkness (244). In addition, fumarate,
malate, and succinate are satisfactory substrates for E. gracilis var. bacil-
laris at pH 3.0-3.6 (228). Occasional failures to use Krebs-cycle acids have
been observed but these cases probably should be reconsidered. Experi-
mental data for Tetrahymena pyriformis (516) indicate that permeability
of the surface to the substrate is a major factor to be considered. Such a
factor may explain the reported inability of Polytoma uvella to use
malate and pyruvate (456), and that of Astasia klebsii to oxidize succinate
or citrate at a significant rate (83). The need for carbon dioxide, estab-
lished for several phytoflagellates (471), might suggest that carboxylation
occurs as an essential part of the cycle but such an assumption is yet to
be confirmed experimentally. The data for Zoomastigophorea also are
fragmentary. Trypanosoma lewisi oxidizes several of the intermediates
rather slowly (403), although the basal "medium" used for such tests may
not be the most favorable one for reactions which depend upon a variety
of giowth-f actors. Trypanosoma cruzi and the species of Leishmania from
man form succinate as one of the products in oxidation of glucose and
levulose (57).

DIGESTION

Digestion in holozoic species occurs typically within vacuoles which
enclose the food after ingestion. The mechanical features of ingestion vary

Physiology 461

with the species and with the type of food. Ingestion in Amoebida com-
monly involves extension of pseudopodia or formation of "food cups" to
engulf the food. A food cup may be quite deep, as in Amoeba vespertilio
(233) in which the food is taken in through a temporary "cytostome"
(Fig. 1. 15, C) similar to the permanent structures in more specialized
Protozoa. In shelled types ingestion is limited to an area of naked proto-
plasm. If there is a large enough opening, ingested particles may be
passed into the shell, as in Arcella. If the shell contains only small pores,
as in many Foraminiferida, the fusion of already extended pseudopodia
to enclose trapped food is the essential feature. A comparable process
often follows the adherence of food particles to axopodia in Heliozoida
and Radiolarida. Ingestion by pseudopodial activity has been reported in
various holozoic phytoflagellates and other simple flagellates, while fairly
large particles are ingested without marked pseudopodial activity in
Lophomonas and Tricbonympha (125). In certain flagellates and in typi-
cal ciliates, ingestion is limited to a cytostome. In simple cases, this ap-
pears to be merely a thin region of the cortex. More often, the cytostome
lies at the base of a groove or pit. The oral groove, or the peristomial
area, of ciliates is often equipped with strong cilia, membranelles, or
undulating membranes which drive particles into the cytostome (Chap-
ter I).

Food vacuoles

The wall of the vacuole in Amoeba and similar types is derived
from the surface layer of the body. In ciliates feeding on small particles,
the vacuole develops at the inner end of the cytopharynx as an enlarging
bulb which is eventually pinched off (103, 300). Formation of the vacuole
apparently is stimulated by the passage of solid particles through the
cytostome into the cytopharynx, since ciliates in a non-particulate me-
dium contain few, if any, food vacuoles (340). The ingestion of large
masses, as in the engulfment of Paramecium by Didinium nasutum, is a
less simple process. In Suctorea (Chapter VII), a food vacuole is formed at
the base of a tentacle which is usually attached to the prey and sucks its
protoplasm into the captor's body. Food vacuoles apparently may fuse or
divide. Fusion of small vacuoles (391) and the division of large vacuoles
into several smaller ones (387) have been noted in Amoeba proteus. Also,
the collection of small ingested particles into one mass, which becomes
surrounded by a common vacuolar membrane, has been described in
Ichthyophthirius multifiliis (372).

A continuous "digestive tract," in which successively formed food vacu-
oles remain joined with one another by slender tubes, has been described
in Paramecium and VorticeUa (300). In addition, a coiled "canal," extend-
ing from cytostome to cytopyge, has been described in Colpidium (74).
cyclosis of food vacuoles being merely an optical illusion caused by move-

462 Physiology

ment of food along this canal. Such a canal resembles to some extent the
long sausage-shaped food vacuoles formed by Paramecium in certain salt
solutions (94), but other workers have failed to find a tubular digestive
system in ciliates (103, 182).

After formation of the food vacuole, the contents apparently become
acid sooner or later. As reported in Pararnecium cau datum (103, 522) and
Actinosphaerium eichorni (212), a drop to pH 4.0-4.3 occurs after a time.
In Amoeba, the pH of the vacuolar fluid falls to some point between 4.0
and 6.5 (389). The acidity of the vacuole in P. catidatum is said to ap-
proach that of 0.8N hydrochloric acid; in certain other ciliates, less than
that of 1 X 10-^ N acid (413). The origin of this acid is uncertain, al-
though Mast (389) attributed it to the respiration of ingested organisms
and their later autolytic changes. During later stages of digestion and
absorption, there is a gradual rise in pH, sometimes to about pH 7.0
in old vacuoles containing undigested residues.

Undigested materials are usually eliminated through a definite area
(cytopyge) in Protozoa with a well-developed cortex. In various Peritri-
chida the contents of the old vacuole are discharged into the vestibule
(Chapter VII). In many other ciliates, the cytopyge lies at the surface
somewhere in the posterior half of the body. A similar differentiation also
may be found in such flagellates as Peranema trichophorinn, in which the
supposed cytopyge is a small area in the postero-lateral body wall lacking
the usual cortical inclusions.

Digestion of proteins^^

Protozoa which ingest solid food presumably are equipped with
digestive enzymes, and would thus be expected to produce both endo-
peptidases (proteinases) and exopeptidases (peptidases). Didinium na-
siitum seems to be an interesting exception which depends upon its
ingested prey for a supply of peptidases (101). "Glaucoma" pyriformis pro-
duces an endopeptidase active in the pH range, 2.2-9.6, with an optimum
at about pH 6.0 (311, 312); comparable enzymes of Plasmodium gal-
linaceum are more active at pH 6.5 than at 7.4 (408). Exopeptidases have
been reported in Parameciu^n caudatum, Frontonia sp., and Amoeba
proteus (204). Certain trypanosomes likewise produce exopeptidases and
also an endopeptidase of the kathepsin type, but no enzymes of the pepsin
or trypsin types (302).

The ability to digest proteins also has been reported for Euglena
gracilis (173, 237, 377) and for such a saprozoic flagellate as Leishmania
tropica (498). Digestion in such cases is presumably extracellular but it
is uncertain whether the enzymes are eliminated during life or are re-
leased by disintegration of the flagellates.

^* A brief general discussion of digestive enzymes will be found in Baldwin's (10)
monograph.

Physiology 463

Digestion of carbohydrates

Enzymes catalyzing the digestion of carbohydrates are of two
general types — polysaccharidases, or polysaccharases, acting on cellulose,
starch, and similar large molecules, and the glycosidases, acting on such
small molecules as disaccharides and trisaccharides.

The digestion of cellulose has been reported for several Protozoa
(Table 8. 5) and the same ability may be assumed for species which feed
on plant materials or those which invade plant tissues. The demonstra-
tion of cellulases in certain flagellates of termites (216, 217, 555) and
the wood roach (554), and in certain ciliates of ruminants (218, 219),

TABLE 8. 5. UTILIZATION OF POLYSACCHARIDES

Species

Cellulose Starch Inulin Dextrin Glycogen

MASTIGOPHORA

Eiitrichomaslix colubrorum (47)

Leishmania tropica (102)

Slrigomonas media (365)

■S". muscidarum (365)

S. parva (365)

Trichomonas columbae (47)

T. foetus (47)

T. termopsidis (555)

flagellates of termites (217)

flagellates of wood roach (554)

SPOROZOA

Plasmodium knowlesi (133)

CILIATEA

Chilodon cucullus (162)
Colpidium campylum (272)
Diplodinium denticulatum (219)
D. maggii (219)
D. multivesiculatum (219)
Entodinium caudatum (219)
Eudiplodinium neglectum (219)
Glaucoma scintillans (272)
Paramecium caudatum (162)
P. multimicronucleatum (162)
Sapropkilus ovijormis (162)
Tetrahymena pyrijormis E (114)
T. pyrijormis E (279)
r. pyrijormis GF-J (251)
T. pyrijormis GHH (279)
T. pyrijormis GP (251)
T. pyrijormis H (114)
T. pyrijormis H (279)
T. pyrijormis L (71)
T. pyrijormis R (311)
T. pyrijormis T, T-P, W (279)
T. vorax (272, 279)
Trichoda pura (162)

—

■>

—

464 Physiology

presumably qualifies these organisms as symbiotes of their respective hosts.
In addition to the Protozoa in which other polysaccharidases have been
reported (Table 8. 5), the many species which store and utilize starch
and glycogen doubtless have enzymes capable of splitting these reserve
foods into simple sugars. However, the utilization of stored polysac-
charides may involve phosphorolysis rather than digestive hydrolysis.
Starch, for example, would yield a-glucose-1 -phosphate instead of maltose
or glucose. This situation raises the possibility that some of the phyto-
flagellates which store starch may be unable to use exogenous starch as a
substrate.

The utilization of disaccharides (Table 8. 6) has been reported on the
basis of fermentation reactions, the activity of extracts prepared from
Protozoa, or the effects of sugars on oxygen consumption. Such abilities
also may be inferred for species which digest polysaccharides.

Digestion of lipids

Little is known about the utilization of lipids by Protozoa, al-
though the production of lipases by many species seems probable in view

TABLE 8. 6. UTILIZATION OF DISACCHARIDES

Species

Cellobiose

Lactose

Maltose

Sucrose

MASTIGOPHORA

Eutrichomastix colubrorum (47)

Leishmania tropica (102)

Strigomonas media (365)

— "

—

S. miiscidarwn (365)

;!-

5". parva (365)

—

Trichomonas columbae (47)

T. foetus (47)

T. termopsidis (555)

flagellates of termites (217)

flagellates of wood roach (554)

SPOROZOA

Plasmodium knowlesi (133)

—

CILIATEA

Colpidium campylum (272)

—

Diplodinium denticulatum (219)

D. maggii (219)

D. multivesiculatum (219)

Eudiplodinium neglectum (219)

Glaucoma scintillans (272)

—

Saprophiliis oviformis (162)

Tetrahymena pyrijormis E. GF-J, OF, H,

L (71, 114, 251)

—

r. Pyrijormis E, GHH, H, T, T-P (279)

—

T. pyrijormis W, T. vorax (272)

—

T. pyrijormis W, T. vorax (279)

—

Key: +, utilized; ±, utilized very slowly; — , not utilized; ?, data not reported.

Physiology 465

of the common storage of cytoplasmic fats and oils, and the absence of
evidence that different enzyme systems are involved in storage and utiliza-
tion of lipids. Amoeba proteus apparently hydrolyzes several animal and
vegetable oils after their injection into the cytoplasm (89), and also di-
gests fats in food vacuoles (387). The products of digestion pass into the
cytoplasm and are combined there to form droplets of neutral fat (387).
Unlike amoebae, certain trypanosomes apparently do not produce lipases
(302).

NITROGEN METABOLISM

For certain phytoflagellates no amino acid need be supplied from
external sources. Therefore, the major feature of nitrogen metabolism
presumably is the assimilation of ammonium-N in synthesis of the amino
acids needed for growth. In Chilomonas Paramecium and Polytoma ocel-
latum, these syntheses apparently include all the amino acids which are
absolute requirements for Tetrahymena geJeii (182). Such flagellates may
have some promise in tracing the intermediate stages and the growth-
factors involved in synthesis of amino acids. Perhaps the general tech-
nique of "inhibition analysis" (525) will prove applicable here.

The fact that certain phytoflagellates can grow on a single amino acid
indicates that transaminations may be as effective, in a general way, as
the assimilation of ammonium-nitrogen from an inorganic source. Utiliza-
tion of an amino acid as the sole source of energy has not been demon-
strated and the dissimilation of amino acids has not yet been traced. A
little more is known about the metabolism of amino acids in other Pro-
tozoa. Strains of Tetraliymena pyriformis seem to need eleven amino
acids, ten of which are considered irreplaceable for higher animals (10).
T. pyriformis undoubtedly synthesizes additional amino acids. In a
chemically defined medium stripped to essentials, these syntheses must
involve transaminations with at least certain number of the eleven serv-
ing as nitrogen-donors. However these reactions have not yet been
traced.

The production of ammonia — reported for Bodo caudatus (310), Leish-
mauia tropica (498), Acanthamoeba casteUanii (46), Plasmodium gal-
linaceum (408), Didiyiiiim nasutum (567), "Glaucoma" pyriformis (100,
362), Paramecium caudatum (77), Spirostomum ambiguum (532) — in-
dicates that such species can deaminate amino acids but nothing is known
about the specific dehydrogenases involved.

For Protozoa in pure cultures there is little critical information on
nitrogenous excretory products. The rather general production of am-
monia, and also the failure of tests for urea and mic acid in cultures of
Tetrahymena pyriformis (362) and in washed suspensions of Paramecium
caudatum fed powdered fibrin (77), have suggested ammonia as the
probable excretory product.

466 Physiology

CARBOHYDRATE METABOLISM

The utilization of various carbohydrates by ciliates, trichomonad
flagellates, trypanosomes and related forms is well known. On the other
hand, the significance of sugars in the metabolism of phytoflagellates re-
mains problematical. Quantitative techniques have not demonstrated
utilization of glucose by Chilomonas Paramecium and Chlorogoniiim
euchlorum (327). Furthermore, sugars do not accelerate growth of Poly-
tomella agilis and Polytoma ocellata in peptone media (454), and sugars
apparently cannot replace acetate in the synthesis of reserve carbohy-
drates by phytoflagellates (344). In a few cases, stimulation of growth
has been reported in peptone media supplemented with certain sugars.

TABLE 8. 7. UTILIZATION OF MONOSACCHARIDES

Species

Sugars

MASTIGOPHORA

Eutrichomastix colubrorum (47)
Leishmania donovani (71)
Leishmania tropica (102)
Leptomonas ctenocephali (71)
Strigomonas media (365)
S. muscidarum (365)
S. parva (365)
Trichomonas columbae (47)
Trichomonas foetus (47)
Trichomonas vaginalis (557)
Trypanosoma brucei (29, 30)

SARCODINA

Acanthamoeba castellanii (45)

SPOROZOA

Plasmodium knowlesi (133)

CILIATEA

Colpidium catnpylum (272)
Glaucoma scintillans (272)
Tetrahymena pyriformis E (114)
T. pyriformis E (279)
T. Pyriformis GF-] (251)
r. Pyriformis GHH (279)
T. pyriformis GP (251)
T. pyriformis H (279)
T. pyriformis L (71)
r. Pyriformis T, T-P (279)
r. Pyriformis W (272, 279)
Tetrahymena vorax D (272)
T. vorax PP, V (279)
Trichoda piira (162)

a, B, D, E, I,j, 1

B, E

a, B, D, E, G, I, 1

B, E

a, B, D, E, G, I,j. 1

A, B, D, E, G, I,j, 1

a, B, D, E, G, I, j, 1

A, B, D, E, /, j, L
a, B, D, E, I,j, 1
a, B, D, E,j, 1

B, D, E, G

a, B, d, E, G, h, i,j, I

a, B, d, E, I

a. B, d, E, 1

a, B, d, E, G, h,j, 1

a, B, c, D, E, f, G, h, i, j, k, 1

a, B, D, e, g, h,j, 1

a, B, c, d, E, f, G, h, i,j, k, 1

a, B, D, E, g, h,j, 1

a, B, c, d, E, f, G, h, i,j, k, 1

a, B, D, E, 1

a, B, c, d, E, f, G, h, i,j, k, 1

a, B, d, E, 1

a, B, c, D, E, f, G, h, i, j, k, 1

A, arabinose; B, dextrose; C, fucose; D, galactose; E, levulose; F, lyxose; G, mannose;
H, melizitose; I, raffinose; J, rhamnose; K, ribose; L, xylose. Bold-face capitals indicate
utilization; italicized capitals, slow utilization; small letters, no utilization.

Physiology 467

However, such effects have been minor ones and cannot, with any assur-
ance, be attributed to utilization of sugars as substrates. This situation
has been puzzhng in view of the fact that these flagellates store carbo-
hydrates and evidently utilize such reserves. Perhaps the difficulty lies in
some fundamental deficiency such as the lack of an adequate phos-
phorylating mechanism for utilizing exogenous carbohydrates. Or pos-
sibly the permeability of the body wall is too low for effective absorption.
Low rates of absorption presumably would not be a hindrance in holozoic
species. Consequently, the investigation of polysaccharides and disaccha-
rides as substrates for holozoic Euglenida and Chrysomonadida might
yield significant information.

Utilization of monosaccharides (Table 8. 7) has been demonstrated by
fermentation reactions, by measuring stimulation of growth or oxygen
consumption, and by quantitative sugar determinations. The decom-
position of a monosaccharide^^ involves a series of reactions catalyzed by
a number of enzymes (Fig. 8. 3), the initial step being phosphorylation
of the sugar to glucose-6-phosphate. This reaction precedes the dissimila-
tion of exogenous sugar and often its storage as polysaccharide. In this
connection, it is interesting that hexokinase has not been found in Poly-
tomella caeca (356), and also that hexosediphosphate but not glucose can
be oxidized by Astasia klebsii (83). Although utilization of glucose also
has not been demonstrated for Euglena gracilis, this species does contain
the following compounds which appear in glycolysis: glucose- 1-phosphate,
fructose-6-phosphate, fructose- 1,6-diphosphate, and glycerophosphoric
acid (1).

In organisms equipped with hexokinase, glucose-6-phosphate is pro-
duced and also may be stored, presumably by conversion into glucose- 1-
phosphate and thence into polysaccharide. Or, glucose-6-phosphate may
undergo dissimilation, the next step being conversion into fructose-6-
phosphate. Phosphorylation of this ester yields fructose- 1,6-diphosphate.
The diphosphate then undergoes cleavage into two interconvertible
triose-phosphates. Later reactions are traced to pyruvate in Figure 8. 3.
The series of reactions up to this point yields a certain amount of utiliz-
able energy. Aerobically, pyruvate may be oxidized through the tricar-
boxylic acid cycle, with more efficient utilization of the original free
energy in the glucose molecule. Anaerobically, pyruvate may be converted
into lactate or into ethanol.

Glycolysis has been traced, at least to some extent, in a number of
Protozoa. Trypanosomes apparently vary in their methods of attacking
glucose. In the earlier work, no evidence was obtained for phosphoryla-
tion. More recently, Trypanosoma equiperdum has been shown to phos-
phorylate glucose to fructose- 1,6-diphosphate, which is split into triose-

^■"' For details of glycolysis, discussions by Baldwin (10) and Lardy (308) may be
consulted.

468 Physiology

PHOSPHOHEXO-
ISOMERASE

glucose

11 HEXOKINASE. ATP

glucose- 6-pho5pfx3te

f ru cf ose-6 -phosphaf e

PHOSPHOHEXOKINASE. ATP

fructose -1.6 -diphosphate

ALDOLASE

o-g!yceraldehyde-3-
phosphote

TRIOSEPHOSPHATE-
ISOMERASE

TRIOSEPHOSPHATE DEHYDRO
GENASE,
PHOSPHATE

1,3-diphosphoglyceric acid

PHOSPHOGLUCO-
MUTASE

glucoTe- 1- phosphate

PHOSPHORYLASE,

PHOSPHATE

stored
polysaccharide

^ dihydroxyacetone-
phosphote
A

cx-GLYCER0PH0S~

PHATE
DEHYDROGENASE

PHOSPHOGLYCERIC
TRANSPHOSPHORYLASE.
ADP

3- phospho-D- glyceric acid

PHOS PHOG LYC E RO M UTA S E

2- phospho-D -glyceric acid

ENOLASE

phospho-enol- pyruvic acid

PHOSPHO PYRUVATE
TRANSPHOSPHORYLASE.

ADP

pyruvic acid

L-w- glycerophosphate

PHOSPHATASE.
H,0

glycerol ♦
phosphate

Fig. 8. 3. Dissimilation of glucose.

phosphates (63). Glycolysis is similar in T. evansi (379) and T. hippicum
(194). In the latter, hexokinase, aldolase, triose-phosphate dehydrogenase,
glycerol dehydrogeanse, and glycero}>hosphate dehydrogenase have been
demonstrated. The activity of hexokinase in certain trypanosomes is in-
hibited significantly by arsenicals (194, 379) and in malarial parasites by
quinacrine (22, 534).

The products of dissimilation vary in different species of Trypanosoma
(31). Certain species decompose glucose mainly, or even quantitatively
(194), to pyruvate. Trypanosoma equiperdiun (475) produces pyruvate
and glycerol; glycerol accumulates under anaerobic conditions but is con-
verted almost completely to pyruvate aerobically (475). Sugar metabolism

Physiology 469

of T. evansi (379) and T. hippicum (194) is essentially similar to that of
T. equiperdum. It has been suggested that T. hippicum, which lacks cyto-
chrome oxidase, is dependent upon the host for removal of waste pyru-
vate. However, it might be interesting to test T. hippicum and T. eqiii-
perdutn under conditions which would insure an adequate supply of
thiamine and other growth-factors in vitro. Such species as T. lexvisi (475,
517, 518) and T. rhodesiense (135) produce several intermediates. Succinic
forms about 40 per cent of the acids recovered from T. rhodesiense sus-
pensions in glucose-Ringer's solution, and is also a major product for T.
lexvisi. Both species also produce acetic, lactic, pyruvic, and formic acids,
ethanol and COo, and T. rhodesiense produces glycerol in addition.
Formate and COo appear only under aerobic conditions in T. lexvisi
suspensions. Whether succinate is produced through the tricarboxylic acid
cycle is uncertain. Since succinic dehydrogenase is cytochrome-linked and
T. rhodesiense presumably lacks the cytochrome system (32), this trypano-
some may be unable to oxidize succinate after producing it. This may not
be true for T. lexvisi which is rather sensitive to cyanide poisoning and
presumably contains the cytochrome system.

Certain flagellates of termites decompose glucose anaerobically to car-
bon dioxide, hydrogen, acetic acid, and certain unidentified products.
Lactic and pyruvic acids, acetaldehyde, methyl glyoxal, and ethanol have
not been detected in significant amounts (217).

In Plasmodium gallinaceum, hexokinase has been demonstrated (533),
and glucose, lactate, and pyruvate all seem to be oxidized through the
tricarboxylic acid cycle (535). The oxidation of pyruvate is inhibited by
malonate, with accumulation of succinate. Parasitized erythrocytes oxidize
pyruvate almost completely to CO2 and H^O by way of the Krebs cycle
and accumulate very little acetate. Cell-free suspensions of P. gallinaceum
produce considerable acetate, as well as CO^ and H2O, under aerobic con-
ditions and the acetate is not further decomposed; anaerobically, pyruvate
does not disappear and acetate is not formed (535). In P. knoxvlesi, lactate
is produced from glucose and can be oxidized (371, 570), and the increase
in lactate is more or less parallel to the production of pyruvate (571).

Little is known about sugar metabolism of ciliates. Paramecium cauda-
tiun decomposes glucose to unidentified organic acids which account for
about a third of the sugar utilized (77). Tetrahymetia pyriformis produces
lactic, acetic, and succinic acids from glucose under anaerobic conditions
(550). When T. pyriformis was supplied with glucose and radioactive CO2,
all the radioactive carbon appeared in the carboxyl groups of succinic acid,
indicating that COo is assimilated in the production of succinate (550,
412), as previously reported for Trypanosoma lewisi under anaerobic con-
ditions (518). The oxidation of substrates through the tricarboxylic acid
cycle in ciliates is indicated by the presence of succinic dehydrogenase in
Tetrahymena pyriformis (311) and Paramecium caudaturn (215), and by

470 Physiology

the stimulatory effects of fumarate, succinate, and a-ketoglutarate on
oxygen consumption of the former (513).

Synthesis of carbohydrates and lipids

Many Protozoa synthesize and store carbohydrates and lipids as
visible deposits. Little is known about the relations of particular sub-
strates and other factors to such syntheses. Photosynthesis^" makes an
important contribution in many phytoflagellates, but those without
chromatophores also store carbohydrates. In pure cultures, lipids may
accumulate as the cultures grow older, whereas carbohydrates may be
predominant in young cultures.

The lipids synthesized by Tetrahymena pyriformis have been estimated
quantitatively (511); sterols make up about 0.05 per cent of the total
(514). A mixture of fatty acids extracted from T. pyriformis has shown
bacteriostatic activity against several Gram-positive bacteria in vitro but
not in vivo. Similar material from Chilomonas Paramecium showed activ-
ity against pneumococcus type III in vitro (370). Acetate is an effective
substrate for the synthesis of lipids and carbohydrates by T. pyriformis.
Although arsenite and malonate inhibit oxidation of acetate, they do not
influence synthesis of either carbohydrates or lipids (515).

CONTRACTILE VACUOLES IN
HYDROSTATIC REGULATION

The major function of contractile vacuoles seems to be that of
hydrostatic regulation. Although they probably do eliminate some soluble
wastes, their excretory function is of doubtful importance. The many
Protozoa which lack contractile vacuoles must carry on excretion through
the general body surface or some permeable portion, and the same mech-
anism probably is operative in species with contractile vacuoles.

The nature of the excretory products is uncertain for most Protozoa.
So-called excretion-crystals have been described in various species, but the
chemical nature of these inclusions has been disputed and their excretory
significance has not been demonstrated satisfactorily. In attempts to iden-
tify less problematical Avaste products, Howland (211) was unable to
demonstrate uric acid in the vacuoles of Amoeba, Paramecium and Vor-
ticella, but did detect it in fluid from cultures of Amoeba and Para-
meciurji. Weatherby (565) found urea in culture fluid but not in the
contractile vacuole or cytoplasm of P. caudatum. However, urea has been
reported in the vacuolar fluid of Spirostommn, the low concentration
suggesting that only about 1.0 per cent of the theoretical urea produc-

1" Major experimental investigations on photosynthesis in phytoflagellates are yet to
be completed. Reviews of photosynthesis in general have been published by Rabino-
witch (470) and by Franck and Looniis (132).

Physiology 471

tion could be eliminated by the contractile vacuole (566). Ammonia,
rather than urea, seems to be the nitrogenous waste product for a number
of species.

The assumption that the contractile vacuole is a hydrostatic regulator
is based upon the fact that, in a system involving two fluids of different
densities separated by a semipermeable membrane, water should pass from
the less dense into the denser medium until equilibrium is reached. The
cytoplasm would represent the denser medium in fresh-water Protozoa,
and the occurrence of endosmosis would necessitate a mechanism for pre-
venting excessive dilution of the cytoplasm. The general occurrence of
contractile vacuoles in fresh-water Protozoa and the absence of such struc-
tures in many marine and parasitic species support this assumption. An
osmoregulatory function also is indicated by certain experimental data.
Injection of distilled water into Amoeba dubia increases rate of pulsation
and water output of the contractile vacuole (214). A decrease in frequency
of contraction with increasing salinity of the medium has been observed
in Amoeba verrucosa (582), species of Paramecium (144, 197), Gastro-
styla steinii (197) and Blepharisma undulans (144). In A. verrucosa, pul-
sation ceases at a salt concentration of 1.5-2.5 per cent; in G. steinii, at
1.25 per cent (197). Conversely, the rate of pulsation in certain marine
and parasitic species rises with decreasing salinity, as in Amphileptus
guttula (268), Nyctotherus cordiformis (197), and Balantidium, entozoon
(112). Under similar conditions, appearance de novo of contractile vacu-
oles has been described for Amoeba biddulphiae (583) and Vahlkampfia
calkinsi (203). However, Flabellula mira develops no contractile vacuoles
even in a 1:20 dilution of sea water. This species seems to eliminate water
by way of large food vacuoles which are emptied at intervals (209).

The water eliminated by the contractile vacuole may be traced to sev-
eral sources. Endosmosis may account for much of it in fresh water species.
Such a process demands the maintenance of a difference in osmotic pres-
sure across a selectively permeable membrane. The internal electrolyte
concentration of Arnoeba proteus and various ciliates, determined with
microelectrodes for measurement of intracellular conductivity, is equiv-
alent to 0.01-0.068N KCl (148, 149). The internal osmotic pressure of
Spirostomum ambiguum, determined by the vapor pressure method, is
equivalent to that of 0.15 per cent NaCl (448), and the difference of
osmotic pressure across the body wall of fresh water peritrichs approxi-
mates that of a 0.05M sucrose solution (295). Formation of food vacuoles
is another source of water in holozoic Protozoa, although there is some
compensation in the evacuation of old vacuoles. This source accounts for
8-20 per cent of the water eliminated by contractile vacuoles of marine
ciliates (297). Another source of water is that arising in metabolism, but
the relative amount has not been estimated.

472 Physiology

The vacuolar cycle

In the simplest cases, small vacuoles appear in the cytoplasm and
fuse to form a new vacuole which increases in volume (diastole) and then
collapses (systole) in discharging its contents to the outside. Canal-fed
vacuoles receive fluid during diastole from feeder canals which may per-
sist throughout the cycle. As described by Lloyd and Beattie (320) in
Paramecium catidatum, diastole involves: (1) an early rapid phase, coin-
ciding with contraction of the canals to force fluid into the vacuole; and
(2) a slow phase, in which further distension involves diffusion of water
into the vacuole from the cytoplasm. In systole there are: (1) a prelim-
inary slow phase, in which fluid passes from the vacuole into the canals,
distending them; and (2) a rapid phase, in which the remaining fluid is
expelled from the vacuole to the outside. Fluid of relatively high osmotic
pressure — that derived from the vacuole at the beginning of systole —
supposedly remains in the canals and facilitates withdrawal of water from
the cytoplasm in the next cycle. On the other hand, Gelei (146) believed
that connections between the vacuole and the canals are closed before
systole. This is also the case in Paramecium multimicronucleatum (294).

The frequency of pulsation, in general, is gieater in fresh-water species
than in marine or parasitic forms. Cycles range from 6 seconds to 20
minutes for fresh-water species, 45 seconds to 32 minutes for marine and
brackish water types, and 72 seconds to 16 minutes for endoparasitic
forms (296). Fresh-water species eliminate a volume of water equivalent
to body volume in 4-45 minutes, whereas marine ciliates require 2.75-4.75
hours. In a given species, frequency of pulsation increases as the tempera-
ture rises within non-injurious limits. Temperature characteristics ([jl
values), calculated from the equation of Arrhenius, have been reported
for Spirostomum ambiguum, Blepliarisma undulans, and four species
of Paramecium over the range, 16-26.8° (145).

According to the osmotic theory of diastole, water passes into the con-
tractile vacuole by osmosis from the cytoplasm. This mechanism would
require an osmotic gradient favoring the contents of the contractile
vacuole. Since it is not clear just how such a gradient would be main-
tained, it is difficult to account for diastole on this basis alone (296). The
filtration theory (129, 448) holds that hydrostatic pressure forces water
through the vacuolar membrane. Haye (195) and Kitching (296) have
pointed out that hydrostatic pressure would not be relieved by passage of
water into the contractile vacuole, since this organelle is surrounded by
cytoplasm during diastole. The secretion theory, favored by Kitching
(296), postulates secretion of water into the contractile vacuole by the
membrane. This assumption seems logical enough and it conflicts with no
available data.

The discharge of the contractile vacuole has been explained in two

Physiology 473

general ways: (1) that the process involves contraction of the vacuolar
membrane; and (2) that systole is produced by cytoplasmic pressure on
the vacuole. Although the change from a sol to a gel in the vacuolar
membrane might exert enough contraction to initiate systole (147), this
mechanism could not in itself bring about complete discharge. Cyto-
plasmic pressure theories maintain that contraction of the vacuole is
brought about by pressure of the cytoplasm against the vacuolar wall.
Observations on Amoeba dubia (214) and A. proteiis (382), in which the
vacuole becomes embedded in a zone of gelated cytoplasm just before
systole, suggest that pressure from this zone brings about systole. For such
ciliates as Paramecium, it appears that hydrostatic pressure is exerted by
a more or less fluid cytoplasm and that systole may be initiated by some
other factor, such as adjustment of the vacuole to the excretory pore.
Adjustment to the pore, as a preliminary step, would presumably insure
discharge of a full vacuole rather than a partially filled one.

GROWTH OF PROTOZOA

Individuals and populations

The growth of individual Protozoa has been traced in very few
cases. In some species, a constant growth-rate has been reported, as in
Plasmodium praecox (193) and Ichthyophthirius multifiliis (373); in
others, a decreasing rate which may or may not follow a sigmoid curve.
Investigations on this phase of protozoan growth have been reviewed by
Richards (482).

Cultures of Protozoa are essential for investigating a variety of prob-
lems. Pure cultures, containing one species of Protozoa and no other
microorganisms, are a necessity for tracing many biochemical and physio-
logical activities and for determining basal food requirements. "Species-
pure" cultures, containing one strain of Protozoa and one or more strains
of other microorganisms, also have been used to advantage in many in-
vestigations. The maintenance of such cultures with known bacteria in
known concentrations has insured reproducible experimental conditions.
In the case of Entamoeba histolytica, cultures of this type show promise
for preliminary investigations on growth requirements and amoebacidal
drugs (27, 477, 478). Mixed cultures, containing two species of Protozoa
either bacteria-free (37, 93, 182, 462) or with bacteria, also have been used
in a few investigations. An interesting example is the growth of Enta-
jnoeba histolytica with Trypanosoma cruzi (442, 443). Cause (140, 141,
142, 143) has been interested in the struggle for existence between com-
petitors for the same food supply (e.g., Paramecium caudatum and
Stylonychia mytilus feeding on bacteria) and in captor and prey relation-
ships (Didinium and Paramecium). In the competition between 5. mytilus
and P. caudatum, mutual inhibition was noted, S. mytilus being the

474 Physiology

stronger competitor. The observations of Brown (37), Dewey and Kidder
(93), and Provasoli (462) involve the captor-prey relationship in bacteria-
free media. Loefer (325, 326, 328) was dealing with analogous problems in
his bacteria-free cultures of Paramecium bursaria, in which conditions
optimal for grovv^th of Chlorella paramecii were not those most favorable
to growth of the ciliate-algal partnership.

Experimental data based upon cultures are to be interpreted in terms
of protozoan populations. Growth of populations in microorganisms"
may consist of several phases (Fig. 8. 4): an initial stationary phase, with
no increase in number of organisms; a lag phase, during which the rate

LOG NUMBER
ORGANISMS

o <y
Ti o

•'

»^

*"»

(i)

■D

'*^-.

■ I 1

1 1

TIME

Fig. 8. 4. Generalized growth ciir\e for populations of microorganisms.

of population-growth increases to a maximum; a phase of logarithmic
growth, during which the population increases at a constant rate; a
phase of negative growth acceleration, in which the growth-rate decreases
progressively; a maximal stationary phase, in which the population re-
mains essentially constant; and various phases of death, in which the
density of population decreases. The first two phases are sometimes
lumped together under the one term, lag.

The early phases in growth of populations have been investigated in
Euglena (235, 236) and Tetrahymena (37, 116, 439, 440), while more

"Recent discussions of bacterial growtli have been published by Hinshelwood (202)
and Monod (401).

Physiology 475

extended curves have been traced for Paramecium bursaria (326), Poly-
toma (467), Astasia longa (506), and Tetrahymena pyriformis (190, 263).
The histories of such populations show essentially the same phases as
those reported for bacteria.

Initial stationary phase. The occurrence of this phase in cultures of
T. pyriformis is related to the age of the inoculum. No stationary phase
follows inoculation of fresh media from cultures in logarithmic growth,
but with older inocula, length of this phase varies with age of the stock
(439). A similar relationship has been observed in Chilomonas Para-
mecium (393). The responsible factors remain unknown, although or-
ganisms in old cultures show lower reproductive ability than those in
young cultures. This difference in C. Paramecium has been attributed to
storage of an "X-substance" in excessive amounts. After inoculation of
fresh medium with old flagellates, fission is delayed until the excessive
X-substance diffuses into the medium (393). Another possibility is that
the activity of important enzymes is impaired in old cultures, perhaps by
progressive vitamin or mineral deficiency or by the accumulation of in-
jurious substances. Upon inoculation of fresh medium, the regeneration
or reactivation of essential enzymes would have to precede growth. A
change to a radically different medium might demand the development
of "adaptive" enzymes before growth could occur, or perhaps the environ-
mental selection of types adapted to growth in the new medium. If the
fresh medium contains mainly complex foods, some preliminary digestion
might be a prerequisite for growth. Under certain conditions, changes in
the organisms must be the major factor, since no relationship between
length of the initial stationary phase and size of the inoculum has been
observed in Tetrahymena pyriformis (439).

The lag phase. The occurrence of lag has been explained in various
ways. According to one view, favorable changes are produced by the or-
ganisms in a "biological conditioning" of the medium. This possibility
is supported by the stimulatory effects of old culture fluid added to fresh
media for Chilomonas Paramecium (393, 397), Colpidium striatum (378),
and Tetrahymena pyriformis (187, 273) in pure cultures. An analogous
"conditioning" has been noted in bacterized cultures of ciliates (260,
337). Another explanation is that the inoculated organisms are still re-
covering from damage suffered in the stock culture. Therefore, lag is a
period of physiological recovery leading to the accumulation of metabolic
intermediates essential for synthesis of protoplasm. During lag in cultures
of Tetrahyjnena pyriformis there is marked phosphatase activity, with
liberation of inorganic phosphate into the medium (123). The condition
of the organisms evidently is the important factor in some cases, since
inocula from cultures of T. pyriformis in the logarithmic phase show no
lag while those from older stock cultures usually do (439). Such a rela-

476 Physiology

tionship has been noted also in Cbilomonas Paramecium (222). The
duration of lag in bacterized cultures of ciliates also may increase, up to
a maximum, with age of the inoculum (491).

In addition to changes in fission-rate, changes in individual size may
occur during lag. Inocula containing small T. pyriform,is show a gradual
increase to a mean size which is later maintained during the logarithmic
phase. If the inoculated ciliates are large, the size decreases to about the
same average as that reached by small ciliates (421).

Phase of logarithmic groiuth. In late lag the rate of growth increases
to a maximum as the population enters the logarithmic phase. During
this period the average size of individual organisms as well as the growth-
rate may remain essentially constant, as in T. pyriformis (421). Length
of this phase is influenced by various factors, and within such a genus as
Leishmania (58), may vary with the species in a particular medium. The
initial concentration of food is a major influence in cultures of Astasia
klehsii (82), Glaucoma scintillans (272), and T. pyrijormis (19, 116,440),
although the rate of fission may be independent of food concentration
within wide limits (82, 440), Supplementary thiamine extends the loga-
rithmic phase for Chilomonas Paramecium in an acetate and ammo-
nium-N medium (73), and any essential vitamin, food, or metal presum-
ably could become a limiting factor during this phase of growth. A pure
culture also may accimiulate waste products or undergo other unfavorable
changes which bring the logarithmic phase to an end.

Phase of negative growth acceleration. Such unfavorable changes as
depletion of the food supply or marked changes in pH of the medium
sooner or later become significant and the rate of fission decreases, as in
Euglena (235, 236). Lower rates of oxygen consumption in this phase
have been reported for Chilomonas Paramecium (221), Trypanosoma
cruzi (33), and Tetrahymena pyriformis (431). Changes in the respiratory
quotient for T. pyrifor7nis (431) also indicate qualitative changes in
oxidative metabolism. There is also a gradual increase in size of in-
dividual ciliates in populations of T. pyriformis (421).

Phase of maximal density. Progressive changes in the culture medium
finally check increase in number and the population reaches its maximum.
Maximal density has been correlated with initial concentration of food
in Astasia klehsii (82), Glaucoma scintillans (272), Mayorella palestinensis
(473), Parainecium bursaria (326), and T. pyriformis (19, 116, 272, 440).

For certain organisms at least, the vitamin supply may be a more critical
factor than the total amoimt of food. T. pyriformis shows almost no
growth in a filtered and autoclaved peptone medium which has previously
supjDorted a population of the same species. With added thiamine and
riboflavin, however, this used medium supports populations approximat-
ing those obtained 'with fresh peptone (181). Maximal density also may
be limited by adverse changes in pH, as noted for Chilomonas para-

Physiology 477

meciiim in an acetate and inorganic-salt medium. As the medium becomes
increasingly alkaline, growth ceases and death of the flagellates soon fol-
lows (73, 222). Periodic addition of acetic, hydrochloric, or lactic acid
increases maximal density two- to four-fold (222).

Duration of the stationary phase may depend upon a variety of factors.
The thiamine content of the medium is important for T. pyriformis (190,
552), and the pH of the mediinn is a limiting factor for C. Paramecium
(222). It is somewhat uncertain just how the population is maintained
during this phase. Fission may continue at a rate which balances the
losses from death, or the life of individual organisms may be prolonged.

Phases of death. Little is known about this phase in protozoan popu-
lations. Morphological changes often accompany the decline in popula-
tion, and a gradual decrease in individual size to about half the maxi-
mum, observed in the maximal stationary phase, has been traced in T.
pyriformis (421). Death may be accelerated by a sharp drop in pH, re-
lated to thiamine deficiency in a medium containing sugar (552). For
some species the decline in numbers is described by a fairly smooth curve;
in other cases, the curve is more or less irregular. Populations of Para-
mecium hursaria show a steady decline over a period of three weeks or
more in certain media (326). Populations of T. pyriformis, in a casein-
peptone medium, have decreased in two major steps separated by a
period of several weeks in which the population remains almost constant.
Following the second step, in which most of the ciliates die, a small popu-
lation may persist at least six months longer (190). The longevity of
such small populations is related to the available thiamine. T. pyriformis
lives for about four months in a certain gelatin medium, while added
thiamine extends life of the populations to 11-12 months. With peptone
culture fluid which has previously supported growth, supplementary
thiamine extends life of the cultures from a maximum of one week to a
minimum of at least nine months (185).

Size of the inoculum in relation to growth. There are three possible
relationships between the initial density of popvdation and the rate of
growth. (1) The rate of growth may be independent of the initial density
under a given set of conditions. (2) The growth-rate may be higher with
large than with small inocula. (3) The growth-rate may vary inversely
with initial density of popidation.

A relationship of the first type has been noted in pure cultures of T.
pyriformis (439). With optimal bacterial concentrations, a similar rela-
tionship has been observed in species-pure cultures of Stylonychia pusta-
lata (16).

A relationship of the second type involves the so-called allelo catalytic
effect of Robertson (488, 489, 490, 492, 493). According to Robertson's
views, fission is stimulated by a nuclear autocatlyst which is liberated only
during fission. Once fission has occurred, the autocatalyst which reaches

478 Physiology

the cytoplasm during nuclear division soon passes into the culture me-
dium, there to accelerate later fissions. When the inoculum contains more
than one organism, the liberation of more catalyst would cause mutual
stimulation of fission, or allelocatalysis. Accordingly, the fission-rate va-
ries more or less directly with size of the inoculum. An apparent allelo-
catalytic effect has been reported for bacterized cultures of certain ciliates
(258, 436), Chilomonas Paramecium (393), and Mayorella palestinensis
(474). The case of C. Paramecium has been questioned (178) because in-
terpretations were based upon terminal counts without any information
concerning the earlier history of populations.

Various explanations have been proposed for the Robertson effect. Cut-
ler and Crump (78, 79, 80, 81) believed that Robertson's findings resulted
from failure to control the bacterial flora of his cultures. The importance
of the bacterial concentration also has been stressed by Johnson (258)
who showed that, in cultures of Oxytricha fallax, the initial concentra-
tion of bacteria may determine whether a culture is to show a Robertson
effect. Another possibility is that the initial pH of Robertson's poorly
buffered medium was not optimal for his ciliates, which could change the
pH toward the optimum (64). On this basis, two ciliates should produce
such a change more rapidly than one and cause an allelocatalytic effect.
Jahn (240) has suggested that, in similar fashion, the oxidation-reduction
potential of the medium might be responsible for an allelocatalytic effect.

An inverse relationship between growth-rate and initial density was
observed by Woodruff in Paramecium aurelia and P. caudatum. The more
rapid reproduction with lower initial densities was attributed to less rapid
accumulation of waste products (580). A comparable relationship has
since been reported for cultures of P. aurelia, P. caudatum, and Pleuro-
tricha lanceolata (167), Stylonychia pustulata, P. caudatum (88), and
Euglena sp. (235). This so-called W^oodruff effect is variously attributed
to the accumulation of waste products in the medium, exhaustion of a
scanty food supply, and changes in oxygen tension and pH away from the
optimum.

Initial pH of the culture medium

The observed relations to growth indicate that pH of the medium
influences utilization of food and synthesis of protoplasm, perhaps
through effects on solubility and ionization of substrates and on permea-
bility of the organism to components of the medium. Activities of extra-
cellular enzymes also may be influenced by pH of the medium. The
"internal" pH may be relatively independent of environmental pH, since
immersion of Amoeba dubia in liquids at pH 5.5 and 8.0 induces no
change in cytoplasmic pH (54) from the normal level of about 6.9 (479).
However, the activity of a proteinase from Tetrahymena pyriformis varies
with pH of the medium (312). Likewise, the rate of oxygen consumption

Physiology 479

by Trypanosojna rhodesiense decreases as pH of the medium falls, and
both changes can be prevented by buffering the medium (64). Accelerat-
ing effects of carbohydrates on growth of Tetrahymena pyriformis are
marked below pH 7.0, but are insignificant in alkaline media (114). The
effect of plant auxins on growth of Euglena gracilis varies with pH of
the medium and stimulation is greatest at pH 5.6 (118). The rate of
locomotion in Amoeba proteus is related to pH of the medium (208,
449), and pseudopodial activity in ingestion of food may be influenced
likewise. Also, the rate at which food vacuoles are formed in Colpidium
increases from pH 4.5 to 6.0, and then decreases to zero at pH 8.0 (399).
For Protozoa in general growth in pure cultures has been reported

TABLE 8. 8. GROWTH-pH RELATIONSHIPS OF VARIOUS
PROTOZOA IN PURE CULTURES

Species pH Range Optimum

MASTIGOPHORA

Astasia klebsii, peptone (82)

Chilomonas Paramecium, peptone (324)

C. Paramecium, peptone, acetate (324)

C. Paramecium, heteroautotrophy (453)

Chlorogonium elongatum, peptone (324)

C. elongatum, heteroautotrophy (453)

C. euchlorum, peptone (324)

C. euchlorum, heteroautotrophy (453)

Euglena anabaena, peptone (172)

E. deses, peptone (172)

E. gracilis, peptone (2)

E. gracilis, peptone (104)

E. gracilis, peptone (237)

E. gracilis var. bacillaris (331)

E. klebsii, peptone (105)

E. mutabilis, peptone (84)

E. piscijormis, peptone (105)

E. stellata, peptone (105)

E. viridis, inorganic (508)

Polytoma uvella (453), heteroautotrophy

Polytomella caeca (346), peptone

Trichomonas vaginalis (254)

SARCODINA

Alayorella palestinensis (472) 6.4-7.2 6.8

CILIATEA

Colpidium campylum (272) — 5.4

Glaucoma scintillans (272) — 5.6-6.8

Paramecium bursaria (328) 4.9-8.0 6.8

Tetrahymena pyriformis E (113) 4.5-8.5 5.5; 7.4

T. pyriformis GY-] (252) 4.9-9.5 5.1-6.0

T. Pyriformis GP (252) 4.0-8.9 4.8-5.3

T. Pyriformis U {\\A) . 4.5-8.5 5.5; 7.4

r. pyriformis W (272) — 5.6-8.0

Tetrahymena vorax (272) — 6.2-7.6

3.2-8.2

4.2-6.0

4.2-8.4

4.8-5.1; 6.8-7.1

5.8-8.4

7.0

5.7-6.7

—

4.9-8.7

7.6

5.7-8.5

—

4.9-8.7

7.4

5.7-8.5

—

4.5-8.3

6.9

5.3-8.0

7.0

3.0-7.7

6.7

3.5-9.0

—

3.9-9.9

6.6

2.5-8.8

—

5.5-7.5

6.5

2.1-7.7

3.4-5.4

6.0-8.0

—

4.5-8.0

5.5

4.0-7.2

—

7.1-8.5

—

2.2-9.2

—

4.9-7.5

5.4-5.8

480 Physiology

between the pH limits 2.1 and 9.9 (Table 8.8). Survival for at least short
periods may be possible within a wider range, such as pH 2.3-11.0 for
Euglena gracilis (2), 2.0-9.65 for E. gracilis var. bacillaris (331), and 1.4-
9.6 for Polytomella caeca (346). Euglena mutabilis apparently can survive
in polluted waters at pH 1.8 (306), and in pure cultures, for at least 12
days within the range, 1.4-7.9 (84). Growth throughout most of the gen-
eral range seems to have been observed only in Euglena gracilis and
Polytomella caeca, and the specific range varies considerably in other
species.

The pH optimum also varies from species to species and within one
species under different conditions. Unfortunately, it is sometimes uncer-
tain just what a reported "optimum" means in terms of protozoan growth.
The apparent optimum may depend upon the time of observation, as in
Euglena gracilis which showed heaviest growth at pH 6.6 after 8-9 days,
but at pH 1.1-1 A after seven weeks (237). Present knowledge of growth-
pH relationships should be extended by tracing growth curves in media
at different pH levels. Most of the available information does not elimi-
nate the possibility that within reasonable limits, a pH above or below an
apparent optimum may retard growth without modifying the eventual
density of population.

The growth of Astasia longa in acid media throws some light on such
questions (507). Growth in peptone medium at pH 3.7, for example, is
rapid for the first fev; days and then ceases for a period of 3-5 weeks.
Later, a second period of growth produces populations comparable in
density to those obtained much sooner at higher pH levels (Fig. 8. 5).
This resumption of growth apparently cannot be attributed to the slight
rise in pH (0.1) during incubation. Only the first phase of growth is
observed in a medium at pH 3.1 and second transfers in medium at the
same pH show no significant growth after four months. A delayed growth
phase seems to be limited to distinctly acid media since it has not ap-
peared within the pH range, 6.0-9.6. A particularly interesting feature
of these populations is the early increase in acid media, even at a pH
level which inhibits later growth. The data suggest the possibility
that inocula from a healthy culture may contain enough critical re-
serves to insure a 20- to 25-fold increase in number, in an unfavorable
environment. This reserve apparently is exhausted before the flagellates
are completely adjusted to the new environment, and in media which
are not too acid, a period of "adaptation" precedes the resumption of
growth.

Two periods of logarithmic growth separated by an appreciable sta-
tionary phase — Monod's phenomenon of "diauxie" — have been observed
also in bacteria grown on a mixture of two carbohydrates (401). In such
cases, it has been assumed that the first phase of growth ends with

Physiology 481

exhaustion of the more readily utilized substrate, and that the bacteria
must become adapted to the second sugar before growth is resumed.

Relationships between growth and pH are fmther complicated by
the occasional observation of two "optima" at the end of a given period.
Such bimaximal relationships, which remain unexplained, have appeared
in bacterized cultures of Stylonychia pustulata (87) and in pure cultures
of Tetrahymena pyriforrnis (113, 252) and Chilofvonas Paramecium

A-

2-'

LOG. number/ml

; /

<ir/

;l— -

7>/

pH 3.1 (transfer I)

pH 3.1 (transfer 2).

"1 1 1 1 1 r

1 1 1 r

100

DAYS

Fig. 8. 5. Growth of Astasia loiiga (strain J) in relation to pH of the
medium. The curves are based on data of Schoenborn (507).

(324). The two optima are replaced by one in T. pyrijormis (113) and
C. Paramecium, (324) grown in the presence of acetate, and T. pyriformis
also shows only one optimum in certain protein and peptone media
(113, 114, 115, 252). Additional questions are raised by variations of the
apparent optimum with the type of medium (113, 115, 252, 324). This
may be the case in bacterized as well as in pure cultures. For instance,
Paramecium aurelia has been assigned a pH optimum in certain cases,
whereas the fission-rate of this ciliate fed on Serratia marcescens is practi-
cally the same between pH 5.9 and 7.7 (438). Growth of Tetrahymena

482 Physiology

pyriformis on Serratia marcescens also was about the same between pH
4.5 and 8.6, but yields were greatest at about pH 5.0 and 7.4 in similar
suspensions of Klebsiella pneumoniae, Pseudornoans fluorescens and
Proteus vulgaris (253).

Temperature

The biothermal range, or range of temperature permitting growth,
extends from about 54 to aproximately 0° C. for Protozoa. Adaptation
to the higher temperatures within this range is rare, although certain
flagellates (at 54°), shelled rhizopods (at 51°), amoebae (at 50-52°) and
cilia tes (at 46°) have been reported from hot springs (232).

Except for the unusual thermophilic species, active stages are killed
by temperattnes approaching or exceeding 45°. Euglena gracilis, at pH
7.0, is killed within eight minutes at 44° (239); Entamoeba gingivalis,
within 20 minutes at 45° (299); Paramecium caudatum, within nine
seconds at 40° (451); Spirostomum ambigmun, at 36° (524); Colpoda
cuciillus, at 37-45° after exposures of 0.5-10.0 minutes (17). Termite
flagellates are eliminated from their hosts after 24 hours at 36° (67),
and gregarine trophozoites from Tenebrio larvae after six days at 37.5°
(369). Lethal exposures depend upon time as well as temperature, and
the thermal death time at a given temperature also varies with pH of
the mediimi. The resistance of Euglena gracilis to high temperatures is
greatest at pH 5.0 and is less above pH 7.0 than below (239). Paramecium
caudatum, on the other hand, shows greater resistance to 40° above and
below pH 7.0 than at the neutral point (53). Both E. gracilis (239) and
P. midtimicroniLcleatum (98) have shown increasing resistance with in-
creasing density of population. After the maximum is reached, however,
resistance decreases gradually in older cultures of the latter.

Cysts are generally more resistant than corresponding active forms.
Dried cysts of Colpoda cucullus, for example, resist 100° dry heat for three
hours, although moistened cysts die within 30 minutes at temperatures of
49-55°. Excystment is retarded by non-lethal exposures to 37-48° (17).
Somewhat higher lethal temperatures, in 5-minute exposures to moist
heat, have been reported for intestinal parasites: Entamoeba coli, 76°;
E. histolytica, 68°; Endolimax nana, 64°; Giardia lamblia, 64°; Chilo-
mastix 7nesnili, 72°; lodaynoeba biischlii, 64° (18). Unsporulated oocysts
of Eimeria miyairii are quickly killed at 53° (476).

Short exposure to temperatures below 0° C. is often not lethal to
active stages (111, 239, 576). Cultures of Leishmania donovani have re-
mained viable after intermittent exposure to —12° over a period of 10
days (200) and Entamoeba gingivalis may live almost 18 hours at 0° (299).
Fission may continue slowly — for example, a fission every two weeks in
Paramecium caudatum (111) — at temperatures just below zero. Cyto-
plasmic division is more susceptible than nuclear division to extremes of

Physiology 483

temperature in Amoeba proteus, so that binucleate forms are occasionally
seen toward the limits of the range (86). Freezing (111, 239, 576) and
prolonged exposure to sub-zero temperatures (111) are fatal to active
stages of many species, although cysts of Colpoda have survived exposure
to liquid air (546).

Little is known about biothermal ranges of individual species. How-
ever, fission occurs in Amoeba proteus at 11-30° (86); in Astasia longa at
15-30° in peptone media and at 22-30° in ammonium-N media (506); and
in Chilomonas Paramecium between 9.5 and 35° (529). An optimum for
fission has been reported in a few species: Paramecium aurelia, 24-28.5°
(581); Chilomonas Paramecium, 26-30.5° (531); Astasia longa, 30° (506);
Tetrahymena geleii, 28.5° in the range, 7.8-28.5° (441). Euglena gracilis,
in peptone medimn, has shown an optimum of 10° in darkness. With sup-
plementary acetate, the optimum is shifted to about 23° which is approxi-
mately that for growth in light (243).

Temperature coefficients (Qio values) and thermal increments ([x val-
ues) for fission have been calculated in several cases. For Paramecium
aurelia, Qio = 2.7 at 21.5-31.5° (581); for P. aurelia, [j. = 23,000 calories at
12-25° (400); for fission of Amoeba proteus (86), ^ — 16,500 calories at
11-30°, and for cytoplasmic division [x = 20,500 (11-21°) and 7,300 (21-
30°). For Tetrahymena pyrijormis, Q,,, and jj, values vary with the
temperature range: at 7.8-12.3°, Qu. = 9.7 and [x = 35,800 cal.; at 12.3-20°,
Qto = 3.0 and jl = 18,400; at 20-28.5°, Qio = 1-5 and ^ =7,350 (441).
Reported Q^o values (22-28°) for Astasia longa vary with the medium —
2.10 in peptone, 2.17 in acetate and peptone, 1.28 in acetate and ammo-
nium-N, and 8.03 in an inorganic medium (506).

The use of thermal coefficients and thermal increments in biology
has been based upon the assumption that Q^o and [i values are related
to the nature of a reaction, and upon the hope that a study of such
data might furnish clues to the fundamental nature of various biological
phenomena. The Q^,, value is the coefficient of increase in the velocity of
a reaction for each 10° increase in temperature. Qio values are calculated
from the equation,

, ^ 10 (log ki - log k2)

log Q,o = — ^ ^/ ^ ^ ^

tl — to

in which k^ represents the reaction velocity at temperature t^ and ko the
velocity at temperature to. Log k is a linear function of temperature
(Centigrade). For a particular reaction, Q^o values vary with temperature
and usually increase as the temperature decreases. For example, Qio
may be 10 or greater for a given reaction at low temperatures, as com-
pared with 2 or less for a higher range.

The thermal increment, described by the law of Arrhenius, is calculated
from the equation.

484 Physiology

„ .4.6 (log k2 - log ki)

1_1
Ti To

in which Tj and T2 are absolute temperature values. The [x value repre-
sents the heat of activation, or the number of calories required to trans-
form one gram equivalent of "inactive molecules" of the reacting sub-
stance into "active" ones. There is a close relationship between [j, and
Qio values, and the latter may be derived from the former for short
temperature ranges. A O^y value of 2.0 corresponds to a [x of about 13,200;
a Qio of 10, to a [ji, of about 44,000 calories. Unfortunately, the biological
significance of thermal coefficients and thermal increments is uncertain.

Light and darkness

A source of light is obviously important for chlorophyll-bearing
flagellates, in which the relation to photosynthesis doubtless accounts
for various effects on growth. However, Dusi (106) has reported that
under constant illumination Euglena gracilis grew well in peptone me-
dium but poorly in inorganic mediimi, whereas E. klebsii grew well in
inorganic medium under the same conditions. E. viridis, on the other
hand, failed to grow under constant illumination. Temperature as the
significant factor, rather than illumination, apparently was not com-
jiletely excluded in these cases.

Light and darkness also may influence the effects of other factors on
growth. Thus the thermal optimum for Euglena gracilis in peptone
medium is about 10° in darkness and 25° in light (243). Accelerating
effects of certain organic acids are relatively greater in darkness, while
oxalate is slightly stimulatory in light and without effect in darkness
(244). Plant auxins also have accelerated growth of E. gracilis in light
but not in darkness (119).

Even less is known about growth of higher Protozoa in relation to
light. Richards (481), in analyzing data on growth of several ciliates,
noted that the seasonal rhythms reached a peak in July. On this basis,
he suggested that temperature is less important than sunlight when
both are variables. On the other hand, light of high intensity is lethal
to pigmented Blepharisvia undiilans, the effect being attributed to a
photooxidation of the pigment with irreversible damage to protoplasmic
components (159). Indirect effects have been reported for Plasmodiimi
cathcmeriiun. Exposure of the hosts to artificially prolonged periods of
"day" and "night" lengthen the cycle of merogony (26).

Effects of certain toxins and venoms

Bacterial exotoxins are relatively inactive against Protozoa. Ex-
posure of Paramecium aurelia, P. calkinsi, and P. caudatum to diphtheria

Physiology 485

toxin has not affected fission-rate or death-rate (444), although undiluted
culture filtrates containing this toxin may be lethal (559). Tetanus toxin
(150 MLD) and botulinus toxin in various concentrations are without
action on P. caudatujn (445). On the other hand, a thermostable cytolysin
produced by Pseudomonas aeruginosa is lethal to Glaucoma scintillans
(60). Although ricin is inactive, certain snake venoms, in minimal con-
centrations of 1.4-150 [j,g/'ml, are lethal to P. caudatum. Locomotion is
inhibited and rupture of the cortex and disintegration of the ciliate occur
sooner or later. Susceptibiltiy to Crotalus atrox venom varies with the
species. Bursaria truncatella, P. aurelia, and Stenlor coeruhiis are killed
within an hour, Frontonia leucas, Oxytricha fallax, and Volvox after
longer periods, while certain other species are not harmed (445). This
apparent resistance of certain species may be largely a matter of degree.
For example, the MLD (minimum lethal dose) of Crotalus atrox venom
for Coleps hirtus is nine times that for Oxytricha fallax. Sensitivity of
14 species to Cobra venom shows no apparent correlation with sensitivity
to Crotalus venom (446). Adequate doses of antiserum completely pro-
tect Paramecium multimicronucleatum against lethal concentrations of
Cobra venom (447).

EflFects of certain therapeutic drugs

In addition to their action on growth of Protozoa, certain drugs
have shown specific effects on metabolic activities (349). From a practical
standpoint, such results are of interest because they help to plan attacks
against parasites at vulnerable points. As more is learned about food
requirements and metabolic activities, the development of specific drugs
for particular parasites more closely approaches realization. Another
interesting possibility is that the determination of specific effects of
chemotherapeutic drugs may reveal additional tools for the analysis of
protozoan metabolism.

Specific effects of certain therapeutic drugs have been reported for
malarial parasites (349, 406) and trypanosomes. Hydrolysis of proteins
by Plasmodium gallifwceum is retarded by atebrin and quinine (408),
and the oxidation of carbohydrates also is retarded by these drugs (527).
The antimalarial activity of a series of naphthoquinones seems to be
related to their effects on succinic dehydrogenase (128, 199). Oxygen con-
sumption of Plasmodium cathemerium is inhibited by sulfanilamide and
sulfathiazole (562), and that of P. knowlesi by sulfanilamide (68) and
quinine (134). Surprisingly, however, sulfanilamide has no effect on
respiration of P. inui, and little or no therapeutic action, whereas the
drug eradicates infections with P. knowlesi in the same host (69). Triva-
lent arsenicals (halarsol, reduced atoxyl, reduced tryparsamide) are
powerful inhibitors of respiration in Trypanosoma rhodesiense (134).

486 Physiology

Triose-phosphate dehydrogenases of T. hippicujn are sensitive to oxo-
phenarsine (194), and the activity of hexokinase also is inhibited by
arsenicals in trypanosomes (62, 194, 379).

The susceptibility of Protozoa to certain antibiotics varies with the
species. Eiiglena gracilis var. bacillaris remains viable in concentrations
of penicillin at least five times as great as those tolerated by Tetrahymena
geleii and the difference in resistance to streptomycin is of the same
order (330). Tyrothricin, in chickens, has shown parasiticidal activity
against extracellular merozoites of Plasmodium gaJUnaceum (544). Aureo-
mycin likewise has shown activity against Entamoeba histolytica (374),
and comparable effects have been reported more recently for terramycin
(Chapter XI). One of the most interesting effects reported so far is the
bleaching action of streptomycin on certain green flagellates, first reported
in Euglena gracilis var. bacillaris (468). The effect, which involves a loss
of the ability to synthesize chlorophyll, presumably is the result of specific
damage to certain enzyme systems.

As mentioned above for pantothenic acid, pteroylglutamic acid and
nucleic acid derivatives, certain vitamin analogues retard or inhibit
growth and oxygen consumption of several Protozoa. In addition to the
general interest of such findings and their bearing on the determination
of vitamin requirements, the therapeutic value of certain pantothenate
analogues in chickens infected with Plasmodium gallinaceum (28) indi-
cates that results of practical value may be expected in further exploration
of this field.

Effects of carcinogenic hydrocarbons

Stimulation of fission by several carcinogenic hydrocarbons has been
reported for Paramecium (577), but these findings have not been con-
firmed (553). In the only investigation on ciliates in pure culture, Tittler
(551) has obtained no evidence that 3,4-benzpyrene, methylcholanthrene,
or 1,2,5,6-dibenzanthrene significantly influences growth of Tetrahymena
pyriform is.

EFFECTS OF IRRADIATION

Irradiation is a tool of potential value in the study of various prob-
lems. One of the least explored is the possibility of inducing biochemical
mutations in Protozoa, and the prospects grow more intriguing as proto-
zoan food requirements become better known. Effects on rates of fission, as
well as the immediate and pathological effects of irradiation, have in-
terested a number of workers, so that such information is available for
a few species.

Beyond the violet end of the visible spectrum extend the overlapping
ultraviolet, X-ray, and gamma-ray spectra. The ultraviolet spectrum in-
cludes radiation from wave-lengths of about 390 mjji, (3900 A., or Ang-

Physiology 487

Strom units) to 1.5 m^i or less. Rays of 390-200 m\i are transmitted through
quartz and are sometimes termed the "quartz spectriun." Below 200 m[x
lies the Schumann-Lyman-Millikan region in which the rays are absorbed
by water, air, and most other materials. In the Schumann range (approxi-
mately 200-125m[j.), fluorite is used for transmission.

The reported effects of ultraviolet irradiation vary with the wave
length, the dosage, the species, and physiological condition of the organ-
isms (155). In the quartz spectrum, radiation is relatively harmless at
the longer wave lengths. Heavy dosage at 313 m[K is not lethal to Para-
mecium multimicromicleatum (157), and there is almost no effect on
Euglena at 313 and 365 va^. (543). Excystment of Colpoda duodenaria is
slightly retarded at 313 mjj, in a dosage of not less than 30,000 ergs/mm^,
but tripled dosage at 366 mjji, is without effect (153). Peraneyna tricho-
phorum is killed at 253 m[j, but not at longer wave lengths (523). Radia-
tion at 302 m[j, in a dosage of 28,000 ergs/mm^, and also the shorter wave
lengths in lighter dosage, are lethal to P. midtijnicronucleatum (157).
Selective effects of particular wave lengths have been noted. Motor re-
sponses of P. trichophorum are most rapid at 302 m[x (523). Fission of
Paramecium caudatiim is retarded more markedly at 280.4 than by equiv-
alent dosage at 265.4 m^., although recovery from exposure to the longer
wave lengths is more rapid. This difference in rate of recovery is attrib-
uted to greater absorption by nucleoproteins at 265.4. Since absorption
is essentially the reverse for cytoplasmic proteins, fission is delayed to a
greater extent at the longer wave length (152). At a particular wave
length, the specific effects may vary with the dosage. At 280.4 nirj,, im-
mobilization of P. caiidatiim requires about 11,800 ergs/mm^, while fis-
sion is retarded by 2,000-3,000 (154).

Effects of ultraviolet vary also with the species. Fabrea salina is about
six times as resistant as Tetrahymena pyriformis and the latter is twice as
resistant as Blepharisma undulans and Spirostomiim amhiguum to radia-
tion at 253.7 n\]). (151). Differences also have been noted within the genus
Paramecium and among several strains of P. multimicronucleatum (158).
Physiological condition of the organisms also influences susceptibility.
Tetrahymena pyriformis in old cultures is much less resistant than in
young populations (151), and starved specimens of Paramecium are more
susceptible than well-fed ciliates (158). Sensitivity of P. caudatum seems
to be greatest in early stages of fission (201). Sensitivity of Paramecium
also increases with rising temperature within the range, 0-30° (96), and
preliminary exposure to ultraviolet increases susceptibility to high tem-
peratures (23).

Effects on fission also have been noted. Fission of P. caudatum may be
accelerated by light dosages (3, 201), and in the absence of serious injury,
recovery from heavier dosage may be accompanied by accelerated fission
(24). Still heavier dosages retard or inhibit fission (3, 152, 201, 524), and

488 Physiology

successive exposures may interrupt fission of P. caudatuin to form chains
of several individuals (201). The ultraviolet action spectrum for Para-
meciiun has been described by Giese (154).

Certain morphological effects are to be expected. Liquefaction of the
cortex in A^noeha diibia and A. proteus is followed by temporary lique-
faction of the endoplasm and then, after heavier dosage, by gelation of
the endoplasm (196). Comparable changes occur in Spirostomiim am-
biguuni (524). After short exposures the endoplasm becomes fluid and
cyclosis is accelerated, while prolonged exposure causes gradual increase
in viscosity, and finally coagulation and vacuolation. Locomotion is ac-
celerated at first, but ceases as lethal dosage is approached. Fragmentation
of the macronuclear chain is common. The cortex eventually ruptures
after lethal exposure. Vacuolation and cytolysis, following immobiliza-
tion, also have been described in 11 other species of ciliates (151, 157).
Cytolysis has not been observed in Peranema trichophorum , although
immobilization, distortion of the body and coagulation of the endoplasm
are characteristic effects (52.S). Euglena gracilis disintegrates after heavy
dosage with quartz ultraviolet, but green flagellates are less sensitive than
colorless strains of the species (250).

Beyond the ultraviolet, the X-ray spectrum extends into the region of
gamma-rays emitted by radium, decreasing wave length being correlated
with increasing power of penetration ("hardness"). Of the radium ema-
nations (alpha, beta, and gamma rays), alpha-rays are softest and gamma-
rays hardest. X-rays produced at 1,000 kv or more extend into the
gamma-ray region.

The morphological effects of X-rays and radium are similar to those of
ultraviolet. Movement of Paramecium (95) and Colpidijwi (76) is first
accelerated and then retarded in lethal exposure to X-rays, and amoeboid
movement is similarly affected by radium (435). Vacuolation of the cyto-
plasm, upon exposure to radium, has been noted in Amoeba diploidea
(584), A. vahlkampfia (435), Entamoeba histolytica (409), and Spiro-
stomwn aynbiguum. (496), and the effect of X-rays on Euplotes taylori is
similar (38). Cytolysis of Paramecium (95), although not of Peranema
trichophorum (523), is caused by lethal dosage with X-rays, and lethal
exposure to radium induces cytolysis of Spirostomum ambiguum (496).
Heavy dosage with alpha rays (polonium source) causes immobilization
and cytolysis of Polytoma uvella. Lighter dosage may be followed by fis-
sion, but the daughter flagellates undergo cytolysis (206). Sublethal ex-
posure of Eudorina elegans to radium induces deformed daughter colonies
containing less than the normal number of flagellates (169). Unusually
large organisms, resulting from continued growth but retarded fission,
occur in Colpidium colpoda after exposure to X-rays (76), and in Bodo
caudatus (485, 487) and Entamoeba histolytica (409) after exposure to
radium. The sensitivity of Paramecium to X-rays is increased by pre-

Physiology 489

liminary treatment with vital dyes and other reagents (97), and is less
at 15° than at lower or higher temperatures (84a). Paramecium hursaria
seems to be less sensitive to X-rays than its symbiotic algae, which are
sometimes eliminated at certain dosages (572). Lethal effects of X-rays
on Tetrahymena pyriformis in pure culture have been attributed to the
production of H2O2 in culture media, which become toxic whether irra-
diated directly or prepared from irradiated distilled or tap water (547).
Effects of radium and X-rays on growth of populations have been
described for several species. Growth of Entamoeba histolytica, exposed
to gamma-rays primarily or to unscreened radium for 24-48 hours, reaches
a maximum one to several days sooner than in the controls (409). Ex-
posure of P. caiidatum and P. muJtimicronucleatum to X-rays for 10
minutes to four hours has retarded fission for 2-5 days. Longer exposures,
or exposures repeated at intervals of several days, may increase the fission-
rate (191). Bodo caudatiis, exposed continuously to gamma-rays in serial
transfers, shows retarded fission and no acclimatization. Such effects may
persist for several weeks after removal of the radium, although recovery
is complete after three months. In a given transfer, the lag phase is
prolonged almost three hours in irradiated cultures (485), and the period
of greatest sensitivity occurs about 2.0-2.5 hours befoie the first fission in
a new culture (486). Although the generation time is essentially normal
thereafter, irradiated populations cannot catch up with the controls be-
fore the end of the incubation period. Slower growth in the young irra-
diated poptdation is correlated with larger individual size. Irradiation
for part of the incubation period, so as to allow 8-11 subsequent hours of
growth, is followed by acceleration of growth to produce pcjpulations
exceeding 90 per cent of the normal density (487). The production of
ammonia (per culture and per flagellate) by B. caudatiis is increased after
exposures which produce maximal effects on size and fission-rate (31o).

Locomotion

Locomotion in free-living Protozoa is of two basic types: siuim-
?ning, which depends upon the activity of flagella, cilia, or their deriva-
tives; and creeping, which is dependent upon direct contact with a
substratum. Creeping in Amoebida and similar organisms usually involves
pseudopodial activity and is termed amoeboid movement.

AMOEBOID MOVEMENT

Several explanations have been proposed for amoeboid movement
(90, 382, 499). According to one view, locomotion in Amoeba proteus is a
"walking" process in which extended psetidopodia become attached to the
substratum and then contract to pull the body forward (91). A rolling
movement has been attributed to Amoeba verrucosa. A given point on the
surface passes forward on the upper surface, downward at the anterior

490 Physiology

end, remains on the lower surface for a time as the body rolls forward,
and then passes upward at the posterior end to repeat the cycle (248).
Locomotion in Amoeba Umax has been interpreted as "fountain stream-
ing," in which there is a forward streaming of endoplasm through a
tubular layer of ectoplasm. During movement, endoplasm is continually
converted into ectoplasm at the anterior end, and ectoplasm into endo-
plasm posteriorly. According to this interpretation, the flow on the upper
surface is backward, instead of forward as in rolling movement (480),

Mast (382, 384) has resolved the ectoplasm of A. proteus into a thin
elastic pJasmalemma, or surface layer, and a thicker plnsmagel. Between
the two there is usually a hyaline fluid, except where the plasmalemma
is attached to the substratum. During locomotion the plasmalemma flows
forward, as in rolling movement. The plasmagel remains a tube which
is converted into endoplasm (plasmasol) posteriorly and is formed from
plasmasol anteriorly as a pseudopodium grows. Locomotion is attributed
to several processes: (1) The plasmalemma becomes attached to the sub-
stratum. (2) There is a local, partial liquefaction of the plasmagel. (3)
The rest of the plasmagel, which is under tension, forces the plasmasol
against this weakened area to produce a bulge, the beginning of a pseudo-
podium. (4) Posteriorly, the inner surface of the contracting plasmagel is
converted into plasmasol. (5) Anteriorly, the plasmagel tube is continu-
ously regenerated by gelation of the plasmasol as the pseudopodium grows.
The major factor is thus assvmied to be a contraction of the ectoplasm, or
plasmagel of Mast. The nature of this contraction remains uncertain,
although it has been suggested that contraction represents the elastic
recoil of a plasmagel under continuous tension (382), that syneresis of
the plasmagel causes contraction (383, 433), and that the process of
gelation involves or causes a contraction (231, 316).

In locomotion of shelled rhizopods, such as Arcella and Diffliigia, a
developing pseudopodium extends from the mouth of the shell and
swings about freely until it makes contact with the substratum and ad-
heres to it. Contraction of the pseudopodium then pulls the body forward
(91, 383). If movement is to continue, a new pseudopodium is extended to
repeat the process. Locomotion of creeping Foraminiferida, by means of
myxopodia, is similar. The myxopodia are extended, become attached
to the substratum, and then contract to pull the organism toward the
point of attachment. Axopodia also may function to a limited extent in
movement along a substratum. The mechanism in Acanthocystis (434)
apparently involves terminal adhesion of axopodia, followed by a con-
traction which rolls the body toward the point of attachment.

FLAGELLAR LOCOMOTION

The mechanical aspects of flagellar activity have been disputed
and various explanations have been suggested for locomotion in flagel-

Physiology 491

lates. Perhaps the most plausible mechanism is that suggested by Lowndes
(333, 334, 335), whose data indicate that the basic function of the flag-
ellum, at least in uniflagellate species, is to produce rotation of the or-
ganism on its major axis as well as gyration about an axis which marks
the general direction of locomotion. In flagellar activity, waves pass
spirally along the flagellum with increasing amplitude from base to tip,
producing two distinct components of force. The resultant of these two
components, acting on the anterior end of the flagellate, causes both ro-
tation and gyration which, in an elongated organism, supply the force
for propulsion, the principle being that of the screw or propeller. An
additional forward component may be supplied by the flagellum itself if
it is swung backward as in Euglena viridis, but not if it is merely swung
outward more or less at a right angle as in Rhabdomonas incurvxim (334).
In such colonies as Volvox, the flagella are believed to act as propellers,
drawing water toward the points of attachment and thus creating for-
ward components of force. The stroke of the flagellum is so directed that
the Volvox colony usually rotates in swimming, although rapid swim-
ming without rotation also may occur (388). The ingenious experiments
of Brown (36) produced data which agree with the interpretations of
Lowndes, and indicate further that gyration of a flagellum alone also may
produce a fairly effective locomotor force. This possibility may explain
gliding in Peranema trichophorum, which Lowndes (333, 335) apparently
could not reconcile with his observations on other flagellates.

Swimming in ciliates

In two respects, rotation of the body on its long axis and the
usually spiral path of locomotion, swimming in ciliates resembles that
in various flagellates. Therefore, the principle of the screw or propeller
would seem applicable to swimming in ciliates also. However, the cilia
themselves apparently contribute a major forward component of force
in addition to causing rotation and gyration. This is indicated in the
"browsing" movements of ciliates along a surface during feeding. Move-
ment may be slower than in ordinary swimming, and particularly in
various hypotrichs, rotation of the body does not occur. The activity of
cilia, or their derivatives, is solely responsible for such movements. The
analysis of ciliary behavior in moving ciliates is a more difficult problem
than that of tracing flagellar movements. However, the activity of indi-
vidual cilia seems to be quite variable (36, 499), and may even include
spiral, flagellum-like undulations (36). Such a range of activity is presum-
ably correlated with the variety of maneuvers to be observed in ciliates.
The spiral path followed in swimming, as traced by Bullington (39)
in 164 species, shows a width, length, and direction rather characteristic
of each species. Both rotation and gyration are attributed to the com-
bined action of all the body cilia rather than a particular group. In

492 Physiology

ciliates normally tracing left spirals, the cilia beat obliquely backward to
the right for forward movement. When the same ciliate swims backward,
the cilia beat obliquely forward to the left. Although a given type of
spiral is more or less characteristic of a species, five species of Paramecium
(40) and four of Frontonia (41) may follow either right or left spirals,
although swimming is always more rapid in one direction than in the
other. Certain other ciliates swim either in right or in left spirals, but
not in both. A right spiral is characteristic of backward swimming in both
Parajnecmm and Frontonia, and is independent of the spiral followed in
forward locomotion.

RESPONSES TO STIMULI

Reactions of Protozoa to different stimuli vary with the species as
well as with the nature and intensity of the stimulus. Some species may
show no reaction to a stimulus which evokes marked reactions in others.
The responses studied most extensively are motor reactions which usually
tend to move a sensitive organism toward or away from the source of
stimulation with some regularity. The response typically involves the
organism as a whole, and the morphological nature of the response de-
pends upon and is limited by the structure of the organism. In other
words, the response is a stereotyped reaction which depends primarily
upon structural features of the species rather than upon the nature of
the stimulus. The character of the response seems to be one of "trial and
error" (248), rather than an immediate and directly induced orientation
to the stimulus as would be required in the usual concept of tropisms.
In a typical species of Euglena, which rotates on its long axis and also
follows a spiral path in swimming, the reaction to moderate stimulation
usually shows the following pattern (248). Following stimulation, the
gyrations of the anterior end of the body are suddenly widened, presum-
ably by an increase in the transverse thrust of the flagellum, and then
normal swimming is resumed in a new spiral path. If the stimulus is still
encountered, the reaction is repeated until the organism enters a path in
which there is no stimulating effect. If stimulation is intense enough, the
flagellate temporarily stops forward movement or may move backward a
short distance before turning into a new path. The reaction of a swim-
ming ciliate is comparable to that of Euglena. Stimulation causes the
organism to swim backward for a short distance, stop, and then swim
forward in a new spiral. Or backward swimming may be omitted. If the
stimulus is still effective, the characteristic reaction is repeated until the
path of the organism eliminates the stimulating effect. Such hypotrichs
as Oxytricha often creep about on the substratum without rotation of
the body on the long axis. If stimulated while creeping, Oxytricha swims
backward, swerves to the right, then swims forward again. The process
is repeated until the stimulating effect disappears. In spiral swimming

Physiology 493

and in creeping the characteristic gyration or swerving occurs in a par-
ticular direction presumably determined by structure of the body. The
reactions of amoeboid organisms are less complicated in that locomotion
is by "creeping," without the rotation and gyration characteristic of
freely swimming flagellates and ciliates. Changes in direction are brought
about by formation of new pseudopodia at a different point on the body
surface.

Responses to light

The reactions of Protozoa to light have been reviewed by Mast
(380, 388). The stimulating intensity of light varies with the wave length
as well as with the intensity of illumination. Within the visible spectrum,
light at about 485 mjjs, produces the maximal effect on species of Chlamy-
domonas, Euglena, Goyiium, Pfnicus, and Traclielomonas, while light at
535 mjji is most effective for Eudorina, Pandorijin, and Spondylomorum
(381). The stimulatory spectrum for Volvox (309) is similar to that for
Euglena. Many flagellates — species of Euglena, Chlamydomonas, Crypto-
7nnnas, and Goniwn, among others — react so that the path of locomotion
is definitely oriented to the source of light. Others, such as Peranema,
may show merely a shock reaction which is not followed by definite
orientation. Species of Euglena (248, 380) respond to a sudden change in
the intensity of illumination by their characteristic motor reaction, and
the response is repeated until the stigma is equally illuminated at each
point in the spiral path of locomotion. As a result, photopositive speci-
mens swim toward the source, and photonegative specimens away from
the source of light. Illumination of Amoeba proteus (386), which is pho-
tonegative in strong light, causes an increase in thickness of the plasmagel
by inducing gelation of the adjacent plasmasol in the stimulated region.
This increase in elastic strength causes a contraction of the plasmagel in
the stimulated area. Therefore, the formation of pseudopodia in this
region is inhibited and new pseudopodia will tend to develop at the op-
posite end of the body. A small increase in illumination may do nothing
more than retard temporarily the growth of a pseudopodium. The result
of the first type of reaction is a photonegative response, while the second
type produces only a delay in locomotion. The photonegative Stentor
coeruleus (248, 380), one of the few ciliates known to react definitely to
light, shows a typical motor reaction to increased illumination, and the
response is repeated until the organism is equally illuminated through-
out its spiral course and is moving away from the source of light.

Reactions to electric current

Although reactions to the electric current can scarcely be con-
sidered part of the adjustment to natural environments, many Protozoa
show rather specific responses. In the genus Amoeba, reactions vary with

494 Physiology

the species. Amoeba proteus shows a well defined orientation in direct
current and moves toward the cathode, whereas A. dofieini shows no re-
sponse (385). The reaction of A. proteus (168, 385) depends upon an
induced solation at the cathodal surface, resulting in a decreased elastic
strength of the plasmagel in this area. The response of the organism de-
pends upon its orientation when stimulated. Amoebae moving toward
the anode show reversal of protoplasmic flow at the cathodal end, fol-
lowed by cessation of flow at the anodal end. If the current is too strong
and the medium is not acid, disintegration of the organism begins at the
anodal surface, whether the amoeba is moving toward or away from the
cathode. With weaker currents, the direction of locomotion is reversed.
Cilia tes (249, 388) usually react to a direct current by reversal of the
ciliary stroke on the cathodal surface. As a result, the body is turned so
that the organism swims toward the cathode. In a strong but sub-lethal
current, ciliary reversal may be so extensive that the ciliate swims back-
ward toward the anode.

Responses to temperature

Reactions to unfavorable temperatures, as described for various
ciliates, involve typical motor responses similar to those noted under
stimulation of light in certain species. The response is repeated until the
path of locomotion takes the organism into a region with a more favor-
able temperature.

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Heredity in Protozoa'

Inheritance within the strain
Strains, races, biotypes
Tendency toward genetic uniformity
Apparently spontaneous changes
Environmentally induced changes

Genetic effects of syngamy

Syngamy in haploid flagellates
Syngamy in diploid Protozoa

Genetic effects of conjugation
General effects of conjugation
Cytoplasmic lag in biparental inherit-
ance
The micronuclcus in conjugation
Behavior of mating tvpes in conjugation
Behavior of antigenic types in conjuga-
tion

Genetic significance of endomixis, autog-
amy, and cytogamy
P.ndomixis
Autogamy
Cytogamy

Genetic significance of the macronucleus

The cytoplasm in inheritance

The killer trait in Paramecium aurelia
Mating types and cytoplasmic inherit-
ance
Antigenic types and cytoplasmic inherit-
ance

Literature cited

INHERITANCE WITHIN
THE STRAIN

Strains, races, biotypes

PROTOZOAN SPECIES is composed of strains (races, biotypes, or
stocks) which differ among themselves in hereditary traits. Such races
have long been known in Paramecium (24, 25), Difflugia (2.1), Arcella (21)
and C entropy xis (70). Observations on mating types of ciliates (Chapter
II) have shown that a conventional species also may include varieties
which are completely, or almost completely, unable to interbreed. This
situation creates taxonomic problems which cannot be solved until more
is known about mating types and the comparative characteristics of these
different ciliate strains. Racial characteristics are of various kinds. Dif-

^ Re\ lews of protozoan genetics have been published by Jennings (28, 29, 30) and
Sonneborn (76, 81).

506

Heredity in Protozoa 507

ferences in size and fission-rate are well known in various ciliates. Strains
of Tetrahymena in pure cultures have shown minor differences in bio-
chemical activities (Chapter VIII), and in the extent to which they can
become acclimatized to salt solutions (42). Differences in pathogenicity,
noted among strains of parasitic species, may be paralleled by morpho-
logical differences. Strains of Entamoeba histolytica with relatively low
pathogenicity may show a small average size (16). In Plasmodium vivax,
relatively low and high degrees of pathogenicity may be correlated with
slow and rapid reproduction. That these racial characteristics are in-
herited is indicated by their persistence in cultures or in infected animals.

Tendency toward genetic uniformity

Although non-hereditary differences, induced perhaps by environ-
mental factors, may be expected within a race, reproduction by fission or
budding should insure exact duplication of genes from generation to
generation, barring mutations or mitotic accidents. Therefore, genetic
constancy of the race would be expected in the absence of sexual phe-
nomena. In general, this expectation has been realized. Such was the case
in the early work of Jennings (24) on Paramecium. Although separation
of wild populations into several races was usually possible, selection for
size within the race was no longer effective. Similar findings of Ackert
(1) and Jollos (33) also indicated that the race is relatively constant.
However, certain apparent exceptions have been reported.

Apparently spontaneous changes

Selection within the race, continued for many generations, has
produced distinct stocks of Difflugia corona differing in number and
length of spines, diameter and height of the shell, and diameter of the
mouth (27). Comparable effects have been observed in Centropyxis
aculeata (70) and Arcella dentata (21). These results remain unexplained.
Although it is possible that gene mutations were involved, undetected
environmental differences might have been perpetuated under the ex-
perimental conditions of continued selection. In the latter case, the dif-
ferent types probably could be considered results of acclimatization
rather than mutations.

Comparable changes within the race have been reported in a few
ciliates. By opposite selection through more than 150 generations. Middle-
ton (48) established two strains of Stylonychia pustulata differing in rate
of fission. During selection there was a gradual increase in the average
difference, indicating that the effects of selection were cumulative. Since
these differences persisted after conjugation and also through fission for
several months after selection was discontinued, Middleton suggested that
the selection of small variations may be "an effective evolutionary pro-
cedure." Similar changes, involving size, division-rate, and resistance to

508 Heredity in Protozoa

environmental factors, were observed by Raffel (65, 66) in a clone of
Paramecium aurelia. The new types did not revert to normal, even after
conjugation, and were believed to have arisen by gene mutation. The
origin of two unusual biotypes — differing from the parent stock in rarity
of conjugation, lower division-rate, higher mortality, and frequency of
morphological abnormalities — also has been reported by Sonneborn and
Lynch (92) in P. aurelia.

Some of these hereditary changes in ciliates were attributed to endo-
mixis (5, 15, 92). Diller's (11) report of autogamy in P. aurelia was dis-
counted as a possible explanation on the grounds that his evidence for
autogamy was far from convincing and that extraordinary assumptions
would be necessary in relating autogamy to the appearance and subse-
quent disappearance of particular traits (92). The recent conclusion of
Sonneborn (81), that autogamy (and not endomixis) occurs in his strains
of P. aurelia, evidently leaves some of these intraracial changes in ciliates
unexplained for the present. The effects of selection described in Stylony-
chia pustulata cannot be ascribed to endomixis or autogamy because
neither process seems to have been observed in this speties.

Certain morphological changes in ciliates have been interpreted as
mutations. An example is Hance's (19) race of Paramecium caudatum
with 2-7 extra contractile vacuoles, an abnormality inherited in fission
and vmaffected by selection or conjugation. Likewise, hereditary changes
in number of nuclei have been observed in P. bursaria (101). A trun-
cated type of P. aurelia has shown similar behavior, persisting through
more than 400 generations without being influenced by conjugation or
endomixis (9). MacDougall's (43) tetraploid mutant in Chilodonella
uncinatus also bred true.

Environmentally induced changes

A variety of changes may be induced by modification of environ-
mental conditions. Although some cases of acclimatization may represent
merely the selection of a resistant strain from a genetically mixed pop-
ulation, serologically distinct types evidently can arise within a pure line,
as in Trypanosoma brucei (67).

Changes in resistance to chemical agents have been investigated in
parasitic and free-living species. Among the parasites, most of the work
has been done on trypanosomes- in which antigenic modifications, occur-
ring during an infection, are especially interesting. After inoculation of
a guinea pig, for example, with Trypanosoma rhodesiense, the flagellates
increase in number for a time. Suddenly, most of them are killed by a
newly developed antibody. The survivors continue to multiply, so that

* Papers by Dobell (14) and Taliaferro (93) may be consulted for references to the
earlier literature.

Heredity in Protozoa 509

the blood is repopulated by a relapse strain. This relapse strain is re-
sistant to the trypanocidal antibody which is still present in the host and
is still active against the original strain {passage strain). Two explana-
tions have been suggested: (1) the activity of the antibody brings about
selection of a resistant strain (the relapse strain) from an originally mixed
population; (2) as the result of an antigenic change, the trypanocidal
antibody is no longer specific for flagellates which give rise to the relapse
strain. The second interpretation receives support from the fact that an
infection started with a pure line of T. brucei showed the usual develop-
ment of a relapse strain (67). The treatment of different strains of Para-
mecium aurelia with homologous antisera also has induced antigenic
changes which are inherited (89).

Similar phenomena have been observed in chemotherapy of trypano-
somiasis. Most of the flagellates are killed, but a few may survive to pro-
duce a resistant strain — often termed an "arsenic-fast" or "antimony-fast"
strain, depending upon the type of drug, although such a designation
may not be entirely accurate. In tests of several substituted phenylarsen-
oxides, for instance, trypanosomes have seemed to develop resistance to
substituent basic or acidic groups on the phenylarsenoxide molecule
rather than to the arsenoxide group as such (72). This drug resistance
may persist for long periods. Strains of T. rhodesiense have remained
resistant to atoxyl, tryparsamide, and acriflavine for 7.5 years through 900
mouse transfers (52), and to atoxyl for 12.5 years through 1,500 mouse
transfers (17). A strain of T. brucei was still tryparsamide-resistant after
59 transfers through guinea pigs and four through Glossina 7norsitans
(52). A tryparsamide-resistant strain of T. rhodesiense has been produced
also by repeated treatment of the flagellates in vitro. The trypanosomes
were exposed to the drug, washed, and then inoculated into a mouse.
The strain was recovered from the mouse and the procedure was repeated
a number of times, with the result that the flagellates became at least
500 times as resistant as the original stock (103). Among the malarial
parasites, Plasynodium gallinaceum has inherited paludrine-resistance in
five cyclical transfers through mosquitoes without intervening drug treat-
ment (4). The mechanism involved in development of resistance to drugs
is unknown. One suggestion is that resistant trypanosomes have lost their
normal ability to absorb active drugs (23). In addition, differences in
stainability of normal and resistant strains have been demonstrated, and
the development of resistance may accompany shifts in isoelectric points
of various trypanosomal proteins (72).

Although genetic significance has not been considered m many studies
of acclimatization in free-living Protozoa, inherited modifications have
been reported in a few instances. Neuschloss (53, 54) acclimatized Para-
mecium caudatum to quinine, arsenic and antimony compounds, and

510 Heredity in Protozoa

various dyes by exposing the ciliates to gradually increasing concentra-
tions. The developed resistance was specific except for some reciprocal
effects of arsenic and antimony compounds. Similar results have been
obtained in P. aurelia and P. caudatiim by a combination of selection and
acclimatization (33). For example, a strain was grown in a non-lethal
concentration of an arsenical and then subjected to a dosage lethal for
most of the ciliates. The survivors were returned to a non-lethal arsenic
medium for a time before heavy dosage was repeated. As the procedure
was continued, the strain became progressively more resistant. Resistance
was inherited for long periods after a return to normal culture media.
Such modifications — although inherited through hundreds of fissions,
through endomixis (or autogamy?), and in rare cases through conjuga-
tion— eventually disappeared after removal of the stimulus. Accordingly,
Jollos (33, 34) called such changes "Dauermodifikationen," distinguish-
ing them from true mutations. The more recent acclimatization of both
amicronucleate and normal strains of Colpoda steinii to arsenicals indi-
cates that the micronucleus is not necessarily involved in "Dauermodifi-
kationen" (71).

Comparable acclimatization has been reported in Bodo caudatus,
strains of which developed a tolerance to acriflavine in concentrations of
1:500, as compared with the normal susceptibility to dilutions of 1:50,000
to 1:10,000. This resistance was inherited, in decreasing degree, for at
least a year in drug-free media (68).

Morphological modifications have been reported in several cases. Loss
of the kinetoplast, induced in Trypanosoma brucei by inoculating in-
fected mice with certain dyes, became an apparently fixed characteristic
(98). Loss of the parabasal body also was induced in Bodo caudatus by
treatment with acriflavine, but no permanently abnormal strain was ob-
tained (68). Various structural changes have been reported in Chlamy-
domonas debaryana (49). One type could be transformed into another by
maintenance in an appropriate medium for a period varying with the
length of time the original strain had been exposed to the conditions
which produced it. In view of these findings, Moewus suggested that many
of the varieties found in natural populations are merely "Dauermodifika-
tionen" induced by specific environmental conditions.

Morphologically distinct types of Chilodonella uncinatus have been in-
duced by ultraviolet irradiation. These changes, believed to be mutations,
persisted through fission and conjugation (44, 45). Likewise, a physio-
logical change, expressed as a lowered fission rate, has been induced in
P. aurelia by treatment with X-rays (40). Homozygous strains were ob-
tained in autogamy, and the abnormality was transmitted through both
exconjugants in matings between normal and abnormal clones. This in-
duced change was attributed to a micronuclear mutation (41).

Aside from the rare cases which may have involved true mutations.

Heredity in Protozoa 511

the genetic significance of these induced changes remains uncertain.
Jollos considered them the result of cytoplasmic modification rather than
gene mutation — an interpretation with interesting implications. In re-
production by fission, an original mass of modified cytoplasm would
already be diluted several million times at the twentieth generation, and
some of these induced modifications have persisted for several hundred
generations after removal of the stimulus. It is inconceivable that modi-
fied cytoplasm could exert significant effects in such high dilutions. If
"Dauermodifikationen" are strictly cytoplasmic, the modified cytoplasm
obviously must reproduce itself in a sort of cytoplasmic inheritance.

GENETIC EFFECTS OF
SYNGAMY

Syngamy in haploid flagellates

Meiosis appears to be zygotic in Phytomonadida, with the result
that heterozygous vegetative stages are eliminated by persistence of the
haploid chromosome number throughout most of the life-cycle. Since the
genotypic composition of the flagellate is indicated by its phenotype after
division of the zygote, the phytomonads may be favorable material for
the study of biochemical genetics because so many species can be grown
bacteria-free in media of known composition. The induction of mutations
in autotrophic and heterotrophic types might produce physiological
changes which could be analyzed genetically. Experimentally induced loss
of chlorophyll might make possible crosses between green and colorless
strains of the same species. Such matings might supply significant data on
the genetics and biochemistry of chlorophyll formation and perhaps on
cytoplasmic inheritance. Although such aspects of phytomonad genetics
have not been explored, the inheritance of morphological traits has been
traced in a few species.

The first observations were reported by Pascher (55, 56) in two strains
of Chlamydomonas. In some cases, the lines derived from hybrid zygotes
were essentially identical with one parental type or the other. Occasion-
ally, some of the lines showed combinations of parental characteristics
and apparently represented new genetic combinations.

Essentially the same pattern of inheritance was reported by Moewus
(50) in intraspecific and interspecific crosses of Polytoma pascheri and
P. uvella. Linkage of such features as size of the body and length of
flagella was described, and occasional crossing-over was reported. Similar
results were obtained with Chlomydomonas eugametos, C. paradoxa, C.
paupera, and C. pseudoparadoxa (51). Although these observations are
very interesting, they need confirmation because the validity of the data
on crossing-over has been questioned (58).

512 Heredity in Protozoa

Syngamy in diploid Protozoa

A number of Protozoa undergo gametic meiosis and are diploid
throughout most of the life-cycle (Chapter II). Except for the ciliates,
which carry on conjugation instead of syngamy, the genetics of diploid
species is yet to be investigated.

GENETIC EFFECTS OF
CONJUGATION

The significance of conjugation in heredity was discussed by
Biitschli, R. Hertwig, and Maupas long before adequate experimental
data were available. It was suggested that conjugation, in bringing about
biparental inheritance, forms new combinations and thus increases varia-
tion. At the same time, conjugation was believed to level out major dif-
ferences arising in other ways, and in this sense, to limit the range of
variation.

General effects of conjugation

The work of Pearl (57) indicated that exconjugants are less vari-
able than non-conjugants, and that conjugation tends to prevent extreme
variation instead of inducing variation. However, Jennings (26) found
that exconjugants were more variable than non-conjugant lines with re-
spect to fission-rate. Since these differences were inherited, conjugation
in a population apparently gave rise to new biotypes, although the de-
scendants of a single pair were closely similar as a result of biparental
inheritance. The appearance of new combinations after conjugation
within a population was reported also in later investigations (7, 32, 64).
The effects may vary with the strain of Paramecium aurelia, variation
being increased in some strains but not in others (90).

The other general effect of conjugation is the production of similarities
through biparental inheritance in single pairs of exconjugant lines. In
tracing the effects of hybridization on viability, body-length, and fission-
rate of Paramecin??! aurelia, Sonneborn and Lynch (92) found that some
lines resembled one parental type, some resembled the second, and others
were intermediate. Inbreeding showed that the intermediate types were
heterozygous; the others were apparently homozygous. It was concluded
that the inheritance of these traits in P. aurelia is basically mendelian.

Cytoplasmic lag in biparental inheritance

An unusual feature of hybridization has been the occurrence of a
"cytoplasmic lag" in the exconjugant phenotypes of P. aurelia. In the
experiments of Sonneborn and Lvnch (91), the two lines from each pair
of conjugants did not become phenotypically identical until ten genera-
tions or so had passed. A similar lag characterizes inheritance of body-

Heredity in Protozoa 513

size in P. caudatum (10), the original size being retained in a hybrid
exconjugant line for 10-36 generations. Since the two lines derived from
a pair of conjugants were considered genotypically identical, this lag in
appearance of the new phenotypes supposedly represented the time re-
quired for elimination of the old cytoplasm and the production of new
cytoplasm under the influence of the heterozygous synkaryon. Assuming
that the volume of old cytoplasm is halved at each fission, a dilution of
at least 1:1,000 would seem to be required in these cases before the new
zygotic nucleus can assert itself by producing a new phenotype.

1st division

2d division

potent i a
gametic
nuclei

potential
zygotic
nuclei

Fig. 9. 1. Theoretical genetic effects of conjugation in Parame-
cium aurelia, based on Dillcr's (11) account of micronuclear behavior.
In the diagram, it is assumed that the two conjugants are heterozygous
for some particular trait and that only two haploid nuclei undergo
the third pregamic division. Only the micronuclei are indicated.

514 Heredity in Protozoa

The micronucleus in conjugation

The behavior of the micronucleus and its derivatives must be con-
sidered in relation to the potential genetic effects of conjugation. For
instance, it is often assumed that the two gametic nuclei in a conjugant
are derived from the same parental haploid nucleus. If this is the case,
the nuclear contributions of a heterozygous conjugant to the two zygotic
nuclei of the conjugating pair would be identical. So far as cytological
evidence goes, this is not necessarily true in Paramecium aurelia because
"two to five products of the second division continue to divide" (11),
and thus produce a number of potential gametic nuclei. Therefore, it is
possible that the two successful gametic nuclei of a heterozygous conju-
gant could originate from different nuclei and thus be genetically differ-
ent (Fig. 9. 1). In P. caudatinn also, a variable number of nuclei undergo
the third pregamic division to produce more than two potential gametic
nuclei, and both cross-fertilization and self-fertilization (cytogamy) are
believed to occur in conjugation (12). Two products of the second matu-
ration division normally undergo the third division in Euplotes, so that
there are four potential gametic nuclei (37, 94). Are the functional
gametic nuclei derived from one second-division nucleus or from two?
Genetic data indicate that both methods of origin occur in Euplotes (8).

Behavior of mating types in conjugation

The effects of conjugation on mating types apparently vary with
the species and the variety of ciliates. In variety I of Paramecium bursaria
(31) the descendants of each pair of conjugants have belonged to the
same mating type in most cases. The few exceptional pairs show various
results. In some cases, the two exconjugants may produce clones of dif-
ferent mating types. In other pairings, a single exconjugant has produced
two different mating types. In some cases, these two types were parental
types; in others, they were not. Five crosses, of the six possible for the
four types in variety I, have produced descendants belonging to all four
mating types. Jennings concluded that mating types are controlled by the
genetic composition of the synkaryon and that the appearance of non-
parental types might represent new nuclear combinations. Chen (6) has
suggested that inheritance of mating types in P. bursaria is probably in-
dependent of the chromosomes count, which may vary as much among
races within the same mating type as it does among different types.

Inheritance of mating types in group A of P. aurelia (81, 86) — types I
and II (variety 1), V and VI (variety 3) and IX and X (variety 5) — may
be illustrated by crosses between types I and II (74). Three results are
possible (Fig. 9. 2). All of the exconjugant lines may be type I; all may
be type II; or an individual exconjugant may differentiate into types I
and II, usually at the first exconjugant fission. The third type of result

Heredity in Protozoa 515

was believed to indicate that mating types are controlled by the macro-
nucleus (74), although it is not clear how this can explain the production
of two types from one exconjugant. The possible action of cytoplasmic
factors {plasmagenes) in variety 1 seems to have been dismissed in the
statement that "mating is determined by the genes and is not affected by
whatever initial differences in cytoplasm may have existed" (86).

Mendel ian inheritance has been reported in matings between a per-
manently type I strain and a "two-type" strain in which both types I and
II appear within a clone (74). The hybrid exconjugant lines usually
showed the two-type condition, indicating its dominance over the one-
type trait. In back-crosses between the recessive (one-type) and the hybrid

Fig. 9. 2. Inheritance of mating types in conjugation of Paramecium
aurelia, group A, mating types I (solid black) and II.

(two-type) progeny, about half of the exconjugant lines showed the one-
type and the rest the two-type condition, as in a mendelian back-cross.
Certain exceptional results were later attributed to cytogamy, which may
occur in about 60 per cent of the conjugating pairs at 27° (81), a tem-
perature within the optimal range (25-30°) for conjugation of variety 1
(87). The method of inheritance, in this "first discovery of inheritance in
Mendelian ratios in the ciliate Protozoa" (74), is not altogether clear.
Individual matings, in crosses between the one-type (type I) and the two-
type strains, doubtless involved type I and type II ciliates. These type II
ciliates presumably were hybrids because the exconjugant lines, instead
of belonging only to type II or type I, included both mating types. The
progeny, in back-crosses to the parental type I (one-type) stock, must have

516 Heredity in Protozoa

been represented only by type II phenotypes which were type II/I hy-
brids, since the back-cross produced both mating types. However, such
assumptions do not explain the occasional origin of two mating types
from a single exconjugant, as reported in the back-crosses.

In group B of P. aurelia — types III and IV (variety 2), VII and VIII
(variety 4), XI and XII (variety 6), and XV and XVI (variety 8) — mating
types usually do not change at conjugation (87). In crosses between types
VII and VIII (Fig. 9. 3), the type VII exconjugant usually produces type
VII, and the other exconjugant type VIII lines. Cytogamy, which seems
to occur occasionally in variety 4 (77), might account for such behavior

C"<0

R E O R G A/N 1 Z A T I 0/N

M M M

Fig. 9. 3. Inheritance of mating types in conjugation of Paramecium
aurelia, group B, mating types \^II (solid black) and VIII.

of mating types. However, both exconjugants may sometimes produce
type VII lines, or both may give rise to type VIII lines. At present, the
combined results cannot be explained logically on the basis of nuclear
behavior in conjugation. Consequently, Metz (47) has insisted that cyto-
plasmic factors afford the only mechanism which can account for the
behavior of mating types in group B.

The inheritance of mating types in Euplotes patella (types I-VI) has
been explained by a system of triple alleles in which the genotypes are
represented as follows: type I, mtimt^; type II, mtiints; type III, mtgrntg;
type IV, mtjTntj; type V, mtzmts; type VI, mt^mt^. Crosses between types
I and II yield types I, II, IV, and V, while IV x VI crosses yield only type
I (38). The crosses I x III, II x VI, and III x IV, also have produced the
expected results (60). An interesting feature of E. patella is that amicro-

Heredity in Protozoa 517

nucleate strains have retained their type characteristics and have shown
mating reactions with other amicronucleate E. patella (37). Mating types
in this ciliate are correlated with the production of specific substances
which are released into the culture fluid and can induce conjugation in
certain combinations. Induction of conjugation by a given substance is
restricted to strains which cannot produce that substance, and may occur
within a clone previously showing only one mating type. For instance,
mating-type substance 1, from a type IV culture, induces conjugation
within a clone of type III, V, or VI (Chapter II). The relation of this
phenomenon to the results obtained in crosses of two mating types is
not yet clear.

Behavior of antigenic types in conjugation

At least four, and possibly six, antigenic types have been identified
within killer stock 51 of P. aurelia and its variants (82, 86). In crosses
between antigenic types A and B (82, 89), three different results may be

«)•■«)

R E p R G A /N I Z \A T I O /N

H 0( M 01

>n c. P 5

r\ c — E O

O — Q

Fig. 9. 4. Inheritance of antigenic types A (solid black) and B in con-
jugation of Paramecium aurelia.

expected (Fig. 9. 4), (1) The progeny of the type A conjugant remain
type A and those of the other remain type B, even through autogamy.
(2) The type A conjugant gives rise to type A lines, while the type B
conjugant produces some type A, but mostly type B. (3) Descendants of

518 Heredity in Protozoa

the type A conjugant remain type A, while the other conjugant produces
mostly type A and few or no type B lines. If a different type B strain is
mated with type A, the proportions may be exactly reversed. The behavior
of micronuclei affords no apparent basis for all these results.

GENETIC SIGNIFICANCE OF ENDOMIXIS,
AUTOGAMY, AND CYTOGAMY

Endomixis

As described in Paramecium aurelia (102), endomixis involves:
(1) a periodic disintegration and resorption of the macronucleus; (2)
division of the micronuclei to produce eight daughter nuclei, all but two
of which usually disintegrate; (3) a fission which produces two ciliates,
each with a functional nucleus; (4) division of this nucleus to produce
four, two of which become macronuclei; (5) division of both micronuclei
at the next fission to restore the normal equipment.

From the genetic standpoint, the significance of such a process is un-
certain. The absence of meiosis and nuclear fusion should eliminate
recombinations of genes, and the genetic implications of macronuclear
replacement under such conditions are unknown. Although genetic effects
have been attributed to endomixis in P. aurelia (5, 66, 92), more recent
data afford no evidence for the occurrence of endomixis in this species
(81). The "endomixis" induced by Sonneborn (73) in P. aurelia appar-
ently represents some other type of nuclear reorganization.

Autogamy

In contrast to endomixis, autogamy in P. aurelia (11) involves not
only replacement of the macronucleus, but also meiosis and subsequent
fusion of haploid nuclei in the same ciliate. Certain other details are of
possible genetic significance.^ The fact that more than two potential
gametic nuclei are usually produced suggests that the new^ synkaryon may
be either homozygous or heterozygous (Fig. 9. 5). The former condition
would result if both gametic nuclei are produced from the same haploid
second-division nucleus, or if the original diploid micronuclei were homo-
zygous. If the stock is heterozygous and the gametic nuclei have different
origins, the resulting synkaryon would often be heterozygous. In other
words, there is no cytological assurance that autogamy invariably results
in a homozygous synkaryon. In contrast to this lack of cytological evi-

^ Two, three, four, or five nuclei may undergo the third prezygotic division, the
division which produces the potential gametic nuclei. "I do not have clear-cut cases of
just a single one of the eight nuclei going ahead to form the gametic nuclei, but prob-
ably this condition does occur at times." Furthermore, ". . . in most cases at least
four potential gametic nuclei are formed in the region of the paroral cone" (11).

Heredity in Protozoa 519

dence, it has been concluded from genetic data that the two gametic
nuclei in P. aurelia are always genotypically identical (81).

Genetic changes in autogamy have been reported in Paramecium. A
clone of P. bursaria may differentiate into two mating types after autog-
amy (31). After autogamy in variety 1 of P. aurelia, all of the progeny
usually belong to either mating type I or mating type II, and heterozygous

Fig. 9. 5. Theoretical genetic effects of autogamy in a heterozygous
ciliate, based on Diller's (II) description of autogamy in Paramecium
aurelia. In the diagram, it is assumed that four nuclei undergo a sec-
ond pregamic division and that only one haploid nucleus of each type
completes the third division.

Strains apparently become homozygous (35, 74). A lag has been noted in
the transformation of heterozygous type II into type I. The change occurs
at different times in different ciliates, so that cultures come to contain
organisms of both types and may show mating reactions. Such a lag was
not observed in the change from heterozygous type I to type II. In addi-
tion to the usual production of all type I or all type II, both mating types
sometimes arise after autogamy. These unusual cases are not readily ex-
plained since the lines arising from one ciliate all contain micronuclei
and macron uclei derived from the same synkaryon.

Cytogamy

In addition to autogamy, cytogamy is believed to occur in P.
aurelia (81). Cytogamy (Chapter II) is essentially incomplete conjuga-

520 Heredity in Protozoa

tion in which the exchange of pronuclei is inhibited and each "conju-
gant" undergoes autogamy (99). The occasional occurrence of cytogamy
in Euplotes patella also is suggested by genetic data (38, 60); autogamy,
as described for other ciliates, does not occur in this species (36). Cytog-
amy has the same genetic significance as autogamy.

GENETIC SIGNIFICANCE OF
THE MACRONUCLEUS

The role of the macronucleus in heredity remains problematical,
although Sonneborn at one time believed that in P. aurelia, "the pheno-
type is controlled exclusively by the macronuclear genes" (81). In addi-
tion, the detection of macronuclear mutations has been discussed and
conditions for their appearance have been postulated (81). However,
such mutations apparently have not been demonstrated.

The supposed macronuclear control of phenotypes in P. aurelia seems
to be based upon the correlation of macronuclear "regeneration" with
certain results in crosses of killer and no?i-killer strains (discussed below).
Macronuclear regeneration was induced by exposing conjugants (variety
1) to temperatures of 38.0-38.5° for not less than 3-5 hours following
"fertilization" (75). Such treatment seems rather rigorous, since P. cauda-
tum may be killed in nine seconds at 40° (59). Among various abnor-
malities (81), there was a retarded division of differentiating macronuclei.
In the postconjugant fissions, some ciliates received new macronuclei and
others only the fragments of the old macronuclei. The latter developed
macronuclei from the fragments, each of which became a complete nu-
cleus. The resulting macronuclei were distributed in subsequent fissions
until the normal nuclear situation was restored.

In applying this process to the study of genetic problems, Sonneborn
(80) crossed non-killers (kk) with homozygous killers {KK). Macronuclear
regeneration was induced in the exconjugants derived from the non-
killers. These were supposed to have received from their mates an excess
of kappa, a cytoplasmic factor essential to development of the killer con-
dition. The zygotic nucleus of each exconjugant was a Kk genotype, and
the macronuclei derived from the synkaryon had the same genotype. The
non-killers which regenerated their macronuclei had to use fraginents of
the old kk macronucleus. Only ciliates with new Kk macronuclei de-
veloped into killers. Furthermore, non-killers {Kk micronuclei and kk
macronuclei) produced no killers after autogamy, although some must
have developed KK micronuclei and macronuclei. Their supply of kappa
presumably was exhausted before autogamy occurred. Accordingly, the
presence of gene K in the macronucleus was considered essential to the
continued production of kappa in the cytoplasm. It might be interesting
to extend these observations to such a combination as Kk micronuclei

Heredity in Protozoa 521

and regenerated KK macronuclei, or kk micronuclei and regenerated Kk
macronuclei, since the production of kappa is inhibited at temperatures
above 33.5° (62, 83).

It has been suggested that the macronucleus controls mating types in
group A of P. aurelia (76, 81). This assumption does not explain the
origin of two mating types from one ciliate after autogamy, a phenome-
non implying formation of two genotypically different macronuclei from
one zygotic nucleus. The least improbable explanation, according to
Sonneborn (81), involves macronuclear mutation, an assumption which
cannot be tested experimentally at present.

THE CYTOPLASM IN
INHERITANCE

There is a growing tendency to relate inheritance of certain traits
in Paramecium aurelia to cytoplasmic factors which may, in different
cases, be dependent upon or independent of nuclear genes. These char-
acteristics include the killer trait, mating types, and antigenic varieties.
Cytoplasmic inheritance is, in a rather real sense, a phenomenon familiar
to all protozoologists in the self-perpetuation of blepharoplasts in flagel-
lates and basal granules in ciliates. Therefore, there is nothing very
startling in the possibility that self-perpetuating cytoplasmic particles
may induce the appearance of specific substances which show physio-
logical activity without assuming the concrete form of new organelles.
The current investigations on ciliates are being followed with much in-
terest and with the hope that future developments may furnish logical
explanations for some of the unsolved puzzles in protozoan genetics.

The killer trait in Paramecium aurelia

A killer strain of P. aurelia, as described in variety 4, gives off into
the culture fluid a substance, paramecin, which is lethal to sensitive
strains but without effect on killers (74, 77, 78, 79). As an antibiotic sub-
stance, paramecin is interesting in that it shows differential effects within
a single variety. Different strains of P. aurelia seem to produce different
quantities and different kinds of paramecin (13, 62, 74, 77). A single
particle of paramamecin (stock 51, variety 4), produced by a killer about
once every five hours, may be enough to kill a sensitive ciliate (2), whereas
Sonneborn (84) has found that as many as half of the sensitive ciliates
may survive when exposed singly or in small numbers to 10,000 or more
particles of paramecin. Paramecin seems to be a desoxyribonucleoprotein
which is inactivated by pepsin, chymotrypsin, and desoxyribonucleases
and shows a sensitivity to high temperatures comparable to that of
various enzymes (95, 96, 97).

The ability to produce paramecin is said to depend upon the presence

522 Heredity in Protozoa

of a dominant gene K and a self-reproducing plasmagene, kappa. Non-
killer strains may be genotypes KK, Kk or kk which lack the factor kappa.
Although gene K may occur in the absence of kappa, maintenance of
kappa in the cytoplasm depends upon the presence of gene K, perhaps
in the macronucleus as well as the micronucleus. Sonneborn (81) sug-
gested that kappa particles are distributed as single molecules throughout
the body. Later calculations of Freer (61), however, indicate a size of
0.3-3.0[j. for kappa particles. Comparable particles, present only in killer
strains, have been identified as granules stainable by Feulgen and Giemsa
techniques and containing desoxyribonucleic acid (61, 63). Treatment of
P. aurelia with nitrogen mustard inactivates kappa and at the same time
reduces the number of these granules (18). Kappa particles apparently
lose the power to reproduce at temperatures above 33.5° (62, 83). Some
of these characteristics suggest close chemical similarity between kappa
and paramecin. Under optimal conditions, kappa in variety 2 of P.
aurelia is quadrupled daily. Consequently, it is possible to decrease
the kappa content by increasing fission-rate, or even to eliminate kappa
completely and permanently from strains of variety 2 (62). This appar-
ently is not possible for the kappa of variety 4 which increases fast
enough to keep up with rapid fission (79, 83). By depressing the fission-
rate, the kappa content of a low-kappa strain may be increased pro-
gressively, at a rate which apparently varies with the strain or with the
kind of kappa. Mutations of kappa have been reported in variety 4 of
P. aurelia. The mutant plasmagenes stimulated production of a new kind
of paramecin with a different lethal action on sensitive cilates (13).

The behavior of kappa in autogamy and conjugation has been de-
scribed (77, 79). After autogamy in a A^A-kappa line, the persistence of
the killer trait for a few generations in homozygous recessive (^^-kappa)
lines suggests that kappa can maintain the production of paramecium
even without gene K. Sooner or later, however, the recessives become non-
killers, presumably because kappa cannot increase in the absence of gene
K. The disappearance of kappa thus shows a "lag" analogous to that
described for inheritance of size in conjugation. If gene K is reintroduced,
by crossing the recessive with a homozygous killer {KK) strain, before all
the kappa has been lost, the hybrid (J'i^A'-kappa) descendants remain
killers. If the cross, kk x ii^iiC-kappa, is made after the recessive has be-
come a non-killer strain upon exhausting its original supply of kappa,
the heterozygous {Kk) descendants of the non-killer conjugants remain
non-killers. Such results are said to demonstrate that gene K cannot
initiate the production of kappa after it has disappeared from the cyto-
plasm. Therefore, cytoplasmic inheritance is very important in trans-
mission of the killer trait.

It has been reported more recently that, in certain crosses of X/^-kappa
y. kk, both exconjugant lines become killers (A^/f -kappa). This result is

Heredity in Protozoa 523

said to depend upon transfer of cytoplasm from the killer to the non-
killer conjugant (86). When autogamy occurs in such lines, some of the
descendants remain killers, presumably as KK-kappa genotypes, while
the rest become non-killers after kappa has disappeared from the cyto-
plasm.

The status of the killer and non-killer traits is further complicated by
the conclusion that another pair of genes, S and s, and a corresponding
plasmagene, sigina, are involved. The relationships between sigma and
its homologous alleles are comparable to those between kappa and its
related genes. Sigma supposedly has the ability to compete with kappa
and to replace it under certain conditions, but is "not actually a factor
for sensitivity" (82). It now appears that a homozygous killer (KKSS)
strain can yield pure killer and pure sensitive strains differing only in
cytoplasmic factors.

Mating types and cytoplasmic inheritance

The difficulty of explaining inheritance of mating types in variety
4 of P. aurelia (Fig. 9. 3) is responsible for the conclusion that plasmagenes
are involved (47, 84). The usual appearance of the original mating type
in each exconjugant line wotdd have to be explained on the basis of
cytogamy rather than interchange of gametic nuclei, if nuclear genes are
responsible. The production of type VIII from conjugants belonging
originally to types VII and VIII would imply that type VIII is dominant
to VII, if nuclear control exists. Other matings, in which the results are
exactly reversed, would indicate that type VII is dominant to type VIII.
Furthermore, the origin of both mating types from one exconjugant can
scarcely be explained on a micronuclear basis, although a similar phe-
nomenon has been attributed tentatively to macronuclear mutation in
group A (81).

These peculiarities in the inheritance of group B mating types are
attributed to the occurrence or the lack of cytoplasmic transfer during
conjugation (84), although such a process has not been detected in cyto-
logical studies on P. aurelia (11) and P. bursaria (100). According to this
hypothesis, no transfer of cytoplasm has occurred when type VII conju-
gants produce only type VII descendants, and type VIII only type VIII.
If the exconjugant lines are all type VIII, in a VII x VIII mating,
cytoplasm has been transferred from the type VIII to the VII conjugant.
If the results are reversed, cytoplasm has been transferred from type VII
to type VIII. If one exconjugant produces two mating types, interchange
of cytoplasm has been followed by segregation of plasmagenes in post-
conjugant fissions. Sonneborn assumes that there are two kinds of plasma-
genes in variety 4, one controlling type VII and the other type VIII. The
same explanation is believed to hold for other varieties of group B.

524 Heredity in Protozoa

Antigenic types and cytoplasmic inheritance

Hereditary antigenic variations in Paramecium aurelia have been
reported by several workers. According to Harrison and Fowler (20),
such variations may arise spontaneously. The stability of the variant
differs with the strain and has ranged from less than three months to
about four years. Similar variations have been induced in P. aurelia
(stock 51) by exposure to X-rays (82) and to homologous antisera (82,
89). Temporary changes, lost after 1-15 fissions, also have been induced
by exposure to trypsin and by maintenance of cultures at 14° for a
number of generations (39). Antigenic types may be modified in different
ways by different experimental methods. Types B, C, and D may be con-
verted into A by incubation at 32°, and types A, C, and D may be
changed to B by incubation at 12°. All the various types remain stable
at 27° if the food supply is controlled to maintain one fission a day (85).
The antigenic type in variety 4 of P. aurelia is said to depend upon
competition between plasmagenes (85, 89).

In the only experiments with bacteria-free cultures, an antigenic modi-
fication, exhibited as insensitivity to antiserum, has been produced by
exposure of Tetrahymena to homologous antiserum, but the ciliates
reverted to the original type after two transfers in normal culture
medium (69).

The status of antigenic varieties in P. aurelia is not yet settled. Since
there is no evidence for micronuclear control, it has been suggested that
cytoplasmic inheritance determines the observed behavior in conjugation
and autogamy (82, 86). Results obtained in conjugation of types A and
B (Fig. 9. 4) are attributed to a lack of cytoplasmic transfer in some cases,
and to the transfer of large or small amounts of cytoplasm in others.
After an A X B cross, the inbreeding of type A exconjugant lines yields
only type A; that of type B exconjugants, only type B. Cytoplasmic
inheritance is believed to offer the most logical explanation for these
various results.

Possible mechanisms involved in the antigenic transformations of
P. aurelia have been discussed by several workers (3, 22, 89), and Beale
has suggested that the potsulated plasmagenes are the antigens them-
selves (3).

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Heredity in Protozoa 525

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18. Geckler, R. P. 1949. Science 110: 89.

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24. Jennings, H. S. 1908. Proc. Amer. Philos. Sac. 47: 393.

25. 1911. /. Exp. Zool. 11: 1.

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33. Jollos, V. 1921. Arch. f. Protistenk. 43: 1.

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35. Kimball, R. F. 1937. Proc. Nat. Acad. Sci. 23: 469.

36. 1939. Amer. Nat. 73: 451.

37. 1941. /. Exp. Zool. 86: 1.

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42. Loefer, J. B. 1939. Physiol. Zool. 12: 161.

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48. Middleton, A. R. 1915. /. Exp. Zool. 19: 451.

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51. 1936. Ber. deutsch. Botan. Gesellsch. 54: 45.

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53. Neuschloss, S. 1919. PflUger's Archiv 176: 223.

54. 1920. PflUger's Archiv 178: 61.

55. Pascher, A. 1916. Ber. deutsch. botan. Gesellsch. 34: 228.

56. 1918. Ber. deutsch. botan. Gesellsch. 36: 163.

57. Pearl, R. 1906. Proc. Roy. Soc, B. 77: 377.

58. Philip, N. and J. B. S. Haldane 1939. Nature 143: 334.

59. Port, J. 1927. Protoplasma 2: 401.

60. Powers, E. L., Jr. 1943. Amer. Mid. Nat. 30: 175.

61. Freer, J. R., Jr. 1948. Amer. Nat. 82: 35.

62. 1948. Genetics 33: 348.

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64. Raffel, D. 1930. Biol. Bull. 58: 293.

65. 1932. Biol. Bull. 62: 244.

526 Heredity in Protozoa

66. 1932. /. Exp. Zool. 63: 371.

67. Ritz, H. 1916. Arch. Schiffs- u. Tropenhyg. 20: 392.

68. Robertson, M. 1929. Parasitol. 21: 375.

69. 1939. J. Pathol. Bad. 48: 305.

70. Root, F. M. 1918. Genetics 3: 174.

71. Schuckmann, W. v. and G. Piekarski 1940. Arch. f. Protistenk. 93: 355.

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73. Sonneborn, T. M. 1937. Biol. Bull. 72: 196.

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75. 1940. Anat. Rec. 78 (suppl.): 53 (abstract).

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77. 1943. Proc. Nat. Acad. Sci. 29: 329.

78. 1945. Ann. Missouri Botan. Gard. 32: 213.

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80. 1946. Genetics 31: 231 (abstract).

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84. 1948. Amer. Nat. 82: 26.

85. 1948. Proc. Nat. Acad. Sci. 34: 413.

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87. and R. V. Dippell 1946. Physiol. Zool. 19: 1.

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95. Wagtendonk, W. J. van 1948. Amer. Nat. 82: 60.

96. 1948. /. Biol. Chem. 173: 691.

97. and L. P. Zill 1947. J. Biol. Chem. 171: 595.

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Host-Parasite Relationships

Parasitism

Types of parasites

Commensalism as an evolutionary goal

Symbiosis

The evolution of parasites

Host-specificity

Taxonomic distribution of parasitic Pro-
tozoa

Protozoa as hosts
Protozoan parasites of Protozoa
Bacterial parasites of Protozoa
Other plants as parasites of Protozoa

Infections

Transfer of parasitic Protozoa

Geographical distribution of parasites of
man
The Americas
The Mediterranean area
Europe north of the Mediterranean area
Central and southern Africa
Southern and southeastern Asia
The Pacific area

Literature cited

PARASITISM

Xrotozoa which HAVE become adapted to life in or on the body
of another organism, the host, are commonly referred to as parasites.
Parasitism, in a correspondingly broad sense, designates the association
of such a parasite with its host. This is also the original meaning of
symbiosis, as proposed by de Bary,i but recent usage has generally re-
stricted this term to the special relationship of "mutualism" (van
Beneden). Since the problematical benefits of revising accepted termi-
nology probably would not balance the resulting misunderstandings,
the prevailing usage will be followed here.

Types of parasites

Protozoa which normally live on the surface of the host's body

may be called ectoparasites — or ectocommensals, if they neither damage

nor benefit the host. Ectocommensalism, obviously limited to aquatic

hosts, may involve definite attachment of the commensal or merely the

^A concise historical discussion of terminology has been published by Kirby (56).

527

528 Host-Parasite Relationships

adherence of a motile organism to the surface of the host's body. The
latter condition is not easily distinguished from casual association of
a free-living species with a pseudo-host. Many other parasites occur in
such body cavities as the mouth and other parts of the digestive tract,
the mantle cavity of Mollusca, and the cloaca of aquatic vertebrates. So
long as these Protozoa are both harmless and useless, they may be con-
sidered endocominensah, or inquilines (14). Endocommensals may be
expected in terrestrial as well as aquatic hosts. Endoparasites which
participate in symbiosis, an association involving mutual benefits to host
and parasite, are known as symbiotes. Parasites which destroy the tissues
of their hosts or damage them in other ways may be called pathogens.
Such terms as "strict parasite" and "true parasite" have been used in
the same sense.

Although some such terminology is convenient for purposes of discus-
sion, there may be practical difficulties in distinguishing symbiosis from
endocommensalism or commensals from pathogens. It has even been
suggested that in a single host species, a pathogen may occasionally be-
come a commensal, or a commensal may sometimes harm the host. How-
ever, the former change does not necessarily occur in the usual carrier
of a normally pathogenic species. It is quite likely that the carrier shows
no obvious symptoms because an effective although incomplete immunity
has been developed.

Commensalism as an evolutionary goal

The evolutionary aspects of pathogenicity and commensalism have
interested many parasitologists. According to one theory, the evolutionary
goal of the parasite is adjustment to commensalism, an association which
tends to conserve available hosts, and in this sense, favors survival of the
well-adapted parasite. This hypothesis implies that endocommensals, as
the product of long-continued adaptation to a particular species of host,
are phylogenetically older than pathogens invading the same host. Patho-
genic species would represent newly acquired parasites which have not
had time to evolve into commensals.

Certain objections to this hypothesis have been discussed by Ball (4).
So far as experimental data are suggestive, there is little reason for
assuming that mere passage of time is a major influence in the loss of
pathogenicity. Packchanian's (75) results with Trypanosoma brucei in
Peromyscus have shown that a given parasite may cause experimental
infections which range, in different species of hosts, from acute lethal
types to chronic infections followed usually by spontaneous recovery.
Furthermore, investigations on avian malaria have shown that one species
of Plasmodium, upon inoculation into a variety of hosts, may produce
lethal infections in one species, malaria of moderate severity in another

Host-Parasite Relationships 529

and yet fail to induce symptoms in a third, although completing a normal
cycle and producing gametocytes in each type of host. Thus, under
experimental conditions, a parasite may in a single transfer become a
dangerous pathogen or show almost no pathogenicity, depending upon
the host. Therefore, long association between a parasite and a host is
not necessary for the reduction or practical elimination of pathogenicity.
In addition, the present pathogenicity of a parasite would seem to be no
real guarantee of its amateur standing. As applied to the Endamoebidae,
the hypothesis of progressive adjustment to commensalism would imply
that man and certain other primates have each acquired Entamoeba
histolytica much more recently than their other amoebae, which may
approach the status of commensals. It seems just as likely that E. his-
tolytica was pathogenic and certain other amoebae were non-pathogenic
when they first invaded the ancestral primates, and that the various
species have merely retained their original characteristics during the sub-
sequent evolution of their hosts. In this connection, the occurrence of a
histolytica-like pathogen {Entamoeba invadens) in various reptiles (28,
81a, 84) may have some significance.

Symbiosis

As an abstract concept, symbiosis (mutualism) is an interesting
association. However, examples involving Protozoa as the symbiotes are
rare.- The most likely candidates are certain intestinal flagellates of
wood-eating termites and wood-roaches (Cryptocerus). The ability to
digest cellulose has been reported for some of the termite flagellates (43,
91, 92) and flagellates of the wood-roach (22, 91). In addition, the results
of defaunation indicate that both types of hosts are dependent, to a
considerable extent and perhaps completely, upon certain of their
intestinal flagellates (19, 20, 21, 22). The status of the rumen ciliates of
herbivores has been disputed (7). There is some morphological evidence
that ingested cellulose is digested by certain species (96), and the pro-
duction of cellulases also has been reported (44, 45). The results of
defaunation have varied from no significant effects (10) to a decreased
digestion of roughage (101). Growth-rates of lambs have remained normal
in the absence of ciliates (9). If the definition of a symbiote merely re-
quires an organism to be beneficial to its host and not necessarily its
major means of support, the possession of cellulases might qualify some
of these ciliates for participation in symbiosis. If the definition is re-
stricted, as it has been occasionally (37), to an organism which is indis-
pensable to its host, then there is no justification for listing ciliates of
the rumen in this category.

" The literature on flagellates of termites and ciliates of ruminants has been reviewed
by Hungate (45a).

530 Host-Parasite Relationships

The evolution of parasites

Speculations concerning the origin and development of protozoan
parasites have been based upon certain assumptions and upon rather
limited observational and experimental data. Within the phylum, para-
sites are not limited to exclusively parasitic groups but are also scattered
in various orders which contain mostly free-living species. Such taxonomic
distribution suggests that parasitic Protozoa have arisen frequently and
independently from different groups of free-living ancestors.

The origin of ectoparasites from free-living species may be assumed
as a matter of course. Endoparasites also may have arisen directly from
free-living ancestors. Another possibility is that endoparasites have
developed from ectoparasites whose prior origin was favored by the
minor adaptive changes required for the transition to ectoparasitism.

As pointed out by Wenrich (98), it is difficult, with protozoological
data, to support the origin of endoparasites from ectoparasites. Ectopara-
sites include mostly primitive flagellates, certain Peritrichida and a num-
ber of Suctorea. Genera containing both ectoparasitic and endoparasitic
species are rare and it is difficult to trace possible connecting links. It is
more probable that endoparasites have arisen directly from free-living
species. The primary invasion probably led to colonization of the diges-
tive tract in most cases. Invasion of the blood and other tissues by many
species followed eventually in the course of evolution. Opportunities for
entering the digestive tract are certainly abundant enough, although
the primary invader must overcome new environmental hazards and must
also establish an infection if it is to succeed as a parasite. The latter step
involves satisfying food requirements and carrying on reproduction.
Furthermore, the probationary parasite must possess or develop methods
for insuring a safe passage from the first host to new ones if it is to
become anything more than a sporadic invader.

It is often assumed that the original host of certain parasites, which
now have two hosts, was the one termed the intermediate host (or vector),
and that parasitism in the final host may be regarded as a secondary
adaptation. Whether this hypothesis can be applied to the genus Leish-
mania is somewhat uncertain. Among the species found in reptiles,
L. chamaeleonis is an intestinal flagellate retaining the leptomonad form
(100), while certain other species invade the blood of gekkos and are
found also in sandflies (Chapter XII).

The occasional occurrence of sporadic endoparasitism by normally
free-living species suggests that direct transition may not have been too
difficult for some Protozoa. Sporadic paratism by Euglenida has been
reported in tadpoles (36, 97) and millipedes (98). Experimental infection
with Tetrahymena pyriformis has been established in the haemocoel of
insects (47, 65). Natural invasion by this ciliate or related species has been

Host-Parasite Relationships 531

observed in the haemocoel of insects (29, 34, 67, 93), in the gills of
Gammanis pulex (78), in the haemocoel of crabs (79), in the coelom of
sea-urchins (64), and in the digestive tract of slugs (82). Such temporary
invasions may be comparable to the initial step in the origin of endo-
parasitism.

The transition from sporadic invasion to the establishment of natural
parasitism need not have required any marked morphological changes.
This is obvious in many parasitic species which belong to predominantly
free-living groups. Both free-living and parasitic species sometimes occur
within a single genus. Species of Astasia have been reported as parasites
of rhabdocoeles (5), rotifers (95), and Crustacea (1), although others are
free-living. Eiiglena leucops (35), parasitic in a rhabdocoele, has lost its
chlorophyll but resembles free-living Euglenidae in other respects.
"Astasia" chaetogastris, found in lethal infections of an oligochaete, re-
tains the stigma but discards the flagellum of the free-living stage (23).
Another example, Euglenamorpha hegneri (97), occurs in the rectum of
tadpoles as two varieties, one with chlorophyll and the other without.
Loss of chlorophyll can scarcely be considered an adaptation to parasit-
ism, since the same change has occurred in free-living Euglenidae exposed
to darkness and other experimental conditions. Hexamita apparently
represents an extreme case in which free-living species and parasites of
the digestive tract in various invertebrates and vertebrates have been
assigned to one genus. Likewise, members of the ciliate genera Anophrys,
Colpidium, Colpoda, Metopus, and Uronema, which include mostly free-
living species, have been reported as intestinal parasites of sea-urchins.
Even such genera as Balantidium and Nyctotherus, which include para-
sites only, cannot be distinguished from free-living ciliates by morpho-
logical criteria. Obviously, the initial stages in development of endo-
parasitism do not demand appreciable changes in structure. Accordingly,
it may be assumed that the primary adaptations have been physiological
rather than morphological.

There are, however, parasites which have undergone more or less
extensive morphological specialization, and thus seem to show structural
adaptations to parasitism. The absence of feeding organelles in the
Opalinida and Astomina is sometimes considered an example of regres-
sive evolution in ciliates, which are predominantly holozoic organisms.
However, the loss of holozoic habits is not a universal feature of special-
ized parasites. The Cycloposthiidae and Ophryoscolecidae, for example,
include highly differentiated ciliates which are distinctly holozoic. Or-
ganelles of attachment have appeared in such parasites as gregarines, the
peritrich Ellobiophyra donacis (18), various dinoflagellates (16, 74, 89),
and such termite flagellates as Streblomastix (59), and Microrhopalodina
(Proboscidiella) (50). Another common feature is the occurrence of rapid
multiplication at certain periods in the life-cycle, as in merogony and

532 Host-Parasite Relationships

sporogony of Coccidia and Haemosporidia. The dinoflagellate, Amy-
loodinium ocellatiim (74), and the ciliate, Ichthyophthirius midtifiliis
(69), also undergo a period of rapid fission following prolonged growth.
In general, such morphological pecularities should perhaps be considered
adaptive features which have been preserved and augmented through
natural selection.

One of the most interesting phases of adaptation to parasitism, that of
physiological and biochemical modifications, must await exploration
until more is known about food requirements and metabolism of para-
sites. This field of investigation may be expected to yield clues to funda-
mental factors in the evolution of parasites and in the maintenance of
more or less obligatory parasitism.

Host-specificity

The development of host-relationships has shown two general
trends (99). In various instances, small groups of parasites have become
adapted to a wide variety of hosts. Examples are found in different
groups of Protozoa. Species of Trypanosoma parasitize some five hundred
different species of vertebrates (100) and the genus Entamoeba also is
represented in many hosts. The genus Eimeria includes more than two
hundred species distributed among such hosts as annelids, insects, myria-
pods, fishes. Amphibia, reptiles, birds, and mammals (62).

In the second type of development, a small group of parasites has
become restricted to a few hosts and may, in some cases, have undergone
extensive evolution within this limited environment. The Entodinio-
morphina contain many ciliates living in ruminants and in the cecum of
horses. Extensive speciation has been noted in some hosts. For Bos indicus,
13 genera containing about 100 species have been listed (58), while nine
species of Cycloposthium have been described from the horse (42). Cer-
tain genera of Hypermastigida and Trichomonadida also have undergone
extensive speciation in a limited group of termites (53), and the opalinid
ciliates are limited almost entirely to Amphibia (72).

The host-specificity of individual species ranges from well-marked to
relatively slight in different cases. The Coccidia of mammals, in experi-
mental cross-infections, generally show a high degree of specificity (8),
although Isospora felis and /. rivolta can infect both cats and dogs (2).
The malarial parasites of man also show a fairly rigid host-specificity,
except for reports that they occasionally produce mild infections in experi-
mentally inoculated apes. Perhaps to a lesser degree, species of Giardia
may be restricted in their distribution among mammalian hosts (37). At
the other extreme, a species may be adaptable to a wide variety of hosts —
Herpetomonas inuscariun may invade flies belonging to a number of
different genera (6, 24); Trypanosoma brucei occurs in various wild and
domesticated mammals and may be transferred to certain laboratory

Host-Parasite Relationships 533

animals; malarial parasites of birds generally can parasitize a variety of
avian hosts; Balantidium coli occurs naturally in man, apes, monkeys, and
pigs; Toxoplasma, recovered from man, is infective for a number of
mammals (70).

TAXONOMIC DISTRIBUTION OF
PARASITIC PROTOZOA

The Phytomastigophorea are represented by only a few parasitic
species and the authentic cases apparently are limited to two orders. The
Dinoflagellida include about 15 genera of parasites. Parasitic Euglenida
are represented by Euglenamorpha (97) and Hegneria (12) and by several
species of Euglena and Astasia. In contrast to the Phytomastogophorea,
many Zoomastigophorea are parasitic — the orders Hypermastigida and
Trichomonadida and a number of smaller groups are exclusively para-
sitic.

Among the Sarcodina, the Proteomyxida include a few parasitic species
and a number of the Mycetozoida also are parasitic. The Endamoebidae
are all parasitic, and Wenyon (100) has suggested that every vertebrate
species probably will be found to harbor parasitic amoebae. The majority
of these amoebae seem to be endocommensals. However, man is not the
only host of a pathogenic species, since reptiles (81a) also may suffer
from amoebiasis.

All known Sporozoa are parasitic and the majority cause appreciable
damage to their hosts. Certain groups have become adapted to particular
environments within the host. The Gregarinidia live primarily in such
cavities as the digestive tract and coelom of invertebrates. The Coccidia
are mainly invaders of epithelial cells, while the Haemosporidia occur
in blood cells and, as exoerythrocytic stages, in certain other tissue cells
of vertebrates.

Among the Ciliatea, the Protociliatia, Astomina, and Entodiniomor-
phina are exclusively parasitic. In addition, a number of parasitic genera
and species are scattered among the rest of the ciliates. Some parasitic
ciliates, such as Ichthyophthirius multifiliis and Balantidium coli, nor-
mally invade and destroy tissues of the host. Many others appear to be
commensals, while certain ciliates of ruminants have been considered
possible symbiotes. The Suctorea include only a few ectoparasites, pre-
sumably ectocommensals, and a few endoparasites.

PROTOZOA AS HOSTS

In addition to their representation among parasites, Protozoa

also serve as hosts of microorganisms.^ Hyperparasitism, in which parasitic

Protozoa are invaded by their own parasites, is not uncommon (57, 86).

Some combinations, involving algae in free-living Protozoa, are possibly

" An extensive review of this subject has been published by Kirby (57).

534 Host-Parasite Relationships

symbiotic. Various other cases of parasitism may result in destruction of
the protozoan host.

Protozoan parasites of Protozoa

Some of the most interesting of these parasites are Suctorea which,
at one time, were believed to be embryonic stages of ciliates. Species of
Endosphaera (31, 66) differ from free-living Suctorea in the absence of
tentacles throughout the life-cycle. There are also a few parasitic species
of Sphaerophrya which have tentacles in the free-living stage but discard
them upon invading a protozoan host. In addition to Suctorea, various
other parasites of Protozoa are known. Dinoflagellates have been reported
from dinoflagellate (17), ciliate (39), and radiolarian hosts. A species of
Astasia has been observed in Stentor and Spirostojmim (41) and unidenti-
fied Zoomastigophorea have been found in ciliates, Suctorea, and Myxo-
sporida. Small amoebae have been reported from opalinid ciliates (88)
and from Trichodina; Microsporida, from Myxosporida (60), ciliates,
gregarines, and Hypermastigida; several Haplosporidia, from gregarine
hosts (68). Some of these associations are examples of hyperparasitism (86).

Bacterial parasites of Protozoa

Certain bacteria are ectoparasitic on flagellates of termites (25,
32, 48, 52, 54). Fusiform bacilli, adherent lengthwise to the cortex of the
host and often regularly spaced, have sometimes been mistaken for
cortical ridges in Devescovina, Lophomonas, Polymastix, Caduceia, and
Staurojoenia. Similarly attached bacteria also have been reported from
ciliates, including a species of Cyclidium (80). Spirochetes, attached
terminally to their hosts (21, 25, 48), have been mistaken occasionally for
flagella or cilia. Although less common than spirochetes, terminally at-
tached bacilli have been observed on such flagellates as Microrhopalodina
(Proboscidiella) kofoidi (50) and M. inflata (26).

Endoparasitic bacteria also occur in certain flagellates of termites —
Trichonyrnpha (51), Pseudodevescovina (54, 33), and Bullanympha (55).
Both nuclear and cytoplasmic parasites have been reported from Para-
mecium (11, 27), for which invasion of the macronucleus is often fatal.

Other plants as parasites of Protozoa

This group includes such Fungi as Chytridiales which sometimes
occur as cytoplasmic (Sphaerita) and nuclear parasites (Niicleophaga)
in Protozoa. Species of Sphaerita have been described from Euglenida
(30, 46, 73), Amoeba (71), flagellates of termites, species of Entamoeba,
Zelleriella, Nyctotherus, and Diplodiniwn (57). The young form of
Sphaerita is a uninucleate amoeboid stage. Growth and nuclear division
result in a plasmodium, which eventually produces a number of small
spores, or sometimes flagellated "zoospores." Niicleophaga (61) has been

Host-Parasite Relationships 535

reported from Endolimax nana (13), Endamoeba disparata (49), Amoeba
(71), and from flagellates of termites (57).

Certain algae also parasitize Protozoa. Blue-green algae occur in the
testacean, Paulinella chromatophora (77), and species of Chlorella in
Frontonia leiicas (40) and Paramecium bursaria (63, 76, 81). Although
these algal-protozoan associations are often considered examples of
symbiosis, the experimental evidence is not entirely conclusive.

INFECTIONS

Upon reaching a suitable host, a parasitic protozoon which is
not promptly eliminated may give rise to an infection. The establishment
of an infection requires multiplication of the parasite at a rate rapid
enough to overbalance any destructive forces which may be encountered,
and the result is a net increase in parasite population. The ability to
establish an infection in a particular host, or the infectivity of the parasite
for that host, is somewhat variable. To what extent the apparent
infectivity may depend upon individual variations in the internal
environment or in natural resistance of the host is uncertain, although
it may be assumed that such factors are important. For instance, the
change from a normal to a high-carbohydrate diet makes the rat suscepti-
ble to infection with Balantidium coli, a ciliate which ordinarily shows
little or no infectivity for this rodent (38). In such a case, the normal
intestinal environment obviously is a factor limiting infectivity. In other
cases, infectivity may be modified by changes in the parasite. An example
is the loss of infectivity for kittens by two strains of Entamoeba histolytica
which had ceased producing cysts in cultures. Modification of the culture
medium so as to restore the ability to encyst was followed by the recovery
of infectivity (15).

In the terminology of Justin Andrews, protozoan infections may be
described in terms of prepatent, patent, and subpatent periods. The
prepatent period precedes the appearance of parasites in numbers large
enough for detection by routine examinations. The apparent absence of
parasites is often the result of failure to find the few parasites actually
present in the material examined. In a malarial infection, however, the
parasites may be developing as exoerythrocytic stages prior to invasion of
the blood.

The patent period opens with the finding of parasites in material
from the host. During this period, the parasite density usually continues
to rise for some time and the transfer of parasites by vectors is most
likely to be successful in this stage. Eventually, the infection may lead to
death of the host, the number of parasites may be sharply reduced by
immunological reactions of the host, or the surviving parasites may leave
the host upon completion of the life-cycle. The patent period passes into
the subpatent period when the parasites are no longer detectable.

536 Host-Parasite Relationships

The siibpatent period varies in significance. It may represent a marked
decrease in number of parasites as the infection is brought under control
prior to elimination. In tertian malaria, on the other hand, a subpatent
period may parallel continued development of exoerythrocytic parasites.
A new patent period may follow the subpatent period, and the sequence
may be repeated several times before the infection is terminated.

Infections which induce the appearance of definite symptoms in the
host also may be characterized in terms of several clinical periods —
period of incubation, period of symptoms and period of convalescence.

The incubation period, initiated by introduction of the parasites,
ends when symptoms are recognizable. In malignant tertian malaria,
symptoms may appear at or near the end^of the prepatent period. In
various other infections, the correlation between incubation period and
prepatent period is not necessarily close. Parasites are often detectable
some time before the appearance of symptoms. At the other extreme,
characteristic symptoms appear and reach a peak before the end of the
prepatent period in infections with Isospora hominis.

The period of symptoms opens typically with the appearance of mild
(or prodromal) symptoms. As the infection progresses, the symptoms
become progressively more severe and more characteristic of the particu-
lar host-parasite association. During this phase, the parasites are pro-
ducing more or less specific effects, the nature of which varies with the
parasite. Variations in the severity of the effects produced may reflect
differences in resistance of the hosts and in virulence of the parasites
(Chapter XIV). Mechanical irritation may be caused by movements of
intestinal Protozoa, and tissues are destroyed by many parasites. Invasion
of individual cells may lead to extensive destruction of tissues — an epi-
thelium by Coccidia, or blood cells by malarial parasites. Tissues also
may be destroyed without invasion of cells, as in ulceration of the in-
testine by Entamoeba histolytica and Balantidium coli. Whether such
ulceration is brought about solely by histolytic enzymes of the parasites
or partly by mechanical means is uncertain. The production of toxic
substances has been suggested for some parasites, although specific toxins
have not been isolated. However, the production of a potent toxin has
been reported for a free-living dinoflagellate. Gonyaulax catanella appar-
ently is the source of the poison found occasionally in the edible Cali-
fornia mussel (87), and concentrates of this substance have shown a
toxicity of 1.65 mouse units per microgram (83).

The period of convalescence, marked by the gradual disappearance of
symptoms, extends to clinical recovery of the host. In some protozoan
infections, apparent convalescence may be merely a period of latency
during which a low-grade infection persists. Latency may be interrupted
sooner or later by a relapse, in which symptoms reappear following re-
newed multiplication of the parasites.

Host-Parasite Relationships 537

TRANSFER OF PARASITIC
PROTOZOA

Protozoan parasites reach new hosts in various ways. Active migra-
tion may lead to invasion of aquatic hosts — Endosphaera (66) of ciliates,
Amyloodinium (74), and Ichthyophthiriiis (69) of fishes. Contact transfer
is the characteristic method for some parasites — oral contact for Enta-
moeba gingivalis and Trichomonas tenax; transfer in coitus for Tricho-
monas vaginalis, Tritrichomonas foetus, and Trypanosoma equiperdum.
Con tarn inative transfer, in which cysts or spores are ingested with food
or drink, is the usual method for Coccidia and for intestinal flagellates,
amoebae and ciliates.

Transfer by vectors is characteristic of blood parasites. Vectors include
blood-sucking flies (Trypanoso7na gambiense), mosquitoes (malarial para-
sites), bugs [Trypanosoma cruzi), fleas {Trypanosoma lewisi), ticks
{Babesia bigemina), leeches {Trypanosoma rotatorium), and apparently
vampire bats {Trypansoma hippiciim). Transfer by vectors may be a
mechanical process during which the parasites undergo no significant
changes. In other cases, the parasite passes through a phase of the life-
cycle before it is again infective for the final host; this cyclic, or infective,
transfer is characteristic of malarial parasites and various trypanosomes.
Some vectors inoculate the parasites directly into the tissues of the host
during feeding. In contrast to this method, Trypanosoma cruzi is voided
from the hind-gut of its vector and reaches the tissues of the vertebrate
by contamination of a woimd or invasion of a mucous membrane.

The case of Histomonas meleagridis, which causes "blackhead" in
turkeys, seems to be unique in that the flagellates are said to be trans-
ferred in the eggs of an intestinal nematode, Heterakis gallinae (94).

Congenital infections may follow placental or ovarian transfer of
parasites. Placental transfer, involving the passage of parasites through
the placenta, has been reported for Plasmodium vivax, P. malariae, and
P. falciparum of man, and occasionally also for certain trypanosomes in
experimentally infected laboratory animals. Ovarian transfer, involving
the direct invasion of eggs by parasites, occurs in such invertebrates as
female ticks infected with Babesia bigemina.

Lacteal transfer, from females to suckling young, has been described
in a few trypanosome infections, and this possibility should be con-
sidered in interpreting cases of supposedly placental transfer.

GEOGRAPHICAL DISTRIBUTION OF
PARASITES OF MAN

The protozoan parasites of man include species which invade the
vascular, epithelial, and other tissues, and also a number which live
in the lumen of the digestive tract. The digestive tract is parasitized by

538 Host-Parasite Relationships

six species of flagellates — Trichomonas tenax (buccalis) of the mouth;
Giardia lamblia of the small intestine; and Chilomastix mesnili, Re-
tortomonas {Embadomonas) intestinalis, Tricercomonas intestinalis, and
Pentatrichomonas hominis of the colon — and six species of amoebae —
Entamoeba giyigivoUs of the mouth; Entamoeba coli, E. histolytica (which
sometimes invades other organs), Dientainoeba fragilis, Ejidolimax nana,
and lodamoeba butschlii of the colon. One ciliate, Balantidium coli,
sometimes invades the colon, while a coccidian, Isospora hominis, ap-
parently is a parasite of the small intestine. The urogenital tract may
harbor Trichomonas vaginalis, which is often found in the vagina and
urethra in the female, and in the urethra and prostate in the male.
Parasites of the blood and other tissues include species of Leishmania
(L. brasiliensis, L. donovani, L. tropica), Trypanosoma (T. cruzi,
T. gambiense, T. rhodesiense), and Plasmodium (P. falciparum., P. ma-
lariae, P. ovale, P. vivax). The status of Toxoplasma as a natural parasite
of man is somewhat uncertain, in view of the apparent rarity of human
infections and the low degree of host-specificity exhibited by these
organisms.

The intestinal Protozoa of man are probably worldwide in distribution
and are fairly common parasites. The malarial parasites, although most
abundant in tropical areas, extend into the temperate zones. The trypano-
somes of sleeping sickness, on the other hand, seem to be limited to
central Africa by the geographical distribution of their vectors. Species
of Leishmania are much more widely distributed, and Trypanosoma
cruzi has an extensive range in the western hemisphere.

The Americas

In North America the usual intestinal Protozoa are to be expected.
The incidence of E. histolytica, for example, ranges from about 0.2 to
50 per cent of the population in different parts of the United States,
with an average possibly approaching 20 per cent (26a). Malaria remains
an important problem only in the southeastern United States (3).
Trypanosoma cruzi occurs at least as far north as central California, al-
though Chagas' disease has not been foimd in man.

In Mexico and other Central American countries intestinal Protozoa
are probably no less common than they are in North America. Malaria is
important in lowland areas, both coastal and interior, and Costa Rica and
Panama in particular have suffered considerably. Sporadic cases of Chagas'
disease have appeared within this area, and both cutaneous and visceral
leishmaniasis are known from scattered localities.

In the Caribbean area, malaria remains a public health problem in
Jamaica, Haiti, the Dominican Republic, Puerto Rico, and Trinidad.
Most of Cuba is free from endemic malaria although there are some
areas in which the disease is still important. Among the smaller islands,

Host-Parasite Relationships 539

malaria occurs endemically in the Caymans and is common in some of
the Leeward and Windward Islands and in Tobago. To the north of
Cuba, malaria has been reported in some of the southern Bahama
Islands but is uncommon.

In South America, endemic malaria extends along the western coast
from Columbia through Ecuador and Peru into northern Chile. East-
ward, malaria is widely distribtited in Venezuela, the Guianas and Brazil,
except for highland areas. Southward, through Brazil, malaria is endemic
in much of Bolivia and Paraguay and extends well into Argentina.
Chagas' disease apparently occurs throughout much of South America.
The data on incidence are quite incomplete, but recent surveys have
shown that this disease is much more common than was formerly sus-
pected. Cutaneous leishmaniasis also is scattered throughout much of
South America and occasional cases of visceral leishmaniasis have been
reported from Argentina northward to Venezuela. Infections with
intestinal Protozoa are presumably as common in South America as they
are in Central and North America.

The Mediterranean area

Malaria (particularly benign and malignant tertian) is still of
some importance in Spain, Italy, Yugoslavia, Albania, Greece, Turkey,
the Levant States, Transjordan, and Palestine, and has been a serious
problem in the eastern area within the last few decades. Within this
period there have been years in which Greece and Albania, for example,
reported a malarial incidence of about 25 per cent. Along the southern
shore of the Mediterranean, malaria extends westward through climati-
cally favorable areas to Morocco. Visceral leishmaniasis has been reported
occasionally in Spain, Sicily, Malta, Greece, Albania, Yugoslavia, Turkey,
Transjordan, Syria, and Lebanon, while dermal leishmaniasis extends
from eastern Egypt into Palestine and the Levant States, southern Turkey,
and several provinces of Greece. Infections with E. histolytica and other
intestinal Protozoa are known to be common in some parts of the Medi-
terranean area and are probably far from rare in other regions for which
data are unavailable.

Europe north of the Mediterranean area

Malaria extends along the western shores of Europe from Portu-
gal to the Baltic Sea, and in recent years, has occurred also in Finland.
Although malaria is a disease of minor importance in northern Europe,
benign tertian is still fairly common in the coastal regions of Holland.
Malaria occurs also around the Black Sea, where both malignant and
benign tertian may be found, and has extended northwestward for some
distance along the Danube River. Infections with intestinal Protozoa

540 Host-Parasite Relationships

are widely distributed and their incidence seems to vary considerably in
different areas.

Central and southern Africa

Except for such climatically unfavorable areas as the Sahara,
malaria extends throughout most of the continent into the Union of
South Africa. The incidence is high in many regions and a native with no
malarial experience is a rarity in the Belgian Congo and various other
parts of tropical Africa. The distribution of the major types of malaria
varies with the region. Benign tertian is apparently less common than
malignant tertian in the Belgian Congo, Nigeria, the Gold Coast, and
Togo, for example, but may represent 20-30 per cent of the cases in the
Union of South Africa. Quartan malaria is fairly common in Togo, less
common than benign tertian in Nigeria, rare in Kenya Colony and the
Cameroons, and rare or absent in Bechuanaland. Trypanosomiasis (Afri-
can sleeping sickness) extends from Gambia and French West Africa east-
ward to Kenya and as far south as Southern Rhodesia. Kala-azar has
occurred sporadically along the border of the Sudan and Ethiopa but
apparently has not extended westward or southward. Available data
indicate that amoebiasis and other intestinal infections are very common
in many parts of tropical Africa and apparently less common in others.
Madagascar, off the southeastern coast of Africa, is a center of endemic
and widely distributed malaria, malignant tertian being important.
Amoebiasis is at least as common in Madagascar as in most parts of the
mainland.

Southern and southeastern Asia

Malaria extends from the shores of the Red and Caspian seas
across Asia to southern and eastern China, and farther inland, from
the Caspian Sea well into southern Russia. For most of this area, the
real incidence of malaria is unknown. Incomplete data suggest that an
estimate of 2,000,000 cases a year, about a third of them malignant
tertian, would be fairly conservative for India. Malaria also is important
in Thailand, which has experienced a malarial death rate of 3 to 4 per
thousand more than once within the past thirty years. Kala-azar extends
from Turkey and Iraq eastward into India, Burma, Thailand, Indo-
China, and China. The disease is considered an important health problem
in India and China but is apparently rare in Thailand and Indo-China.
Oriental sore extends from Turkey, Arabia, and Iran into India. Amoe-
biasis is probably common throughout the area.

The Pacific area

In Australia, malaria is endemic along the northern coast but
not elsewhere. Amoebiasis is probably general in distribution, along

Host-Parasite Relationships 541

with other intestinal Protozoa. Malaria is widely distributed and of
common occurrence in Sarawak, New Guinea, Borneo, and other parts of
the East Indies and amoebiasis also is known to be common throughout
the region. In Melanesia, malaria is generally distributed in the New
Hebrides and Solomon Islands. In the Philippines, malaria (perhaps
more than a third of it malignant tertian) is common enough to be an
important health problem, and the incidence of amoebiasis also seems to
be fairly high. North of the Philippines, malaria extends into southern
Japan.

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90. Taylor, A. B. and R. L. King 1937. Trafis. Amer. Micr. Soc. 56: 172.

91. Trager, W. 1932. Biochem. J. 26: 1762.

92. 1934. Biol. Bull. 66: 182.

93. Treillard, M, and A. Lwoff 1924. C. R. Ac. Sci. 178: 1761.

Host-Parasite Relationships 543

94. Tyzzer, E. E. 1934. Proc. Amer. Acad. Arts & Sci. 69: 189.

95. Valkanov, A. 1928. Arch. f. Protistenk. 63: 419.

96. Weineck, E. 1934. Arch. f. Protistenk. 82: 169.

97. Wenrich, D. H. 1924. Biol. Bull. 47: 149.

98. 1935. Proc. Amer. Philos. Soc. 75: 605.

99. 1935. U7iiv. Penn. Bull. 35 (No. 58): 23.

100. Wenyon, C. M. 1926. Protozoology (London: Balliere, Tindall & Cox).

101. Winogradow, M., T. Winogradowa-Federowa and A. Wereninow 1930. Zentralbl.

f. Bakt.. II, 81: 230.

Protozoa of the Digestive
and Urogenital Tracts

Protozoa of the human mouth
Trichomonas tenax
Entamoeba gingh/alis

Flagellates of the human intestine
Retortomonas intestinalis
Tricercomonas intestinalis
Chilomastix mesnili
Pentatricliomonas hominis
Giardia lamblia
Flagellosis
Chemotherapy

Amoebae of the intestinal lumen
Endolimax nana
lodamoeba biitsclilii
Dietitamoeba fragilis
Entamoeba coli
Effects on the host

Amoebiasis

The causative organism
Invasion of tissues by Entamoeba

histolytica
Various types of primary amoebiasis
Chemotherapy

Intestinal amoebiasis

Secondary amoebiasis
The search for new amoebacidal drugs
Problems in control of amoebiasis

Balantidiosis

Balantidium coli
Effects on man
Chemotherapy

Coccidiosis

Isospora hominis
Effects on man
Chemotherapy

Trichomonas of the urogenital tract
Effects on man

Laboratory diagnosis of infection
Protozoa of the mouth
Protozoa of the intestine

Wet preparations

Permanent preparations

Concentration methods

Culture methods

Complement-fixation
Trichomonas vaginalis

Literature cited

PROTOZOA OF THE
HUMAN MOUTH

HE HUMAN MOUTH is in somc respects a fairly rigorous environ-
ment for Protozoa. Foods and drinks vary widely in temperature and
chemical nature, and the disturbances involved in the practice of dental
hygiene add further complications. Nevertheless, a flagellate (Tricho-

544

Protozoa of the Digestive and Urogenital Tracts 545

monas tenax) and an amoeba {Entamoeba gingivalis) manage to infect
a significant proportion of the population.

The life-cycles of these two parasites do not include cysts, so that
infections are spread by the transfer of trophozoites. Under experimental
conditions, a trace of moisture has kept E. gingivalis alive long enough
for droplet transfer, and for transfer by way of contaminated cups and
other utensils (93). Transfer by direct oral contact entails much less risk
for the parasite.

Trichomonas tenax

This flagellate probably was first described as Cercaria tenax by
O. F. Miiller in 1774 (94). Many years later, the organism was found
again and described as Tetratrichomonas buccalis Goodey (65). T. tenax
(Fig. 11: I, A-C) shows a size range of about 5-21 x 3.8-7. 6[j.. The im-
dulating membrane is usually rather short and the membrane-flagellum
may not extend beyond the membrane. Autotomy occasionally pro-

Fig. 11. 1. A-C. Trichomonas tenax, parabasal body shown only in B;
x2530 (after Wenrich). D-F Entamoeba gingivalis: small specimen free
from inclusions (D), a smaller rounded form (E), and a large specimen
with many food vacuoles; x2000 (after Kofoid and Swezy).

546 Protozoa of the Digestive and Urogenital Tracts

duces forms with projecting axostyles and disproportionately long mem-
branes (71). Mitosis has been described by Hinshaw (71), and T. tenax
has been compared with other trichomonads of man by Wenrich (175).
Although rarely present in the healthy mouth, infection with T. tenax
may approach an incidence of 90 per cent in cases of advanced pyorrhea
(13, 72, 76). However, a casual relationship to pyorrhea has not been
established (95, 97). In addition to their occurrence in the mouth, the
flagellates have been found occasionally in pus from infected tonsils and,
rarely, in material from the lungs.

Entamoeba gingivalis

This species, described as Amoeba gingivalis by Gros in 1849,
evidently was the first amoeba reported from man. The specific name,
Endamoeba buccaUs, was proposed later by Prowazek (133) who had over-
looked the paper by Gros. Several detailed descriptions have been
published more recently (30, 78, 99, 104), and mitosis has been described
by Stabler (155a) and Noble (128a). Literature on the species has been
reviewed by Kofoid (95).

The amoeba measures 6-60[x in length, usually shows clear pseudo-
podia, and may contain a number of food vacuoles containing leucocytes,
or less commonly, bacteria (Fig. 11. 1, D-F). The amoebae ingest living
leucocytes and consequently are not mere scavengers (30). In cultures,
both leucocytes and red corpuscles are ingested (78). The nucleus, 2-6[jl
in diameter, often shows a central clump of granules, as well as a zone
of coarse granules near the nuclear membrane.

The incidence of infection apparently increases with age, although
the healthy mouth rarely harbors E. gingivalis. In cases of pyorrhea, the
incidence is high and may exceed 90 per cent (72). Such a coincidence is
tempting but there is no conclusive proof of pathogenicity (97). This
amoeba seems to be a natural parasite of monkeys (68, 90, 99) as well
as of man, and experimental infections are possible in dogs with a pre-
existing gingivitis (74).

FLAGELLATES OF THE
HUMAN INTESTINE

The small intestine is invaded only by Giardia lamblia, whereas

the colon may contain Retortomonas intestinalis, Tricercomonas intesti-

nalis, Cliilomastix mesnili and Pentatrichomonas ho7ninis.

Retortomonas intestinalis

(Wenyon and O'Connor) Wenrich

This flagellate, often known as Embadomonas intestinalis (Wen-
yon and O'Connor) Chalmers and Pekkola, has been reassigned to Re-
tortomonas by Wenrich (165) on the basis that Embadomonas Mackin-

Protozoa of the Digestive and Urogenital Tracts 547

non is a synonym of Retortomonas Grassi. Objections to this conclusion
have been presented by Bishop (17).

R. intestiymJis (Fig. 11. 2, H, I) is a small (4-9 x 3-4^) organism with
two unequal flagella. The longer flagellum extends anteriorly, the other
anterolaterally from a pit (the "oral pouch" or "cytostome"). Shape var-
ies somewhat although the anterior end is usually rounded. The cyto-
plasm often contains food vacuoles. Binary fission has been described
by Bishop (17).

The cyst (Fig. 11. 2, F, G), which measures 4.5-7.0 x 3.0-4.5[j., is ovoid

Fig. 11. 2. A-E. Tricercomonas intestinalis: flagellate stages (A, B); uni-
nucleate (C), binucleate (D), and tetranucleate (E) cysts; xl600 (after
Dobell and O'Connor). F-I Retortomonas intestinalis: flagellates from cul-
tures at 37° (H) and 17-20° (I); cysts (F, G); x3600 (after Bishop). J-O.
Chilomoastix mesnili: flagellate showing cytostomal fibril and cytostomal
flagellum (J), x3000 (after Geiman); late fission, new cytostomal fibrils pres-
ent (K), x2850 (after Geiman); uninucleate (L) and binucleate (M) cysts
from Macaca irus, x2850 (after Geiman); polar view of uninucleate cyst
from man (N), xl600 (after Kessel); binucleate cyst, nuclei joined by a
paradesmose (O), xl600 (after Kessel).

548 Protozoa of the Digestive and Urogenital Tracts

to pear-shaped and contains one nucleus; nuclear division apparendy does
not occur (17).

Tricercomonas intestinalis
Wenyon and O'Connor

This species may or may not be identical with Enteromonas
hominis Fonseca. This question has been discussed by Dobell and
O'Connor (52) and by Wenyon (178), and the latter has pointed out that
Enteromonas hominis was described with only three flagella.

Flagellated stages (Fig. 11. 2, A, B) have three anterior flagella and a
fourth which may seem to lie within the cytoplasm and emerge at or near
the posterior end of the body. The size range is 4-10 x 3-6[j.. The cysts
(Fig. 11. 2, C-E) measure 6-8 x 3-4[x and contain 1-4 nuclei (22). A flagel-
late apparendy identical with T. intestinalis has been found in mon-
keys (46).

Chilomastix mesnili

(Wenyon) Alexeieff

This flagellate (Fig. 11. 2, J-O) seems to be specifically identical
with one in apes and monkeys (64). The active stage, 6-20|x in length, has
three anterior flagella and a shorter fourth which usually lies in the cyto-
stomal groove, a depression extending obliquely from near the anterior
end to about the middle of the body. A second groove often arises near
the left anterior margin of the cytostomal cleft and extends posteriorly
in one or two spiral turns. Solid food is ingested through a cytostome at
the posterior end of the cytostomal groove (178). Just beneath the
surface, a cytostomal fibril of uncertain significance extends along the
cytostomal groove. Fission has been described by Geiman (64) and by
Boeck and Tanabe (24).

Encysted stages (Fig. 11. 2, N, O) measure 7-10 x 4.5-6.0jx, contain one
or two nuclei, and often granules and fibrils representing the blepharo-
plasts, cytostomal fibrils and possibly flagellar axonemes. Mitosis has
been described in encysted stages (67, 100).

Pentatrichomonas hominis

(Davaine) Kirby

Two flagellates from the human colon are described in the litera-
ture as Tridwmonas hominis (Davaine) Leuckart, with four anterior
flagella, and Hexamitus ardin-delteili (44), later transferred to the genus
Pentatrichomonas (103, 105) on the basis of its fifth free flagellum. In
confirming observations of Wenrich (171), Kirby (91) concluded that
T. hominis normally has a fifth free flagellum and that the two sup-
posedly distinct flagellates should be recognized as Pentatrichomonas

Protozoa o£ the Digestive and Urogenital Tracts 549

hominis. Trichomonads apparently identical with P. hominis have been
found in monkeys, cats, dogs, and rats (171).

The flagellate (Fig. 11. 3, A-C) measures 8-15x3-5[j.. The undulating
membrane is about as long as the body and there is usually a free
posterior portion of the membrane-flagellum. The fifth flagellum, which
may be trailed posteriorly, beats in a rhythm different from that of the
other four anterior flagella (103). A costa extends beneath the base of
the undulating membrance and the axostyle usually projects beyond the
posterior end of the body. Encysted stages are unknown.

Fig. 11. 3. A. Pentatrichomonas hominis, showing five anterior flagella,
membrane flagellum, nucleus, axostyle and costa; x2530 (after Wenrich).
B. P. hominis, larger specimen with several food vacuoles; costa not shown;
x2400 (after Wenrich). C. Bodian silver preparation of P. hominis, showing
pelta anterior to the nucleus and a dorsal filament extending posteriorly
from the pelta, between the costa and the nucleus; x2040 (after Kirby).
D-G. Giardia lamblia: dorsal view (D), ventro-lateral view (E) of flagellate;
cysts with four (F) and twelve (G) nuclei; x2560 (after Kofoid and Swezy).

550 Protozoa of the Digestive and Urogenital Tracts

Giardia lamblia

Stiles

This flagellate, described by Lambl in 1859 as Cercomonas intesti-
nalis, probably was first seen by Leeuwenhoek in 1681. Although the
organism is often referred to as Giardia iy-itestinalis, Lambl's name had
already been used for a parasite of Amphibia and the correct specific
name is Giardia lamblia. Nuclear division and fission have been de-
scribed (101).

The flagellate (Fig. 11. 3, D, E) measures 9-21 x 5-lltj.. The body is
flattened dorso-ventrally with a rather convex dorsal surface and a more
flattened ventral surface, the anterior part of which forms a concave
"sucker." Two flagella emerge from the posterior pole, while three other
pairs extend from the lateral and anterolateral surfaces. The paired
axostyles, which may appear fused in stained preparations, extend to
the posterior end of the body. Two parabasal bodies, sometimes fused
together, lie near the axostyles in the posterior third of the body.

The cysts (Fig. 11. 3, F, G) measure 8-14x6-10[j, and may contain
2-16 nuclei, axostyles, parabasal bodies, and fibrils which are possibly
flagellar axonemes. The cyst is the stage most commonly found in stool
examinations, since the flagellated forms are discharged primarily during
attacks of diarrhea.

Although it was suspected, at one time, that rodents may serve as
reservoirs, this suspicion has not been confirmed. Experimental infection
of rats has been reported (8), but such infection has been temporary and
has not led to production of cysts.

Flagellosis

The effects of flagellate infections have been evaluated primarily
by correlating clinical observations with incidence of infection. On such
a basis, there is no evidence that Retortomonas intestinalis and Tricerco-
monas intestinalis are harmful. Chilomastix mesnili has been associated
with abnormal stool frequency often enough to arouse suspicion, but
there is no reason for considering this species a serious parasite.

Pentatrichomonas hominis has been found in diarrheic patients (150)
and "Pentatrichomonas ardin-delteili" was first observed (44) in patients
with dysentery and later in cases of dysentery and chronic diarrhea (105).
Therefore, it is often assumed that P. hominis is occasionally a causative
or contributary factor in digestive disturbances. Invasion of tissues —
agonal (110) and possibly postmortem (176) — has been reported, but
probably does not occur in the usual infection.

Infection with Giardia lamblia is frequently correlated with digestive
disturbances, although there is no invasion of tissues. Heavy infections
might interfere with normal absorption, since the flagellates adhere to

Protozoa of the Digestive and Urogenital Tracts 551

the mucosa. Symptoms include chronic diarrhea, attacks of diarrhea
alternating with constipation, chronic stomachache, occasional cramping
and colic, nausea, abdominal tenderness, loss of appetite, chronic head-
aches, and irritability (61, 111, 178).

Chemotherapy

The treatment of flagellate infections is a less pressing problem
and has attracted less attention than the treatment of amoebiasis. Some
of the drugs used for Entamoeba histolytica have been tried also in
flagellate infections, but the results are not always directly comparable.
Atebrin has been used effectively for elimination of Giardia lamblia,
although occasional infections are not cured. Giardiasis in children also
has been treated with bismuth-salicylate, followed by treparsol (111),
and with acranil (16). Diodoquin is said to be active against P. hominis,
and good results with gentian violet in combination with argyrol enemas
also have been reported.

AMOEBAE OF THE INTESTINAL
LUMEN

The human colon may be invaded by Endolimax nana, Dienta-
moeba fragilis, Entamoeba coli, and lodamoeba biltschlii. In addition,
natural infection with Entamoeba polecki, a. parasite of monkeys, has
been observed (88).

Endolimax nana

(Wenyon and O'Connor) Brug

First described as Entamoeba nana (179), this species was later
transferred to the genus Endolimax by Brug in 1918. An apparently
identical amoeba has been reported from monkeys (46).

The amoeboid stage (Fig. 11.4, A), usually observed only in loose
stools, is a small (6-15[j,) sluggish form with clear pseudopodia and food
vacuoles containing bacteria. The stained nucleus often shows no periph-
eral granules, although such can be demonstrated after adequate fixation
(155). The endosome is large, usually irregular but sometimes ovoid or
spherical, and may be central or eccentric. Precystic stages have been
reported as rounded forms without food vacuoles. Mitosis has been de-
scribed (49a).

The mature cysts (Fig. 11. 4, B-E), 5-12x4-6[jl, contain four, or rarely
eight, nuclei. The shape is usually ovoid, and one surface is often more
convex than the opposite side. Stored glycogen may be present in young
cysts but disappears gradually as the cysts mature. Occasionally, small
filaments have been reported as possible chromatoid bodies. The nuclei
of the mature cyst are appreciably smaller than those of the trophozoite,
and the endosome is often eccentric.

552 Protozoa of the Digestive and Urogenital Tracts

Fig. 11. 4. A-E. Endoliwax nana: amoeboid stage (A); cysts from natu-
rally infected monkey (B) and from man (C-E); B, xl830; A, C-E, xl600
(after Kessel). F-N. lodamoeba biitschlii: amoboid stage ingesting food
through tubular "food-cup" (F), x2400 (after Stabler); small (I), medium
(G, H), and large (J) amoeboid stages, periendosomal granules evident in
nucleus, xl685 (after Wenrich); binucleate cyst (K), xl600 (after Kessel);
cysts with three (possibly four) nuclei (L) and one nucleus (M), xl685
(after Wenrich); uninucleate cyst from naturally infected monkey (N), xl900
(after Kessel).

lodamoeba biitschlii

(Prowazek) Dobell

This species apparently was described as Entamoeba biitschlii
by Prowazek (135) and was later transferred to the genus lodamoeba
(45). Occasional use of the name, lodamoeba williamsi, is based upon
Prowazek's (134) earlier erection of the species "Entamoeba wiUiamsi"
for a mixture of Entamoeba coli and perhaps /. biitschlii. Infections with
/. biitschlii have been reported from apes and monkeys (167) as well
as man.

The amoeboid stage (Fig. 11. 4, F-J) measures 4-20[ji, in length. The
organism moves slowly, usually with clear blunt pseudopodia. The
stained nucleus is 2. 0-3. 5;;, in diameter and contains a central or slightly

Protozoa of the Digestive and Urogenital Tracts 553

eccentric endosome measuring a third to half the nuclear diameter.
Nuclear granules may be scattered on a "network" or may lie just within
the membrane. The nucleus has been described by Wenrich (167). Pre-
cystic stages are rounded amoebae without food vacuoles and either with
or without glycogen.

The cyst (Fig. 11. 4, K-N), usually more common than the trophozoite
in stool samples, measures 6-16[x and is nearly always uninucleate. For
example, only 0.2 per cent of the cysts were binucleate in one series of
examinations (159). The cyst may be spherical but is more often irregular.
Inclusions resembling chromatoid bodies of other amoebae have been
seen occasionally (167), but a large mass of glycogen is characteristic.

Dientamoeba fragilis

Jepps and Dobell

In this amoeba the percentage of binucleate forms has ranged
from 9.0 (168) to about 80 (81) in different infections. Occasional speci-
mens contain more than two nuclei (49), sometimes as many as seven
(173). Several detailed descriptions have been published (81, 166, 168,

^<./ :^

'-^..-.iili--^'

Fig. 11. 5. Dientamoeba fragilis, xl600 (after Wenrich). A. Uninucleate
form, interphase nucleus. B. A single prophase nucleus with four chro-
mosomes. C. Feulgen preparation, single nucleus with eight chromosomes.
D. Single nucleus in anaphase. E. Early telophase, with paradesmose. F.
Binucleate form with persisting paradesmose. G. Two nuclei in interphase.

169), and the most recent descriptions of nuclear division are those of
Dobell (49) and Wenrich (172). The literature has been reviewed by
Wenrich (173).

The diameters of rounded amoebae range from 3.5 to about 20tj,. Move-
ment is active, with broad and usually clear pseudopodia. A number of
food vacuoles may be present. The nucleus (Fig. 11. 5) usually shows a
central group of 4-8 granules, four being the most common number. In
mitosis, the division of four chromosomes into eight has been demon-
strated (169, 172) and the central group of granules represents these
chromosomes. A reticular organization of the nucleus also has been ob-

554 Protozoa of the Digestive and Urogenital Tracts

served occasionally (173). A fibril, similar to the paradesmose of tricho-
monad flagellates, sometimes joins two nuclei in D. fragilis (49).

The ability of D. fragilis to encyst remains unproven, although sphe-
roid to ovoid bodies (4-9 x 4-6[x) containing two supposed nuclei have
been identified tentatively as cysts of this amoeba (130).

Entamoeba coli

Losch emend. Schaudinn

This species occurs in monkeys (46, 48) as well as man. The amoe-
boid stages (Fig. 1 1. 6, A) vary from 15 to 40^;. in diameter, with a common
range of 20-30[ji. Locomotion is sluggish, with blunt and often granular
pseudopodia. Food vacuoles contain bacteria and other material from the
intestine but ordinarily no tissue cells. The stained interphase nucleus
shows a rather small and normally eccentric endosome, as well as a
fairly coarse layer of peripheral granules. In addition, finely granular
periendosomal material is stained in the Feulgen technique. The rest of
the nucleus is Feulgen-negative (169a). As would be expected, the typical
nuclear structure is much modified in mitosis (158).

Spheroid precystic forms usually measure 15-18[i., with a range of 12 to
35[j(,. There are no food vacuoles and it is sometimes difficult to distin-
guish precystic E. coli from E. histolytica.

The cysts (Fig. 11. 6, B-E) range from 10 to 38[ji, in diameter, although
the majority measure 15-20[x. Young cysts contain one or two nuclei and
relatively large masses of glycogen. Mature cysts contain eight nuclei, or
sometimes 16 or more (48). Chromatoid bodies, visible in the unstained
cyst as refractile inclusions, are common in young cysts but have usually
disappeared, along with the glycogen, in mature cysts. Chromatoid ma-
terial may appear as splinters, filaments, irregular clumps of splinter-like
bodies, small irregular fragments, or as one or more lobulated masses.

Effects on the host

Pathogenicity of Endolimax nana and lodamoeba biltschlii is
doubtful and perhaps improbable, although Smithies (150) observed
digestive disturbances in all of his patients infected with amoebae. The
report of a fatal infection, apparently with /. biltschlii, seems to be the
only case of its kind on record (43).

Dientamoeba fragilis also is often considered a commensal. However,
heavy infection has been associated with definite illness involving diges-
tive disturbances, chronic fatigue, and loss of weight, and both the infec-
tion and the symptoms were eliminated by chemotherapy (66). Wenrich
also has suggested possible pathogenicity for this species (168), and has
reviewed other reports of this nature (173).

Entamoeba coli is another supposedly harmless species, but various
gastro-intestinal complaints have been noted in infected patients (150).

Protozoa of the Digestive and Urogenital Tracts 555

AMOEBIASIS

This term is restricted, in the present discussion, to infections with
Entamoeba histolytica and the resuhing effects in man.

The causative organism

Entamoeba histolytica Schaudinn ol man apparently is identical
with an intestinal amoeba of monkeys (47). The amoeboid forms (Fig.
11. 6, F) usually measure 20-30[x, with a range of about 8.0 to almost 60[x.
The size apparently varies in different strains. Locomotion of E. histolyt-
ica is much more rapid than that of E. coli, and the pseudopodia of the
former are usually clear. Food vacuoles of E. histolytica in stool samples
may contain red corpuscles or other tissue elements but rarely bacteria or
other material from the limien of the colon. Feeding activities and diges-
tion have been described by Hopkins and Warner (77). The typical
stained nucleus shows a small central endosome and relatively fine periph-
eral granules near the membrane. The Feulgen technique stains only a
zone of small periendosomal granules (169a). Characteristic changes in
nuclear structure occur in mitosis (106).

The relatively inactive, rounded precystic forms usually measure 7-20[j,.
Even before secretion of the cyst membrane, the cytoplasm may contain
glycogen and chromatoid bodies. Origin of the chromatoid bodies from
cytoplasmic vacuoles or globules has been traced in living material (77).
The chromatoid material is usually interpreted as stored food (46).

The approximately spherical cysts (Fig. 11. 6, G-K) measure about
6.0 to 2O1J1,, the range varying in different strains (50, 146, 179). Although
the recognition of hereditarily distinct "large" and "small" races seems
justified, the transformation of a small race (average, 8.5[jl) into a large
race (average, 19.0[x) has occinred after maintenance of a strain in the
laboratory for six years (122). Small races apparently differ from large
races in other respects as well as in size. Small races seem to grow less
readily in standard media (146, 152) and seldom or never show ingested
red corpuscles (62). Differences in pathogenicity also have been correlated
with differences in size.

Chromatoid bodies and glycogen are usually seen in young cysts, but
the glycogen and later the chromatoids disappear as the cysts reach ma-
turity. The uninucleate cyst sometimes contains so much glycogen that
the nucleus is displaced toward the surface. The chromatoid bodies are
typically rod-like, often with rounded ends. Both size and shape are vari-
able, and the inclusions may form clumps instead of being scattered
through the cytoplasm. There are commonly a few large (5-10[;l) plump
rods. At the other extreme, there may be as many as 30 or so small bodies.
The mature cysts usually contain four, and rarely eight or more nuclei.
After elimination from the intestine, immature cysts apparently do not

556 Protozoa of the Digestive and Urogenital Tracts

Fig. 11. 6. A-E. Entamoeba coli: A. Rounded amoeboid form, numerous
food vacuoles, xl600 (after Kessel). B. Cyst ^\ith large glycogen vacuole, a
few chromatoid bodies, two nuclei in division; xl600 (after Kessel). C. Cvst
with twelve nuclei; schematic (after Brooke). D. Typical cyst from natu-
rally infected monkev; xl8r)0 (after Kessel). E. Cyst with eight interphase
nuclei and several chromatoid bodies; xl600 (after Kessel). F-K Entamoeba
histolytica: F. Amoeboid form with ingested red corpuscles; xl600 (after
Kessel). G. Cyst with large glycogen "vacuole," several sinall chromatoid
bodies, nucleus in early division; xl600 (after Kessel). H-K. Cysts of vari-
ous sizes; xl600 (after Kessel).

develop further at room or refrigerator temperature and do not undergo
excystment (29a). Cysts of E. histolytica are often passed intermittently,
sometimes at intervals of a week or so, whereas cysts of E. coli are more
likely to be found at any examination.

Excystment requires 12-18 hours. The amoeba becomes active and then

Protozoa of the Digestive and Urogenital Tracts 557

a small portion of the cytoplasm emerges through a pore in the mem-
brane. Eventually, the organism surges back and forth until it squeezes
out as a multinucleate stage (33). The details of growth, nuclear division
and plasmotomy vary somewhat, but eight uninucleate amoebae are
usually produced from each excysted stage before the normal cycle of
fission is resumed (33, 46).

Invasion of tissues by E. histolytica

Invasion of the wall of the colon is heaviest in regions where
stasis of the contents occurs most frequently (31, 125) — cecum, ascending
colon, rectum, sigmoid flexure, and the appendix. In very severe infec-
tions the colon may be attacked throughout its length, but no single
region is invaded invariably. Consequently, the proctoscopic finding of
no rectal ulcers cannot guarantee freedom from amoebae in other parts
of the colon.

It is generally assumed that invasion of the tissues may involve me-
chanical penetration by pseudopodial activity and the destruction of
tissue cells by cytolytic enzymes. The relative importance of these two
factors has been disputed. Epithelial necrosis with no apparent mechan-
ical penetration has been seen in kittens (118), whereas penetration in
monkeys has been attributed primarily to mechanical activities (69).
Meleney and Frye (121) concluded that in kittens as well as man, lysis of
tissue cells and mechanical penetration are both significant factors,
whereas Craig (41) has stressed the cytolytic activity of E. histolytica in
human amoebiasis. The interpretation of cytolytic activity is based upon
the histological appearance of invaded tissues and upon the reported ex-
traction of an active cytolysin from E. histolytica in cultures (37).

Development of the amoebic ulcer has been discussed by various
workers (41, 52, 69, 136, 137, 140, 178). The amoebae apparently may in-
vade the tissues by crawling into the crypts of Lieberkiihn or by attacking
the more superficial mucosa. As pictured by Wenyon (178), invasion of
an intestinal gland and multiplication of the amoebae is followed by de-
generation of gland cells and loosening of the tissues so as to block the
duct, and there may be a slight nodular elevation of the mucosa. The
earliest lesions reported in human autopsy material are inconspicuous
"pinpoint" lesions in individuals reporting no symptoms of amoebiasis
(58). The early lesion, if it does not open into the intestinal lumen, may
be considered an amoebic abscess which will later rupture to form a
small flask-shaped ulcer. After penetrating the epithelium, the amoebae
may migrate along the basement membrane or may pass through into
the underlying connective tissue. Increase in number of amoebae is ac-
companied by local necrosis of tissue cells and rupture of capillaries, and
the margin of the ulcer is gradually undermined. This ulcer of the colon
differs from the typical bacterial ulcer in that there is no tendency for

558 Protozoa of the Digestive and Urogenital Tracts

developing fibrous tissue to limit the area of invasion. Instead, there is a
gradual transition from the surrounding normal tissue to the completely
necrotic tissue at the margin of the ulcer. The amoebae are usually most
numerous in the intermediate zone. If secondary bacterial invasion oc-
curs, as is the case fairly often, typical inflammatory reactions modify the
histological picture considerably.

Extension of the ulcer may involve increase in depth and in diameter.
Penetration may continue through the muscularis mucosae and sometimes
even to the serosa, to be followed occasionally by perforation, or by local
adhesion of the colon to some adjacent structure. Individual ulcers may
heal spontaneously after a time, with a resultant fibrosis of the gut wall
and a variable amount of epithelial regeneration. In chronic infections,
this fibrosis, primarily of the submucosa and muscularis, may lead to
extensive thickening of the colonic wall, either locally or sometimes
throughout much of its length.

Complications include perforations of the colon or the appendix, ab-
scesses of the appendix, perirectal abscesses, adhesions of the colon,
fistulae of amoebic origin, and sometimes amoebic granuloma of the
colon simulating carcinoma. A number of these complications, as en-
countered in a group of 20,000 patients, have been listed by Musgrave
(125).

Secondary sites of infection may be established by migration of E.
histolytica from the colon into the ileum, or more commonly, by circula-
tory transportation of the amoebae. Upon entering the capillaries of the
portal system, the amoebae would pass first to the liver. From this organ,
they might be carried to the heart and to the lungs, and then perhaps
back to the heart and out in the systemic circulation.

Amoebic abscess of the liver is the most common secondary lesion, al-
though the incidence has varied from less than 1.0 to about 50 per cent
in different groups of patients. Liver abscess may follow acute primary
amoebiasis or may develop in patients with no previous history of diar-
rheic amoebiasis or dysentery. Factors influencing the occurrence of liver
abscess are unknown. Such abscesses may be multiple or single, small or
large, and occur most frequently in the right lobe of the liver. Complica-
tions may result from rupture of a liver abscess into the peritoneal cavity,
or following adhesions, into the pleural cavity, into the stomach, or
thiough the body wall. Considerable progress is being made in the recog-
nition of hepatic amoebiasis in its early stages (151), and such early
symptoms as hepatic enlargement and tenderness have been correlated
with laboratory diagnoses. Since these early conditions seem to be cleared
up by chemotherapy, their recognition and characterization represent
a real advance in the control of secondary amoebiasis.

A pulmonary abscess may be initiated by rupture of a liver abscess
into the pleural cavity, or by transportation of the amoebae through the

Protozoa of the Digestive and Urogenital Tracts 559

pulmonary circulation. Other secondary invasions have been reported in
the skin (56), lymph glands (96), bone marrow (102), brain, spleen, and
urinary bladder. Inflammation of the uterus and vagina, with a bloody
mucous discharge containing E, histolytica (123), and invasion of the
uterine submucosa (142) have also been reported.

Various types of primary amoebiasis

Although some workers still favor the theory that in the asympto-
matic individual, E. histolytica lives in the lumen of the colon as a com-
mensal (117), there is justification for the opinion that even the "carrier"
does not escape at least some damage to the tissues (39, 41, 58, 82). There
is still no conclusive proof that E. histolytica can live in the human colon
without actual invasion of tissue. The status of the so-called small races,
which are often believed to have little tendency to invade human tissues
(146), remains indefinite in spite of the fact that the small races have not
been found in the more severe types of intestinal amoebiasis. The spon-
taneous transformation of a small race into a large race (122) has added
to the uncertainty.

In patients with symptomatic primary amoebiasis, various degrees of
severity may be recognized. Many cases are mild in character, others show
recurrent diarrhea in addition to symptoms seen in mild cases, and typical
amoebic dysentery occurs only in the more severe cases.

The characterization of mild cases, as seen in various geographical
areas (26, 27, 40, 41, 125, 145), stresses the variety of symptoms and the
confusing clinical picture. Boyers (26) has encountered more than 1,900
complaints in about 700 patients. One very common feature is fatigability,
which may develop into a condition of chronic fatigue. Constipation,
either recurrent or chronic, is usually more common than diarrhea. Other
symptoms include dull headaches, nervousness, irritability, sleepiness
during the day, restlessness, aches in the muscles or in the regions of the
joints, abdominal distention by gas, "chronic indigestion," and other
obscure digestive disturbances. The clinical picture sometimes suggests
chronic appendicitis.

In the diarrheic type, recurrent and sometimes prolonged attacks of
diarrhea accompany many of the symptoms present in mild cases.

In amoebic dysentery the stools contain appreciable amounts of blood
and mucus. Bowel movements may range from five "or six to 30 or more
per day, so that loss of weight and dehydration become extensive in
severe cases. A mild fever may develop, and various symptoms of the
diarrheic cases often appear in aggravated form. The onset of acute
amoebiasis may be sudden in individuals with no previously recognized
symptoms, or there may be a gradual transition from a mild or diarrheic
case to typical dysentery. Various complications arise if E. histolytica
becomes established secondarily in the liver or other organs.

560 Protozoa of the Digestive and Urogenital Tracts

The factors responsible for development of severe amoebiasis have not
been determined. Although the appearance of precipitins and comple-
ment-fixing antibodies indicates an immunological response, the signifi-
cance of such factors in the host-parasite relationship is uncertain.
Differences in severity of amoebiasis may be correlated with differences
in diets (2, 55), but the relation of specific dietary deficiencies to the
development of severe amoebiasis remains to be established. The bacterial
flora of the colon may be a contributory factor occasionally, as indicated
by observations on experimentally infected rats (156) and kittens (33,
126). Such a bacteriostatic agent as penicillin has shown therapeutic
activity in infected rats and may also have a prophylactic effect when
administered before inoculation with E. histolytica (156).

Chemotherapy

Intestinal amoebiasis. Completely effective treatment involves
elimination of the infection. Therefore, the results can be determined
only by periodic laboratory examination of the patient for at least six
months, and preferably a year or more, after treatment. Even an ideal
drug would not maintain a perfect record in such tests because it is im-
possible to eliminate all chances of reinfection. Consequently, the best
that can be expected is a high percentage of "permanent" cures.

During treatment and for a short time afterward, the diet of the patient
with a mild case should omit roughage and intestinal irritants. The
patient with an acute case is usually limited to liquid foods and is pref-
erably kept in bed during treatment. The choice of orally administered
drugs varies with the physician. The more commonly used types fall into
three groups, arsenicals, quinoline derivatives, and alkaloid derivatives
(1, 3, 40, 41). Some of the newer antibiotics form a promising fourth
group.

The arsenicals include stovarsol (acetarsone, or acetylamino-hydroxy-
phenylarsonic acid) and carbarsone (4-carbaminophenylarsonic acid).
Carbarsone seems to be the best of various arsenicals (1), whereas stovar-
sol has been considered somewhat dangerous for routine clinical use (3).

Several quinoline derivatives have given good results. Chiniofon (yat-
ren, quinoxyl, anayodin) is therapeutically satisfactory without producing
serious toxic effects (40, 41). Vioform (iodochlorhydroxyquinoline) seems
to be more active than chiniofon and produces only minor toxic effects
(3). Diodoquin (5,7-diiodo-8-hydroxyquinoline) is a more recently intro-
duced drug which seems to be quite effective.

Widely used alkaloid derivatives include kurchi alkaloids and emetine
(the active agent of ipecacuanha). Kurchi alkaloids, from the bark of an
Indian tree, show no marked toxicity, but the amoebacidal activity is
somewhat less than that of other widely used drugs (3). Emetine-bismuth-

Protozoa of the Digestive and Urogenital Tracts 561

iodide, although generally considered therapeutically effective, has ac-
quired such a reputation for toxicity that it is undesirable for treatment
of mild amoebiasis (8, 40, 41). However, oral dosage with emetine-hydro-
chloride in enteric-sealed tablets has given good results in a small group
of patients, some of whom were children (149). This method apparently
permits dosage with emetine at levels high enough for amoebacidal effec-
tiveness without any serious danger to the patient.

Aureomycin, in contrast to penicillin, seems to be decidedly amoebaci-
dal and has produced apparent cures in cases of intestinal amoebiasis
(114). Likewise, terramycin is proving to be effective in treatment of
primary amoebiasis (123a). In addition to the usual amoebacidal drugs,
supplementary treatment with penicillin or a sulfonamide, such as sulfa-
guanidine, may be beneficial when intestinal amoebiasis is aggravated by
secondary bacterial invasion (1).

Secondary amoebiasis. Treatment of secondary invasions is a more diffi-
cult problem than the treatment of intestinal amoebiasis and should be
started as early as possible. For hepatic amoebiasis, emetine-hydrochloride
apparently is the most effective drug available at present (86), although
preliminary results with chloroquine are quite encouraging (34, 116).
Many hepatic cases, in which treatment w^as begun early, have been cured
by emetine alone. In more advanced hepatic invasion, aspiration of
abscesses may be necessary in conjvuiction with chemotherapy. Emetine-
hydrochloride is injected subcutaneously or intramuscularly, the former
method being less painful. The effectiveness of emetine in liver abscess
apparently depends upon the rapid concentration and prolonged reten-
tion of the drug in the liver following the usual injection (129). Un-
fortunately, emetine-hydrochloride is highly toxic and its effects are
cumulative, so that cautious administration is essential.

The search for new amoebacidal drugs. The need for more effective
drugs has led to the testing of many new compounds. Preliminary screen-
ing has involved two general procedures: tests for amoebacidal activity
in cultures, and tests for therapeutic value in infected laboratory animals.
Until pure cultures are available, the results obtained with cultures must
be interpreted cautiously. If culture tubes are plugged with cotton, the
failure of E. histolytica to grow in the presence of a drug might reflect
nothing more than a rise in oxidation-reduction potential of the medium
following bacteriostasis. Therefore, petrolatum seals, or other devices for
maintaining anaerobic conditions, are essential in such tests (28). The
use of monkeys (25, 89) in testing amoebicidal drugs has the advantage
that these animals often have natural infections with E. histolytica. How-
ever, E. polecki, which also occurs in Macaca mulatta, must not be con-
fused with E. histolytica in the interpretation of results (88). Dogs
maintained on a fish diet are susceptible to experimental infection with

562 Protozoa of the Digestive and Urogenital Tracts

E. histolytica, and there seems to be fairly good correlation between the
canine and the human response to known amoebacidal drugs (160).
Young rats also have been used to advantage (85).

Problems in control of amoebiasis

The transfer of E. histolytica is a simple inatter. All that is neces-
sary is for viable stages, voided in the feces of an infected individual, to
reach the mouth of another host and be swallowed. The control of in-
testinal amoebiasis involves nothing more than preventing the comple-
tion of this sequence. The fact that no immediate solution of the problem
is in sight depends not only upon the biological characteristics of E.
histolytica but also upon human behavior. That the combination is still
beyond control by current public health practice is attested by the wide-
spread distribution of E. histolytica.

The encysted stage of E. histolytica is well adapted to its normal
method of transfer. At temperatures below 22° cysts may remain viable
for 1-6 weeks under favorable conditions, with time of survival showing
an inverse relationship to temperature (29a). Cysts also are viable for at
least several hours after ingestion by flies and passage through the insects
(131, 179). Pollution of the soil with cysts is an important source of in-
fection whenever human excrement is used as fertilizer. Under suitable
experimental conditions, cysts remain alive in soil for at least eight days
at 28-34° (14). Consequently, uncooked vegetables from contaminated
soil are potentially dangerous. Treatment of such vegetables Avith dilute
acetic acid may be an efl:ective prophylactic measure (15).

The infected individual may distribute cysts widely, as in the "general
pollution of the environment" noted in a revealing survey of a children's
home (79). Cysts were recovered from the hands of children, from soiled
clothing, from the bottom of a laundry chute, from damp sand in a play-
box, from a wading pool, and from the floor of the pool after drainage.
In general, any conditions under which sanitary precautions are relaxed
or neglected will contribute to infection. Crowding in asylums, prisons,
and other institutions may be a contributory factor, especially when
coupled with carelessness or ignorance. A recent outbreak in an eastern
state hospital is illustrative. Investigation showed that a toilet used by
kitchen attendants was without soap and paper, that attendants caring
for amoebic patients spent part of their time working in the kitchen, and
that carriers of E. histolytica had been serving as cooks and kitchen
helpers.

Since cysts may remain viable for 15-45 minutes under the fingernails
(4), the infected food-handler has often been considered a major source
of infection (40, 41), both within the family and in hotels and restaurants.
Although there are no adequate data and at least one statistical study has
failed to support such transmission (147), the burden of proof would seem

Protozoa of the Digestive and Urogenital Tracts 563

to lie upon those who wish to consider this factor unimportant in the
epidemiology of amoebiasis. Whether or not it can be assumed that food-
handlers are important, the control of amoebiasis at this point would
require laboratory examinations at intervals, as well as rigid enforcement
of sanitary regulations. The sheer numbers of individuals involved in
handling food, the time required for thorough examinations, and the
scarcity of experienced laboratory personnel make even a single survey
of all food-handlers an utter impossibility. This situation leaves educa-
tional measures as the only practical supplement to adequate sanitary
codes.

A source of pure drinking water is another important requirement.
That amoebiasis can be spread through polluted water was demonstrated
in the Chicago hotel outbreak of 1933 (29), although faulty plumbing
rather than inadequate purification of drinking water was involved. In
the purification of mimicipal water supplies, rapid sand filtration after
preliminary chemical coagulation and sedimentation is reasonably etlec-
tive in removing cysts of E. histolytica (12). Although it is not certain
that filtration is completely protective, an efficiently operated filtration
plant is probably the best safeguard for a large population. The efficiency
of chlorination alone vaaies with the concentration of free chlorine, with
temperature and pH of the water, and with the amount of organic matter
present. Varied results have been obtained on experimental scales. Stone
(157) found cysts of E. histolytica no more resistant than Escherichia coli,
being killed within 20 minutes by chlorine at 4-10 ppm, whereas Morton
(124) believes that even under ideal conditions, chlorine at 30 ppm for
30 minutes would be necessary to kill all cysts. Conservative opinion holds
that routine chlorination, as currently practiced, cannot prevent the
spread of amoebiasis by water supplies. Treatment with high concentra-
tions ("superchlorination"), followed by removal of enough chlorine to
restore potability, is recommended (128).

BALANTIDIOSIS

Balantidium coli

(Malmsten) Stein

This ciliate is the only one definitely known to be parasitic in man.
The active stage (Fig. 11. 7, A, B), measures 30-200 x 20-70[j,. The cyto-
stome is well developed and functional, as are the two contractile vac-
uoles. Food vacuoles may contain bacteria and other material from the
colon, or sometimes red corpuscles and other tissue elements. A cyto-
logical study of B. coli, with special reference to the fibrillar system, has
been published by McDonald (113).

The cysts (Fig. 11. 7, C), which reach a diameter of 60-65[jl, are the
largest ones encountered in human stools. Food vacuoles are usually

564 Protozoa of the Digestive and Urogenital Tracts

eliminated and the most conspicuous feature is the macronucleus. Cysts
containing two ciliates have been seen occasionally, but their significance
is uncertain. Conjugation, but not encystment, has been observed in
cultures (80).

The infection is usually localized in the colon, although invasion of

Fig. 11. 7. A-C. Balantidium rali: A. Somewhat contracted specimen
from a stained preparation, showing macronncieus, several food \acuoles,
fibrils extending from gnllet to cortex; material apparently being dis-
charged from the cytopyge; x520. B. A more elongated specimen, showing
two contractile vacuoles and other structures; x600 (after Wenyon). C. Cyst
(stained preparation), bilobed macronucleus; x520. D-F. Isospora hominis:
oocysts with undivided zygote (D), with two sporoblasts (E), and with two
spores containing developing sporozoites; xl030 approx. (after May). G.
Trichomo7ias vaginalis, typical undulating membrane, costa, axostyle, nu-
cleus, parabasal body, and parabasal filament; x2400 (after Wenrich). H.
T. vaginalis, showing group of blepharoplasts and other structures (except
parabasal body); schematic (after Powell).

Protozoa of the Digestive and Urogenital Tracts 565

the ileum occurs occasionally. Both active ciliates and cysts may be ex-
pected in stool samples. The incidence of infection seems to vary widely
in different parts of the world and is apparently quite low in the United
States (163, 180). Host-specificity is evidently less rigid than that of
various other intestinal Protozoa. This ciliate apparently occurs in the
pig, as well as in man and various other primates, and it has been sug-
gested that B. coli may be a natural parasite of the pig.

Effects on man

In some cases, the symptoms are insignificant enough to suggest
a pseudo-carrier condition. At the other extreme, there may be recurrent
attacks of diarrhea, and in severe infections, a chronic dysentery. The
stools may contain much mucus and sometimes blood and pus. General
symptoms include colic, loss of appetite, occasional nausea, general weak-
ness, and fatigability. In long-standing cases, loss of weight may be
noticeable. Ulceration of the colon, which may be extensive in acute
cases, resembles that produced by E. histolytica. The ciliates penetrate
the mucosa where they often occur in groups; in deeper ulcers they may
even invade the muscle layers. Perforation of the colon and extension of
the infection from the colon to the lower ileum also have been reported.
The ciliates have been seen in blood and lymph vessels of the gut and
also in adjacent lymph glands, but secondary foci of infection apparently
are not established.

Chemotherapy

A number of drugs have been tried in balantidiosis, often with
unpredictable results. Carbarsone, however, has been effective in a num-
ber of cases (163, 180, 181).

COCCIDIOSIS

Although other species have been reported in rare instances, Iso-
spora homiyiis (Rivolta) Dobell (45a) is the only coccidian known as a
frequent parasite of man. The name, Isospora belli, also has been pro-
posed for this coccidian (177). Human coccidiosis was formerly considered
rare and more than half of the earlier cases had been reported from the
Mediterranean area, especially along the easterii shores and in the Balkan
countries (108, 115). During World War II, Isospora hoyninis was found
to be widely distributed, particularly in tropical areas. Cases have been
reported in Indo-China, India, Dutch East Indies, the Philippines, Japan,
Hawaii, China, Tonkin-China, southern Russia, Palestine, Argentina,
Brazil, Uruguay, Venezuela, Mexico, and Cuba, as well as in the Mediter-
ranean region. The incidence of infection in U. S. troops evacuated from
Okinawa was about 0.75 per cent (108).

566

Protozoa of the Digestive and Urogenital Tracts

Isospora hominis

The life-cycle has not been traced completely although develop-
mental stages, possibly of /. hominis, were described by Virchow in 1860
and by Eimer in 1870. Oocysts also have been recovered from the small
intestine by aspiration through Miller-Abbott tubes (108).

The oocyst (Fig. 11. 7, D-F) measures 25-33 x 12-16[j,. In freshly passed
stools, the zygote is usually undivided. A dividing zygote or two sporo-
blasts may be seen occasionally, and in cases of constipation, oocysts may
be passed with sporoblasts enclosed in spore membranes (108). The num-
ber of oocysts passed in the stools may increase and decrease in irregular
cycles of several days each (10), or the number may increase gradually
to a maximum and then decline steadily to zero (119). Development of
sporozoites, four in each of the two spores, has been observed in stools
kept at room temperature for 24-48 hours (120), although maturation
may require 60-72 hours at 70° F. (10). Passage of oocysts, which usually
begins as symptoms are abated, may continue for several weeks and
sometimes for two months or longer (108, 120).

Effects on man

Since intestinal Coccidia invade epithelial cells, tissue destruction
is inevitable. Although some cases are so mild that specific symptoms are
not evident (120), infection often leads to digestive disturbances with a
diarrhea persisting for several weeks. Symptoms in severe cases may in-
clude abdominal cramping, nausea, and lack of appetite (10). A typical
case, accidentally contracted in the laboratory, showed an incubation
period of six days, diarrhea for 22 days, and then normally formed stools
at the end of another week (35). A similar course has been described
for an experimentally induced infection (119). No relapses have been
reported.

Chemotherapy

No adequate treatment has been described. Recent data indicate
that standard courses of emetine, atebrin, quinine, carbarsone, tetra-
chlorethylene, chiniofon, and diodoquin have no significant effect on
duration of the infection (10, 108). However, the therapeutic and pro-
phylactic activity of sulfamerazine and sulfaguanidine in Eimeria tenella
infections of chickens (57) might suggest the possible value of such drugs
in human coccidiosis.

Protozoa of the Digestive and Urogenital Tracts 567

TRICHOMONAS OF THE UROGENITAL TRACT i

Trichomonos vaginalis Donne was the first trichomonad flagellate
to be described from man. Cytological descriptions have been published
by Reuling (141) and Powell (132), and the species has been compared
with other trichomonads of man by Wenrich (170). The flagellate (Fig. 11.
7, G, H) measures 10-30 x 5-15[x. In material fresh from the vagina, occa-
sional flagellates contain leucocytes or more rarely bacteria, but the
majority show no food vacuoles. Food vacuoles are common, however, in
flagellates from bacterized cultures (132). The undulating membrane
usually does not extend into the posterior third of the body except after
atitotomy (18, 132). In view of the evidence that T. vaginalis is morpho-
logically distinct from P. hominis of the intestine (170), it is interesting
that agglutinin tests have failed to demonstrate antigenic differences
(112).

Pure cultures of T. vaginalis have been available for a number of years
and are being used to advantage in the study of growth requirements and
physiological characteristics of the flagellate (Chapter VIII).

Effects on man

Trichomonas vaginalis seems to be absent or else rarely present in
the normal vagina but is to be expected in many cases of vaginitis. It is
probable that this flagellate is one of the causative factors in vaginitis and
that infections may be correlated also with increased morbidity after
childbirth (19, 20). Infection is often accompanied by a definite leucor-
rhea and a vaginal condition resembling that in acute gonorrheal vagi-
nitis. Infections induced by inoculation of pure cultures also have led to
vaginal irritation and abnormal discharges (161). Although the evidence
for pathogenicity may not be entirely conclusive, it is strong enough to
justify prompt treatment of the patient, especially in pregnancy.

In the male, infection with T. vaginaNs commonly accompanies a non-
gonorrheal urethritis. The incidence of infection, which has approached
37 per cent in some groups (60), is higher than was formerly suspected.
Present indications are that the male is an important transmitter of
T. vaginalis and that the female may become a reservoir for venereal
infection.

^ Morphology and biology of Trichomonas vaginalis, growth requirements and culture
media, clinical aspects of infections, and therapeutic measures have been reviewed in
the following monograph: Trussell, R. E. 1947. Trichomonas vaginalis and trichomo-
niasis (Springfield: Thomas).

568 Protozoa of the Digestive and Urogenital Tracts

LABORATORY DIAGNOSIS OF
INFECTION 2

Protozoa of the mouth

The examination of fresh smears and stained preparations will
often demonstrate infections with Entamoeba gingivalis and Tricho-
monas tenax, although culture methods seem to be much more reliable.
Several satisfactory media have been described for E. gingivalis (54, 78,
92, 98) and for T. tenax (13, 72, 73, 76, 109).

Protozoa of the intestine

Wet preparations. Material to be examined for active stages should be
reasonably fresh, preferably stools passed in the laboratory shortly before
examination. Survival of trophozoites of Entamoeba histolytica shows an
inverse relationship to temperature — 2-5 hours in stools stored at 37°,
6-16 hours at 22-25°, and 48-96 hours at 5°. Cysts can survive for longer
periods, while Pentatrichomonas hominis may live as long as two weeks
at 22° (162).

Microscopic examination of fresh material, especially on a warm stage,
is useful for detection of amoeboid forms and may serve for complete
identification of active flagellates. For encysted flagellates and amoebae,
temporary staining techniques are widely used. Lugol's iodin solution,
undiluted or in 1:5 dilution (52), Donaldson's iodin-eosin stain (53), and
D'Antoni's stabilized iodin stain (7) have given satisfactory results. In
addition, a rapid stain which differentiates amoeboid stages in wet
preparations has been introduced by Velat, Weinstein, and Otto (164).

Permanent preparations. Although experienced workers may have no
serious difficulty in identifying most intestinal Protozoa by means of
fresh smears and the iodin stain or comparable techniques, permanent
preparations are useful for purposes of confirmation and for permanent
records. In addition, physicians without laboratory facilities, or without
experience in identification of Protozoa, may find it convenient to fix
smears made directly from stool samples and then ship the smears to a
laboratory for staining and diagnosis. One of the various hematoxylin
techniques is usually preferred for permanent preparations.

Concentration methods. Although direct smears are satisfactory for
routine examinations, it is advantageous to concentrate the Protozoa
when their presence only in small numbers is suspected. A portion of the
stool may be mixed with physiological salt solution and then filtered
through cheesecloth. The filtrate is centrifuged, the supernatant liquid
is discarded, and the sediment is mixed with fresh salt solution and again

^ Adequate descriptions and evaluations of laboratory techniques will be found in
Craig's (42) comprehensive monograph.

Protozoa of the Digestive and Urogenital Tracts 569

centrifuged. Washing is continued until the supernatant fluid is clear.
The sediment is then examined for Protozoa. For concentration of cysts
only, the zinc-sulphate flotation method (59) is usefvxl.

Culture methods. In the detection of mild infections, culture methods
may be successful when direct examination of stools is negative. A variety
of culture media^ have been developed and a number of them have
proven useful in diagnosis. Craig (40) has pointed out that in the use of
culture media for E. histolytica, individual experience with a medium
probably counts as much in the long run as the particular type of culture
medium.

The effectiveness of culture methods for detecting E. histolytica in
stools containing cysts has been increased by adding streptomycin to the
medium. The retardation of bacterial growth, as well as that of Blasto-
cystis hominis, apparently facilitates growth of the amoebae after excyst-
ment (153). Perhaps the most satisfactory diagnostic medium will prove
to be one which inhibits growth of bacteria more or less completely. The
development of such a mediimi should be possible when more is known
about the growth requirements of the intestinal amoebae and flagellates.

Complement-fixation. The practical application of complement-fixa-
tion to diagnosis of amoebiasis was first reported by Craig (37, 38). Fol-
lowing the introduction of a commercially prepared antigen, this test is
being used on a progressively wider scale. Complement-fixation seems to
be of value in the diagnosis of mild primary amoebiasis, and with certain
modifications, in the detection of early hepatic amoebiasis (Chapter 14).

Trichomonas vaginalis

The examination of an ordinary wet preparation or hanging-drop
is often adequate for the detection of T. vaginalis. In the diagnosis of
mild infections, in following the effects of treatment, and in detecting
trichomonads in centrifuged urine specimens, culture methods (18, 109,
148) are more efficient. Some of the more recently developed media (83,
84, 154, 161) are designed for growth of T. vaginalis in bacteria-free cul-
tures. The medium of Kupferberg, Johnson, and Sprince (107), now
available commercially, requires only the addition of serum and penicillin
for diagnostic use.

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^ Satisfactory culture media have been described for the following intestinal Protozoa
of man: flagellates — Chilomostix mesnili (9, 21, 24, 63, 70, 75), Retortomonas intestinalis
(17, 70, 75), Pentatrichomonas hominis (9, 63, 70, 75, 148, 174), Tricercomonas intes-
tinalis (70); amoebae — Dientamoehn fragilis (9, 49); Endolimax nana (9, 51), Entamoeba
coli (9, 48, 51, 148), E. histolytica (9, 23. 32, 36, 51, 63, 127, 138, 144, 148), lodamoeba
biltschlii (9); dliates—Balantidiutn coli (11, 80, 139, 148).

570 Protozoa of the Digestive and Urogenital Tracts

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Protozoa of the Digestive and Urogenital Tracts 573

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XII

The Blood Flagellates

Leishmaniasis

Visceral leishmaniasis
Distribution
The causative organism
Symptoms and pathology

Oriental sore
Distribution
The causative organism
Symptoms and pathology

Mucocutaneous leishmaniasis
Distribution
The causative organism
Symptoms and pathology

Transmission of leishmaniasis

Laboratory diagnosis

Chemotherapy

Control of leishmaniasis

Trypanosomiasis

African sleeping sickness

Distribution

The causative organisms

Symptoms and pathology

Diagnosis

Chemotherapy

Control of sleeping sickness
Chagas' disease

Distribution

The causative organism

Vectors and reservoir hosts

Symptoms and pathology

Diagnosis

Therapy and control

Literature cited

LEISHMANIASIS

T„

.HE FLAGELLATES causing human leishmaniasis belong to the
genus Leishniania Ross. To this genus there have been assigned also
certain flagellates of reptiles. Among these are L. chamaeleonis, which is
an intestinal parasite retaining the leptomonad form (71), and also L.
ceramodactyli (5) and L. hemidaclyli (66), which are blood parasites of
gekkos and infect sandflies. L. ceramodactyli develops in the posterior
station. L. hemidactyli develops in the anterior station like the species
found in mammals, and this is the case also for L. tarentolae (6a) from
the blood of gekkos.

The three parasites of man are usually given specific rank: L. donovani
of visceral leishmaniasis, L. tropica of oriental sore, and L. brasiliensis
(L. peruviana, L. tropica var. americana) of muco-cutaneous or American
leishmaniasis. The lack of obvious morphological differences has led
some workers to the opinion that all three parasites are merely strains

574

The Blood Flagellates 575

or races of a single species (31). On the other hand, the specific status
of the three types has been defended on the grounds that the clinical,
pathological and epidemiological differences seem to be genetically stable

(34a).

Visceral leishmaniasis

Distribution. In the Eastern Hemisphere, kala-azar has occurred en-
demically in certain parts of India, southern U.S.S.R., Burma, Indo-
China, Central China, Turkestan, Iraq, along the Mediterranean shores,
and in the Sudan. Cases have occurred also in the Western Hemisphere
— various parts of Brazil (55), Argentina, Bolivia, Colombia, Paraguay,
Peru, and Venezuela. In American areas, visceral leishmaniasis has been
reported in both infants and adults. Both young and old are susceptible
in India also, while in the Mediterranean area, children under four years
have been attacked almost exclusively.

The causative organism. Kala-azar was at various times considered an
unusual malaria and a serious type of hookworm disease before the cau-
sative organism was discovered. L. donovani seems to have been observed
by Cunningham in 1885, by Firth in 1891, and by Borowsky in 1892.
Although Borowsky recognized the organisms as Protozoa (30a, 54), his ob-
servations remained unknown to Leishman and Donovan who described
the parasites independently in 1903. After various assignments to the
Sporozoa, the affinities of L. donovani were demonstrated when Rogers
(56a) found the flagellate stage in cultures.

L. donovani occurs in the mammalian host primarily as leishmanial
stages in lymphoid-macrophage (reticulo-endothelial) cells of the spleen,
liver, bone marrow, intestine, and lymph glands. Occasionally, leish-
manial forms occur also in mononuclear and polymorphonuclear leuco-
cytes in the blood. The leishmanial stage may be ovoid or approximately
spherical (Fig. 12. 1, I, J). The spherical forms are usually 1-3[jl in diam-
eter. Ovoid stages generally measure 2.0-5.0 x 1.5-2.5[a. Identification is
based upon the presence of both nucleus and kinetoplast. The latter is
often elongated and may be more or less perpendicular or sometimes
tangential to the nucleus. In well-stained specimens, an axoneme some-
times can be traced from the kinetoplast to the periplast.

Leishmanial stages multiply in the macrophages, which eventually rup-
ture to release the parasites. Most of them apparently are picked up by
macrophages, but some reach the blood stream from which they can be
ingested by the vector, a species of Phlebotomus. Surviving leishmanial
forms change into leptomonad stages in the insect. These multiply in the
midgut, and after several days, appear also in the foregut. The lepto-
monads of the foregut are believed to be infective for man.

The growth of L. dojiovani in cultures is similar to that in the sandfly.
After inoculation into a suitable medium the leishmanial stage grows,

576 The Blood Flagellates

F G

Fig. 12. 1. A-E. Flagellated forms of Leishmania donoi'njii from cul-
tures; x2500; (A, B after Laveran; C-E, after Wenyon). F-H. Leishmania
tropica, leishmanial forms from sores; x5500 (after Weuyon). I. L. donovani
in large mononuclear cell; from blood. xl350 (after Laveran). J. L. dono-
vani in spleen; semidiagrammatic. xlO.S5 (after Xattan-Larrier). K. L. dono-
vani in liver; semidiagrammatic, xl035 (after \attan-I.arrit-r).

develops a flagellum, and becomes a leptomonad form (Fig. 12. 1, A-C)
measuring 7-20ij, in length. Active leptomonads are usually abundant in
cultures after 48-72 hours at 22-25°. Old cultures may contain non-flagel-
lated stages resembling the original leishmanial forms.

The organism causing American cases of visceral leishmaniasis, found
in dogs and cats as well as in man, has been considered a new species,
L. chagasi (14). However, the flagellate may be identical with L. donovani
of kala-azar (2, 59).

The Blood Flagellates 577

Symptoms and pathology. Kala-azar is a chronic or sub-acute disease
characterized by enlargement of the liver and spleen and by an irregular
fever, anemia, and leucopenia. Mortality is high in untreated cases. The
disease is more severe in epidemic outbreaks than in intervening periods,
and effects on a population may be aggravated by famine and by other
diseases. The incubation period varies from about 10 days to two or three
months as a rule (10), although incubation periods in U. S. troops during
AVorld War II ranged from three weeks to 19 months (42).

A common early symptom is a high fever preceded by rigors. The fever
may become continuous or may be intermittent and irregular. Early en-
largement of the liver and spleen is typical, and the swelling usually
increases during the course of the disease. After emaciation becomes
marked in children, splenomegaly often produces a swollen protruding
abdomen. Grayish discoloration of the skin, especially over the forehead
and temples and sometimes around the mouth, is characteristic of chronic
cases. Eventually, a low fever becomes more or less continuous and ex-
tensive loss of weight occurs.

The spleen, liver, and bone marrow are affected in practically all cases.
The extent to which other organs are involved is determined by length
and severity of the infection. Normal splenic tissues may be partly re-
placed by macrophages, and fibrous tissue of the reticulum may increase.
Effects on the liver are similar. Kiipffer's cells, which increase in number,
are often loaded with leishmanial forms, and there may be an increase
in fibrous tissue with marked cirrhosis in chronic cases. In the bone
marrow, as much as three-fourths of the normal tissue may be replaced
by packed macrophages, so that the production of blood cells is greatly
reduced. Consequently, some degree of anemia is to be expected, although
the red cell count seldom falls below 2,500,000. In addition, there is fre-
quently a leucopenia with a count well below 4,000, and a granulopenia
also has been noted. In the intestinal mucosa, multiplication of macro-
phages sometimes distends the villi.

Multiplication of macrophages in the lymph glands, kidneys, testes,
lungs, heart, and adrenals is usually not extensive; accordingly, the para-
sites are to be expected primarily in isolated phagocytes. However, adreno-
cortical hypofimction, presumably a result of tissue destruction, has been
observed and may be correlated with skin pigmentation and low blood
pressure (15).

Skin nodules, similar to non-ulcerating lesions of oriental sore, some-
times appear in treated patients about two years or so after clinical re-
covery (1, 9). The nodules, usually small, appear most commonly on the
face and neck. Beneath the thin epithelium there is an oedematous
dermis showing atrophy of connective tissue. Surrounding this area there
is a zone of fibroblasts and multiplying macrophages, the latter often
containing parasites. Such lesions may form a lasting reservoir of infec-

578 The Blood Flagellates

tion and may explain the sporadic occurrence of kala-azar year after year
in households and other small groups (48).

In summary, the essential pathological characteristic of kala-azar in
man, monkeys and hamsters (40) is the increase in lymphoid-macrophage
cells. The macrophages ingest L. donovani but are imable to prevent their
multiplication after ingestion. Consequently, it is uncertain "whether the
reticulo-endothelium is valuable, as the only defense the body has, im-
perfect as it is, or deleterious, as being the most suitable location for the
parasites" (70).

Oriental sore

Distribution. Classical oriental sore, as seen in Eurasia and Africa,
is a widespread type of leishmaniasis. In Europe, the disease has been
known in Spain, Italy, Greece, and rather rarely in France. In Africa,
oriental sore has been fairly common in Egypt, the Sudan, Algeria, French
Congo, and Nigeria. In Asia, the disease seems to be endemic in Arabia,
Asia Minor, Mesopotamia, Persia, subtropical parts of the U.S.S.R., and
parts of India.

The causative organism. L. tropica was recognized as the causative
organism when Wright described the parasites in 1903. Morphologically,
this flagellate (Fig. 12. 1, F) is essentially identical with L. do?wvani.
Like the latter, it is usually found inside macrophages in man.

The invertebrate phase of the cycle, as traced in Phlebotomus papatasii,
is initiated by metamorphosis of the ingested leishmanial forms into
leptomonads in the midgut. With continued multiplication, the infection
gradually extends into the pharynx and mouth cavity. In at least a few
sandflies, the flagellates eventually reach the anterior part of the epi-
pharynx. The last step apparently is essential for transfer to man.

Symptoms and pathology. The incubation period ranges from a few
days to several months, and sometimes even three or four years. The skin
lesion begins as a small pimple, resembling the swelling which sometimes
follows insect bites. The pimple grows and may eventually develop into
a nodule an inch or more in diameter. Clinically, the non-ulcerating
nodule, the superficial flat ulcer, and the deeper boil may be distin-
guished. The non-ulcerating lesion, after a year or so, dries up to a scab
which drops off, leaving a scar. More commonly, the surface of the nodule
breaks down to form an ulcer. Secondary invasion by bacteria usually
occurs, and the ulcer may grow to a diameter of several inches. Sections
through a sore show an oedematous dermis with many macrophages,
pressure from which may cause local destruction of hair follicles and sweat
glands. Patients often show only one or two lesions, although sometimes
more (even a dozen or so). There is usually little constitutional disturb-
ance. However, lymph glands near sores may become swollen, and fever
sometimes occurs.

The Blood Flagellates 579

Muco-cutaneous leishmaniasis

Distribution. Muco-cutaneous leishmaniasis, generally more severe
than classical oriental sore, has been reported from Argentina, Bolivia,
Brazil, British Honduras, Colombia, Costa Rica, Ecuador, French Guiana,
Panama, Paraguay, Peru, Uruguay, and Venez.uela.

The causative organism. L. brasiliensis is very similar to L. tropica in
morphology and likewise is found mainly in macrophages in man. The
insect phase of the cycle is similar to that in L. tropica.

Symptoms and pathology. The skin lesion develops much as in oriental
sore. The non-ulcerating type grows from the primary papule into a
slightly elevated reddened area with a rough surface from which a liquid
oozes and dries into a crust. This liquid usually contains parasites and
thus may start a new sore on contact with a break in the skin. The ulcerat-
ing lesion becomes excavated centrally and secondary invasion by bacteria
often occurs. Neighboring lymph glands are often swollen, and general
symptoms may include fever, chronic headaches and aches in the joints.
The mucous membranes also are sometimes involved. Ulcers may develop
in the nose and mouth and, more rarely, in the vagina. An ulcer originat-
ing in the nose may spread downward over the upper lip into the oral
cavity, or the nostrils may become plugged and the nasal septum pro-
gressively destroyed. Depending upon their location, mucosal lesions may
eventually destroy the sense of smell or hearing, or may cause blindness.

Transmission of leishmaniasis

Transmission of oriental sore by direct contact has long been
known. In fact, natives of certain regions in India formerly made a prac-
tice of inoculating material from sores into the skin of young children.
This crude vaccination led to development of a sore on an unexposed
part of the body and, it was hoped, to prevention of more conspicuous
sores later in life. The general opinion is that L. tropica may invade a
sound mucous inembrane but cannot penetrate unbroken skin, and it is
likely that L. brasiliensis has similar abilities. Contact transfer of kala-
azar also may be possible, since the flagellates have been demonstrated
in nasal secretions of patients.

Bedbugs, fleas, mosquitoes, lice, houseflies, and ticks have all been
suspected, at one tiine or another, of transmitting leishmaniasis to man.
There is no convincing evidence that any one of them normally serves
as a true host. However, occasional mechanical transfer may be possible,
as in the transmission of L. tropica by Stomoxys calcitrans in Lebanon.
Investigations on sandflies have been more successful. By 1924 it was
known that Phlebotomus argentipes develops a flagellate infection after
feeding on kala-azar patients. In 1927, hamsters were infected by inocula-
tion with flagellates from sandflies (29). In the same year, it was found

580 The Blood Flagellates

that flagellates from naturally infected sandflies induced typical oriental
sore when inoculated into man (4). After feeding upon these lesions, sand-
flies developed flagellate infections. These flagellates, in turn, induced
typical sores upon inoculation into man. The second crop of sores again
was infective for sandflies. In later work on kala-azar, hamsters were in-
fected by oral introduction of L. donovani (63) and later by ingestion of
infected sandflies (65). In 1931, L. donovani was transferred to a hamster
by the bite of a sandfly under experimental conditions (64). Some of the
earlier difficulties in producing heavy mfections of sandflies have been
largely eliminated by better diets for the flies, and kala-azar can now
be transferred readily to hamsters (67, 68). Such techniques also have
made possible the vector transfer of kala-azar to human volunteers (69).
Similarly, the experimental transfer of L. tropica by sandflies has been
facilitated by adding salt to the diet of the flies (3).

Diagnosis

Blood films, and smears of other tissues (splenic pulp, bone mar-
row, liver, lymph glands) obtained by puncture methods, may be ex-
amined directly for L. donovani. Spleen smears are probably positive in
at least 80 per cent of the actual infections, and sternal puncture is
equally reliable (49). The results of the two methods agree closely (30),
and sternal puncture has the advantage of being less dangerous. Thick
blood smears are somewhat less reliable than smears from the spleen and
bone marrow. In any case, diagnosis may be difficult in early stages of
kala-azar, and prolonged search of smears may be necessary. In diagnosis
of dermal leishmaniasis, the parasites are best detected in material aspi-
rated from the periphery of the lesion, from the tissues just beneath the
ulcer, or from non-ulcerated nodules. L. brasiliensis is most abundant in
the early lesions of the skin and mucosa and may be found also in lymph
glands adjacent to sores. It is usually difficult to recover the flagellates
from old bacterized sores.

Culture methods, for the experienced worker, are generally more reli-
able than tissue smears in diagnosis of kala-azar and may be preferable
where facilities are available. Aseptic precautions are required, and for
best results, blood agar slants may be inoculated with leucocytes centri-
fuged from 5-10 ml of blood. Upon incubation at 22-25° C, a heavy
growth of flagellates may be expected within 72 hours. With the addition
of penicillin for the control of bacteria, culture methods also appear to
be satisfactory for demonstrating L. tropica m bacterially contaminated
lesions (43). This technique may prove useful likewise in diagnosis of
kala-azar.

Several indirect tests for kala-azar are based upon the characteristic
increase in serum globulins. In Brahmachari's test the addition of 2-3
volumes of distilled water to one of kala-azar serum precipitates the

The Blood Flagellates 581

globulins. In Napier's aldehyde test a positive serum forms an opaque
coagulum upon addition of a small amount of formalin. Chopra's anti-
mony test is a ring test in which a flocculent precipitate develops in the
zone of contact between a positive serum and a solution of urea stiba-
mine. Although such tests are no substitute for demonstration of the
parasites, they have been useful as presumptive tests in field surveys. A
non-specific complement-fixation test, using an antigen prepared from a
human strain of Mycobacterium tuberculosis, has given good preliminary
results with kala-azar, although false positives may be expected occa-
sionally in pulmonary tuberculosis (60, 61).

Chemotherapy

Prior to 1915 there was no reliable cure for kala-azar and untreated
cases had shown a mortality of about 90 per cent in India. Following the
introduction of tartar emetic (11), there was a marked change for the
better. Introduction of urea stibamine by Brahmachari in 1922 led to
further improvement. By 1925, the death rate in India had dropped to
about 10 per cent of the cases; in 1928, to about 7 per cent. Neostibosan,
introduced a few years after urea stibamine, is more or less comparable
in effectiveness. Several more recently tested drugs appear to have real
value. Stibatin (sodium antimony gluconate) has given satisfactory re-
sults and has fairly low toxicity. Stilbamine seems to be effective against
relapsing kala-azar, although its rather high toxicity is an undesirable
feature. Stilbamidine, unfortunately too toxic for routine use, has pro-
duced clinical cures in about 98 per cent, with a relapse rate of only
about 4 per cent. This drug is useful in antimony-resistant and antimony-
sensitive cases. Pentamidine isothionate is comparatively harmless to the
patient but is not quite so effective as stilbamidine — about 94 per cent
clinical cures, with relapses in about 16 per cent. Penicillin has given
satisfactory results with "cancrum oris" (noma), a sort of oral gangrene
which is a frequently fatal complication of kala-azar.

In dermal leishmaniasis, intravenous or intramuscular injection of
leishmanicidal drugs is usually combined with local treatment for con-
trol of bacteria. Since the lesions of oriental sore often tend to heal
spontaneously, it is difficult to evaluate the effects of chemotherapy.

Control of leishmaniasis

The usual vectors of leishmaniasis are small Diptera belonging to
the genus Phlebotomus. These sandflies, or owl midges, are active mainly
in twilight and darkness. The females are blood-suckers, usually attacking
warm blooded animals. Four or five days after a blood meal, the eggs are
laid in moist shaded locations — animal burrows, caves, crevices among
rocks, cracks in the soil, or at the bases of hollow trees. The eggs hatch
in about a week, and two months or more are required for development.

582 The Blood Flagellates

Sandflies are found throughout the year in the tropics. In the cooler
climates, the adults apparently do not live through the winter. Instead,
hibernation presumably occurs in the last larval instar. The control of
sandflies by widespread attacks on breeding places is not, for obvious
reasons, a practical method of control. However, the use of residual DDT
sprays on suspected shelters and breeding places has produced striking
results on a limited scale (27). Similar treatment of buildings with DDT
is an effective protection (28). In addition, sleeping nets, fine-mesh screen-
ing and insect repellents are useful in preventing contact of sandflies
with human beings.

The importance of animal reservoirs is uncertain. Natural infections
with flagellates resembling L. donovani have been reported in dogs, cats,
horses, and sheep. L. tropica also occurs naturally in dogs, and the canine
strain is infective for man (6). Certain rodents (gerbils, sousliks), found
infected in Middle Asia (U.S.S.R.), also may serve as reservoirs (32). Al-
though these lower mammals obviously are reservoirs, it is not known
how extensive a part they play in the maintenance of endemic leish-
maniasis.

TRYPANOSOMIASIS

Species of Trypanosoyna are found in fishes, amphibia, reptiles,
birds, and mammals, and the great majority appear to be non-pathogenic
in their natural hosts. The trypanosomes of mammals include a few
pathogens, such as T. briicei of nagana, T. equinum of mal de caderas,
T. eqiiiperdum of dourine, T. evansi of surra, and the types which attack
man — T. cruzi of Chagas' disease, and T. gambiense and T. rhodesiense
of sleeping sickness. Life cycles and methods of transfer differ within this
small group. Metacyclic, or infective, stages of T. cruzi develop in the
hindgut ("posterior station") of the vector and are deposited on the skin
of the vertebrate by a visiting insect. The flagellates may become estab-
lished in the vertebrate if they reach a mucous membrane or a break in
the skin. Such species as T. cruzi and the non-pathogenic T. lewisi are
probably the most primitive of the mammalian trypanosomes, since the
invertebrate phase of the cycle resembles that of the presumably ancestral
herpetomonad flagellates. Metacyclic stages of T. gambiense, T. rhode-
siense, and T. brucei develop in the salivary glands of the vector and are
transferred to the mammalian host by inoculation. On such bases, these
species are believed to be more highly evolved than the T. lewisi group.
Mechanical transfer by tabanid and stomoxid flies is characteristic of T.
evansi and T. equinum, which are apparently related to the T. brucei
group. Differentiation of these species may have followed their introduc-
tion into regions free from their natural vectors. Such a possibility is not
too remote, because mechanical transfer of T. gambiense is considered
possible for a short time after the flagellates are ingested by the insect

The Blood Flagellates 583

host. T. equiperdum, possibly evolved through a similar accident of
distribution, is now transmitted through direct contact in coitus.

Current differentiation of species is based, in some cases, upon host
relationships instead of conventional morphological features. For ex-
ample, T. briicei, T. gambiense, and T. rhodesiense are morphologically
indistinguishable. The same is true for T. equiperdum and T. evansi. In
such cases, the homologues could be, and probably should be, considered
specialized strains of a single species (31).

The range in pathogenicity of trypanosomes has interested various
workers in possible explanations for pathological effects. The sugar con-
sumption theory (58) assumed that the consumption of sugar by trypano-
somes might reduce the blood sugar of the host too rapidly for the liver
to maintain a normal blood level. The resultant strain was supposed to
cause a breakdown of liver function, leading to fatal intoxication. In
spite of a few cases in which blood sugar levels have been low consistently
(56), marked reduction in sugar levels generally occurs only in the last
days or hours of a lethal infection.

Another theory (7) holds that death of the host is caused by asphyxia-
tion, supposedly the result of a pulmonary oedema following partial ob-
struction of capillaries by agglutinated trypanosomes. A third suggestion
(35) is that lactic acid, produced from sugars by trypanosomes, interferes
with normal tissue oxidations. The lactic acid concentration of the blood
usually does show an increase during the terminal stages of a fatal in-
fection, but the concentrations reported are considered too low for
appreciable injury to the host. Approximate doubling of the serum potas-
sium level has been reported before death of rats infected with T.
equiperdum (78), while in other cases no correlation has been observed
between the time of survival and the tolerance to potassium (57a). Tryp-
anosomal toxins also have been suggested as an explanation for patho-
genicity. Although there is no evidence that trypanosomes produce true
exotoxins, it is possible that endotoxins (in the bacteriological sense)
might harm the host.

African sleeping sickness

Distribution. There are two varieties of this disease, Gambian and
Rhodesian. The Gambian variety has ranged from about 15° N. to
15° S. latitude but is more common in the western than in the eastern
part of this zone in Africa. Within its range, the disease occurs primarily
along rivers and near lakes, in correlation with the distribution of its
major vector, Glossina palpalis. In the eastern part of its range, Gambian
sleeping sickness extends into the territory of the Rhodesian variety.
The latter, which is less widely distributed, has been known in North
and South Rhodesia, Portugese East Africa, Nyasaland, Tanganyika Ter-
ritory, northeastern Mozambique, Uganda Protectorate, and the southern

584 The Blood Flagellates

Sudan. Within these areas, high temperatures (75-85° F.) favor develop-
ment of the trypanosomes in tsetse flies, while temperatures below 70°
are unfavorable. A few outbreaks of epidemic proportions have been
recorded, but the disease is generally sporadic in occurrence.

Fig. 12 2. Trypanosoma gamhiense. A-D. Stages in Glossina palpalis
(x3300, after Robertson): A. Stage in mid-gut after two days; B. Stage in
gut after 2-3 weeks; C. Crithidial stage in salivary glands; D. Metacyclic
trypanosome in salivary glands. E-I. Stages in blood of man; x2200 ap-
prox.: E. Slender form (after Laveran); F. Slender form (after Bruce);
G. Intermediate form (after Bruce); H. Broad form (after Laveran); I.
Broad form (after Bruce).

The causative organisms. Trypanosoma gamhiense Dutton, first seen
by Forde in 1901, is the causative organism of Gambian sleeping sickness;
T. rhodesiense Stephens and Fantham, of the Rhodesian type. Although
the two parasites differ in virulence, and to a considerable extent in
geographical distribution, they cannot be distinguished with certainty
from each other or from T. brucei. Some workers believe that T. rho-

The Blood Flagellates 585

desiense is a more virulent strain of T. gambiense (17), and there have
been suggestions that the two types are interconvertible (26).

T. gambiense (Fig. 12.2) measures 10-40[x in length and contains a
small spherical kinetoplast. Three forms have been described: slender
flagellates with a long flagellum, intermediate forms with a short flagel-
lum, and broad forms with no "free flagellum. It has been suggested that
such polymorphism is correlated with sexual phenomena, but the evi-
dence is inconclusive (Chapter II). In man, trypanosomal stages appear
in the blood, where they undergo fission. Eventually the flagellates may
get into the cerebrospinal fluid, the invasion often bringing on "sleeping
sickness."

Glossina palpalis is the major vector, but G. tachinoides is important
in some parts of West Africa. The fly sucks the trypanosomes into the gut
along with a meal of blood. For a few hours, at least, the trypanosomes
apparently remain unchanged. If conditions are favorable, the flagellates
survive and undergo fission, but only a small percentage of the tsetse flies
actually become infected after ingesting T. gambiense. After about two
weeks, the digestive tract contains many slender trypanosomes. The
infection gradually extends into the foregut and some of the flagellates
usually reach the salivary glands by the end of the third week. Here
the trypanosomes become attached, change into the crithidial stage
(Fig. 12.2) and divide rapidly for two or three days. The flagellates then
develop into metacyclic trypanosomes infective for the vertebrate host.
The insect phase of the cycle lasts from three to five weeks, depending
upon environmental conditions.

In the transfer from tsetse fly to vertebrate, two methods are possible:
mechanical transfer of the flagellates shortly after ingestion; and cyclic
transfer, in which the flagellates pass through developmental stages in the
vector before they are again infective for the vertebrate. Within a day or
so after ingestion, completion of the insect cycle becomes essential for
infection of the vertebrate.

The life cycle of T. rhodesiense is quite similar to that of T. gambiense.
However, the insect phase of the cycle — in the usual vector, Glossina
morsitans — requires only about two weeks for completion.

Symptoms and pathology. In Gambian sleeping sickness, the bite of
an infected fly often causes a local irritation Avhich normally disappears
after a few days. Following an incubation period, ranging from tw^o
weeks to a year or more, an irregular recurrent fever usually is the first
noticeable symptom. Although the fever is sometimes mild, temperatures
of 105-106° may be observed. After a time, physical weakness becomes
marked and other symptoms include anemia, rapid pulse, severe head-
aches, enlargment of the cervical lymph glands ("Winterbottom's sign"),
and loss of weight. Itchy skin eruptions are fairly common, although less
noticeable in natives than in foreigners. Enlargement of the liver and

586 The Blood Flagellates

spleen is said to increase with rise in temperature and to decrease as
the temperature falls. In some cases, the disease may seem to end at this
stage. However, the frequency of spontaneous recovery — if it actually oc-
curs— is uncertain, since there are indications that the infection some-
times becomes latent.

On the basis of later history, three general types have been dis-
tinguished (39). In a mild variety, an equilibrium seems to be established
with the patient in poor physical condition. In view of the lowered re-
sistance to other diseases, even this mild form contributes significantly
to depopulation. A severe acute type is characterized by marked toxemia
and often by oedema and may lead to death without involvement of the
central nervous system. The third type is the classical form with a sleep-
ing sickness stage. The second and third types become predominant in
epidemics, whereas the mild type is otherwise the most common. In
later stages of the third type, invasion of the central nervous system is
followed by sleeping sickness. This stage often develops in untreated
cases after a more or less prolonged period, sometimes several years.
Physical weakness and languor become more and more pronounced and
loss of weight is often striking. The patient falls asleep at irregular
intervals. Convulsive movements of the limbs, sometimes followed by
temporary paralysis, become evident. Mania may develop, and mental
and physical symptoms sometimes resemble those of paresis. The spells
of sleep gradually become more frequent, and death is usually the out-
come in untreated cases.

Anemia is a common feature and becomes more marked in later stages.
Leucopenia, with a relative increase in mononuclears, is common, but a
leucocytosis may occur instead. Red corpuscles in fresh blood smears
may undergo agglutination at room temperature, a phenomenon attrib-
uted to an increase in "autoagglutinin." Swelling of the lymph glands is
often produced, even in early stages, by multiplication of lymphocytes in
the germ centers. Multiplication of cells may occur also in the endo-
thelium of lymph channels. Hemorrhages frequently develop, and the
degeneration of lymphatic tissue may be followed by invasion of fibrous
tissue. Enlargement of the spleen is more or less proportional to the
degree of anemia and to the parasitemia. Endothelial proliferation, in-
crease in number of phagocytes, increase in thickness of the capsule, and
some degeneration of splenic tissue have been observed. Endothelial
proliferation also has been observed in vessels of the lungs, liver and
kidneys, sometimes leading to obliteration of capillaries in the lungs and
kidneys. Lymphocytic infiltration has been noted in the heart, peri-
cardium, liver, digestive tract and skin. Aggregates of macrophages,
sometimes found in the skin, may contain ingested trypanosomes.

The typical lesions depend upon the presence of trypanosomes in the
tissues. Extravascular distribution of the flagellates in experimental infec-

The Blood Flagellates 587

tions has been noted in the kidney, brain, inner ear, fetal heart, lymph
glands, wall of the stomach, and the choroid plexus. Chronic inflamma-
tion of the brain is not observed until after the trypanosomes have
reached the cerebrospinal fluid. Capillary hemorrhages sometimes occur,
and there is a proliferation of neuroglia and endothelial cells, the latter
sometimes even in the arteries. Some of the neurons may degenerate
and atrophy of dendrities is observed occasionally. The spinal cord is
usually affected less severely than the cerebral cortex.

Rhodesian sleeping sickness is similar in many respects to the Gambian
variety but is generally more acute. Fever is more evident in early stages,
while early enlargement of lymph glands is less common than in the
Gambian variety. Also, the mortality is higher in Rhodesian sleeping
sickness, although mild cases supposedly of the Rhodesian type have been
reported occasionally.

Diagnosis. During the acute phase, laboratory diagnosis is compara-
tively easy. The trypanosomes can usually be detected in fresh or stained
blood films and in material obtained by puncture of enlarged lymph
glands. In later stages fewer flagellates are present in the blood, so that
examination of several blood films, or preferably thick smears, may be
necessary to detect the parasites. Smears from centrifuged blood may be
positive when thin films or thick smears are negative. During the sleeping
sickness stage, blood examination is much more likely to be negative.
Therefore, the examination of cerebrospinal fluid, obtained by lumbar
or cisternal puncture, may be necessary if other methods fail.

Chemotherapy. A number of drugs have been used in treatment of
Gambian sleeping sickness, some with fair success and others with good
results. In the experience of Kellersberger (34) with more than 9,000
cases, Bayer 205 was effective in early stages but useless after the central
nervous system was involved. Atoxyl also was not curative in later stages.
Tryparsemide showed more activity in early stages and also was the most
effective drug in later stages, even arresting a few apparently moribund
cases. However, this drug is quite toxic and dosage is usually spread
over a period of two or three months. In treatment of early cases, p-arseno-
phenyl butyric acid (24), germanin and pentamidine all seem to be
fairly effective. Orsanine seems to be active in cases showing involvement
of the central nervous system. Good results have been obtained also with
melarsen oxide which may be given orally or by injection and has shown
low toxicity and relatively rapid action. The trypanocidal activity of
tryparsemide, mapharsen and stilbamine involves effects on hexokinase
and other enzymes of trypanosomes (16).

Chemotherapy has been less successful in Rhodesian sleeping sickness.
In recent years, apparent cures have ranged from 16 to 48 per cent during
various outbreaks in Tanganyika Territory. Particular drugs may differ
in their activity against T. rhodesiense and T. gambiense. Bayer 205

588 The Blood Flagellates

seems to be more effective against T. rhodesiense, while the reverse ap-
parently is true for tryparsemide and atoxyl.

An important complication of chemotherapy has been the tendency
of trypanosomes to develop a resistance to arsenicals or antimonials
(Chapter IX). However, the terms, "arsenic-fastness" and "antimony-fast-
ness," sometimes applied to such phenomena, may not be entirely ac-
curate. Resistance may be developed against substituted phenyl groups
rather than against arsenic or antimony as such.

Control of sleeping sickness. Man apparently is the main source of
infection with T. gambiense, and in many areas, the human reservoir is
more than adequate for the maintenance of sleeping sickness. In various
parts of British West Africa, for instance, a general incidence of 1-6 per
cent has been observed and some villages show a much higher percentage
of infection. Distinct reduction in the incidence of human infection has
been obtained in West Africa by surveys and treatment of populations.
In addition, pentamidine has shown some promise in mass prophylaxis,
but there are serious practical difficulties in carrying out such programs
on a wide scale.

Lower animals also may serve as a source of human infection. Domestic
and wild animals apparently become infected with T. gambiense occa-
sionally and may act as reservoirs, but there is little evidence that wild
mammals are especially important in this respect. However, it appears
that various types of game, particularly antelopes, may serve as reservoirs
for T. rhodesiense, and cattle (72) also are known to become infected.
Recent progress in chemotherapy and prophylaxis, on a practical scale,
seems to promise not only elimination of the reservoir problem in
domestic animals, but also the general control of trypanosomiasis in
cattle and other domesticated herbivores.

Direct attack on tsetse flies helps to control sleeping sickness and also
to reduce the incidence of nagana, a serious disease which is caused by
T. brucei and has prevented the maintenance of domestic animals in
densely infested areas. Measures effective in controlling the flies vary
with the haunts and habits of the different species.^ Four species are
known to be important in transmission of sleeping sickness. The Gambian
variety is spread by Glossina palpalis, and in some areas at least, by
G. tachinoides. Rhodesian sleeping sickness is transmitted by G. morsitans
and G. swynnertoni. Glossina palpalis is seldom found far from water,
unless carried off while feeding on some animal (33), and G. tachinoides
has similar habits. Consequently, these species can be controlled in some
areas by clearing out bushes and low trees along rivers in the vicinity
of villages and river crossings. Such a cleared strip, extending for a half
mile or more on each side of a bridge or ford, offers fairly good pro-

^ The literature of the past forty years, dealing with ecological relationships, feeding
habits and breeding habits of tsetse flies, has been reviewed by Jackson (33a).

The Blood Flagellates 589

tection. Certain other species, such as G. morsitans and G. swynnertoni ,
have a wide range over bush land and must be controlled by other
methods. Destroying or driving out game may be effective, with reduction
in numbers or even practical elimination of tsetse flies in the area. This
method has been practiced successfully in Southern Rhodesia for the
control of G. morsitans. Aside from the decrease in sleeping sickness, the
reduction in incidence of nagana has favored the maintenance of cattle.
The local use of DDT sprays also seems to have some value. The spraying
of domestic animals not only protects them from flies, but may help also
in reducing the numbers of tsetse flies. In addition, the spraying of DDT
from airplanes has been effective in practical tests over bushy areas of a
few square miles.

A rather interesting delayed effect on the tsetse fly population may
be produced by hybridization. It seems that mating will occur readily
between subspecies or species in certain combinations, leading to hybrid
offspring which are often sterile or of low fertility. Biological warfare of
this type has been suggested as a possibility in controlling tsetse flies.

Chagas' disease

Distribution. This disease was first described by Chagas (12) in
Minas Geraes, Brazil, and for many years this appeared to be the only area
in which cases were at all common. Prior to 1937, only 113 cases had
been reported outside Brazil, although these were distributed through
Argentina, Guatemala, Panama, Peru, El Salvador, and Venezuela (77).
Increasing interest, coupled with the extensive use of xenodiagnosis and
complement-fixation tests, has revealed that the disease is far from rare in
South America. In Chile (50), xenodiagnosis has shown an incidence of
12 per cent in 12,581 individuals; complement-fixation tests, 17 per cent in
8,142. Surveys of smaller groups have indicated a comparable or higher
incidence in other areas: Argentina, 23-42 per cent; Bolivia, 6-31 per cent;
Brazil, 15-51 per cent; Uruguay, 6 per cent; Venezuela, 27-46 per cent.
Cases also have been reported in Colombia, Ecuador, Mexico, and
Paraguay. Such data accentuate the need for intensive surveys throughout
the known range of Trypanosoma cruzi. Perhaps Chaga's disease, once
considered geographically as well as historically Brazilian, will prove to
be more nearly an ail-American disease.

The causative organism. Trypanosoma cruzi is unusual in that, in the
vertebrate, the trypanosomal form invades various tissue cells in such
organs as the heart, striated muscles, central nervous system, thyroid
and lymph glands, bone marrow, ovaries, and testes. Invasion is fol-
lowed by metamorphosis into the leishmanial stage. Repeated fission then
occurs, so that the host cell is distended into a relatively thin membrane
("cyst") enclosing leishmanial stages (Fig. 12.3), the number of which
varies with size of the host cell (41). Metamorphosis into trypanosomal

590 The Blood Flagellates

forms, through an intermediate crithidial stage, is followed by rupture
of the "cyst" to liberate the flagellates into the body fluids. A minimum
of four or five days is required for this phase of the cycle.

In tissue-culture infections (38, 41, 57), trypanosomes of the type
usually seen in the blood also have been observed about the fifth day
after inoculation with T. cruzi. Muniz and de Freitas (47), using peri-
toneal fluid as a culture medium, have obtained stages similar to those
normally seen in the vertebrate. The presence of tissue cells apparently
is not necessary for transformation of the trypanosomal into the leishma-
nial form. On the other hand, metamorphosis into the trypanosomal
stage, observed in whole peritoneal fluid, does not occur in cell-free
fluid. Regular intervention of a crithidial stage between the leishmanial
and the trypanosomal forms in the vertebrate has been questioned by
Elkeles (25) who failed to find crithidial forms in his material. Also
interesting is the report that crithidial stages from cultures mostly dis-
integrate in normal serum, while leishmanial and trypanosomal forms
are not affected (44). However, all stages in the classical vertebrate cycle
have been observed in chick embryo tissue cultures (41).

Trypanosomal stages in vertebrate blood are ingested by an insect
vector and apparently change into leishmanial forms in the midgut.
Fission of leishmanial forms may occur, but metamorphosis into crithidial
forms takes place within a day or two. In the crithidial phase, multiplica-
tion occurs for some time before small metacyclic trypanosomes are
derived from crithidial stages in the hindgut. Completion of the insect
cycle requires about two weeks. In cultures, transformation of trypano-
somal into crithidial stages seems to depend upon some substance present
in washed erythrocytes, peptone and meat infusion. Hematin apparently
is not the significant factor (46). This "metamorphosis-inducing factor"
presumably would be required also in the digestive tract of the vector.

Transfer of the parasites to the vertebrate host involves discharge of
metacyclic trypanosomes from the hindgut as the bug ingests another
meal of blood. Large numbers of flagellates, sometimes as many as
2,500/mm3 (21), are deposited in the fecal material. If the trypanosomes
reach a break in the skin or the wound made by the insect, infection may
result. Or infection may follow contamination of intact mucous mem-
branes (the conjunctiva and the oral, rectal, and vaginal mucosae). In
addition to the usual transfer by vectors, rodents are known to eat
triatomids and may acquire the infection in this way. Such transfer would
be favored by the occasional survival of T. cruzi for several weeks in dead
insects (75). Carnivores probably can become infected by eating infected
rodents. Other direct methods apparently include lacteal and placental
transfer in mammals (23). Furthermore, there is always a possibility of
transferring the flagellates by blood transfusion in man.

Vectors and resenjoir hosts. The reported range of T. cruzi extends

The Blood Flagellates 591

from 41° S. (Patagones, Buenos Aires, Argentina) to 38° N. latitude
(Pinole, California). Within this range, the trypanosome has been re-
ported from a number of mammalian hosts and from a variety of insects.
In addition to Trypanosoma cruzi, the similar T. rangeli has been re-
ported from man, dogs, and reduviid bugs in South America (55a).

Panstrongylus megistus (Triatoma megista) was the first insect identi-
fied as a vector of T. cmzi (12). Since 1909, more than 30 species of

Fig. 12, 3. Trypanosoma cruzi. A. Stages in the digestive tract of the
vector; schematic (after Lent). B. Trypanosoma! stage from blood, x2800
(after Wenyon). C. Leishmanial forms in heart muscle, semidiagrannnatic,
xll50 approx. (after Chagas). D-H. Metamorphosis of the leishmanial into
the trypanosomal form, semidiagrammatic, x2300 approx. (after Wenyon).

triatomid bugs — belonging mostly to the genera Panstrongylus, Rhodnius,
and Triatoma — have been found naturally infected, the incidence rang-
ing from 16 to 92 per cent in different areas. Infected insects have been
observed in Argentina, Bolivia, Brazil, Chile, Colombia, Ecuador, French
Guiana, Guatemala, Honduras, Mexico, Panama, Paraguay, Peru, El
Salvador, Uruguay, Venezuela, and also in Arizona, California, and
Texas. In the United States, insect infection was first reported for
"Trypanosoma triatomae" in Triatoma protracta of California. Subse-

592 The Blood Flagellates

quently, infections have been reported in T. uhleri (37), T. longipes
(74, 76), T. protracta (76) and Paratriatoma hirsuta (76) from Arizona,
and in T. gerstaeckeri (51), T. protracta (74) and T. heidemanni (53)
from Texas. The incidence of infection has approximated 33 per cent
in representative triatomids of Texas (68a). The strain of T. criizi from
T. heidemanni is infective for man under experimental conditions (53).

The vectors of practical importance are the bugs which have become
adapted to life in association with man. Such bugs infest the primitive
huts and cabins common in rural South and Central America. During the
day the triatomids normally hide in cracks in walls, in bedding and
in furniture, as well as outdoors in piles of rubbish. Feeding on man or
other accessible mammals is usually a nocturnal activity.

The ecological distribution of most infected triatomids indicates that
T. cruzi is a natural parasite of various wild mammals which constitute
a permanent and extensive reservoir. The cat was the first reservoir host
to be recognized (12). Natural infections have since been reported in
anteaters, armadillos, bats, dogs, ferrets, foxes, opossums, porcupines, and
squirrels in various parts of Central and South America. In the United
States, the San Diego wood rat of California was the first recognized
reservoir (73). More recently, armadillos, house mice, opossums, and
wood rats have been found infected in Texas (52); wood rats and white-
footed mice, in Arizona (76).

Symptoms and pathology. Chagas' disease^ may occur in either an acute
or a chronic form. The acute type is most common in children under
10 years of age. The chronic form is the predominant type in adults, and
perhaps 70 per cent of these cases occur in the age group, 20-50.

Following an incubation period, which has lasted 10-12 days in experi-
mental human infections (13), characteristic symptoms appear. The acute
case usually begins with a fever which is often moderate and may be
irregular or remittent. Another common early symptom is facial oedema,
sometimes accompanied by a conjunctivitis so severe that one eye cannot
be opened. This effect has been attributed to invasion of T. cruzi by
way of the conjunctiva. In severe cases, the oedema may become extensive,
involving the extremities and sometimes most of the body. Adenitis is
characteristic and often includes the submaxillary, preauricular, cervical,
inguinal, and axillary glands. There is usually a detectable swelling of
the liver and spleen, especially the former. A progressive anemia and a
rapid pulse are commonly noted. Physical weakness, loss of appetite,
diarrhea and headaches are frequently noted in children. Occasional
cases, usually fatal, show symptoms of acute meningoencephalitis. The
cardiac signs are usually not well defined and do not show the marked
changes in rhythm reported for many chronic cases. However, there is

*A comprehensive discussion of symptoms and treatment has been published by
Laranja, Dias, and Nobrega (38a).

The Blood Flagellates 593

sometimes an acute myocarditis which may lead to heart failure. Mortality
in this form of Chagas' disease often exceeds 10 per cent.

The acute form of the disease runs a fairly short course. In surviving
patients, the trypanosomes usually disappear from the blood after a few
weeks and the oedema and other symptoms gradually subside. The
temperature often drops to normal. However, a low fever may persist
for some time after termination of the acute phase. Although there may
appear to be clinical recovery, the infection may persist, even for as
long as 16 years (22). In other words, a patient may simply progress from
the acute into the chronic form of the disease.

The usual chronic case is the cardiac type and, for practical purposes,
almost every chronic case may be considered a potential heart patient.
Symptoms vary with the extent of damage to cardiac muscle. While
myocardial damage is progressive, it is usually so slow that several years
may be required to produce indications of heart failure. Symptoms may
be mild or almost unnoticeable, the only evidence of cardiac damage
being that obtained with the electrocardiograph. Commonly, however,
such symptoms as dyspnea, palpitation, and abdominal pain in the
upper right quadrant are observed. Cardiac enlargement is often notice-
able, and irregularities in heart rhythm are common. Prognosis of the
cardiac case depends upon progress of the infection. Patients with slight
or moderate enlargement of the heart may be expected to live for several
years. For those with marked enlargement, the outlook is rather un-
certain. Mortality in chronic cases approaches 10 per cent, and the
majority of deaths occur before the age of fifty.

Pathological effects include degeneration of the invaded cells as well
as a cellular infiltration and eventual fibrosis of the invaded tissues.
Lesions of the heart, brain and liver are most characteristic, although the
flagellates have been found in most organs of the body in acute cases.
The heart shows a diffuse myocarditis. Among the muscle fibres there
is extensive infiltration of lymphocytes and macrophages, sometimes with
wide separation of the individual fibres, some of which show fragmenta-
tion and degeneration. Groups of leishmanial forms may be found either
in the muscle fibres or in large mononuclear cells and monocytes. Cellular
infiltration of the epicardium and endocardium is noted occasionally.
Multiplication of T. cruzi may also occur in skeletal muscle, which shows
much the same changes as cardiac muscle. Damage to the brain is ob-
served in some cases. Parasites may be found in neuroglia cells and
in large mononuclears in centers of inflammation scattered through the
nervous tissue. In the liver, the flagellates have been found in Kiipffer
cells. Fatty degeneration of liver tissue is sometimes noticeable and en-
largement of the liver is fairly common. The spleen also may be enlarged
to some extent, but parasites have been detected less commonly than
in the liver. Leishmanial stages also have been found occasionally in the

594 The Blood Flagellates

thyroid, adrenal glands, ovaries, and testes. Enlargement and congestion
of lymph glands are common effects but the flagellates seem to be absent
or rarely present in lymphatic tissue.

Diagnosis. Microscopic detection of T. cruzi is often easy enough in the
early acute stage. In addition, the precipitin test is useful in diagnosis
of early cases with mild symptoms (43a). In chronic cases, flagellates occur
in the blood in small numbers at most and the examination of blood
smears is usually negative. Culture methods have been used, and their
practical value may be increased by the addition of bacteriostatic agents
(penicillin, streptomycin) to suitable media (58a). Inoculation of blood
into laboratory animals may give good results, and xenodiagnosis also
is often effective. In xenodiagnosis, trypanosome-free insects are allowed
to feed upon suspected human cases. The subsequent appearance of
T. cruzi in the triatomids justifies a positive diagnosis. The success of
xenodiagnosis depends upon an adequate number of trypanosomes in
the blood, and some workers feel that the results are positive in an
unfortunately small percentage of chronic cases. Precipitin tests appar-
ently have little value in chronic cases (43a), but accurate diagnosis is
possible with complement-fixation tests (Machado-Guerreiro reaction) in
which the test antigen is prepared from cultures of T. cruzi (Chapter
XIV). Results already obtained in thousands of tests (18, 23, 45, 50) sug-
gest that complement-fixation is superior to other diagnostic procedures
and should be the method of choice for suspected chronic cases.

Therapy and control. Chemotherapy has been generally ineffective
and the problems of treatment are complicated by the usual occurrence
of the most acute cases in young children. A quinaldine compound,
Bayer 7602 Ac, is one of the few which have shown some activity against
T. cruzi. The drug has been tried in acute cases, but the results are
scarcely extensive enough for accurate evaluation. General methods of
treatment are those used for heart patients — rest, dietary control, and
other methods indicated for relief of heart failure.

Control of Chagas' disease is essentially an economic problem. Preva-
lence of the disease in rural areas of Central and South America is
mainly attributable to the infestation of native cabins and huts with
triatomid bugs. Long range control must depend upon improvements
in rural housing, since well built houses with adequate screening are
effective barriers to the vectors. In short-term control, the persistent use
of insecticides, although only an emergency measure at best, can be
reasonably effective on a limited scale.

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2. Adler, S. 1940. Mem. Inst. Oswaldo Cruz 35: 179.

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The Blood Flagellates 595

6. and 1930. Atin. Trop. Med. & Parasitol. 24: 197.

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24. Earle, H. 1946. Publ. Health Rep. 61: 1019.

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27. Hertig, M. and G. B. Fairchild 1948. Amer. J. Trop. Med. 28: 207.

28. and R. A. Fisher 1945. Bull. U. S. Army Med. Dept., No. 88: 97.

29. Hindle, E. and W. S. Patton 1927. Nature 119: 460.

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31. 1943. Biol. Rev. 18: 137.

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33. Hunt, A. R. and J. F. E. Bloss 1945. Trans. Roy. Soc. Trop. Med. i- Hyg. 39: 43.
33a. Jackson, C. H. N. 1949. Biol Rev. 24: 174.

34. Kellersberger. E. R. 1933. Amer. J. Trop. Med. 13: 211.
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36. Kofoid, C. A. and I. McCulloch 1916. Univ. Calif. Publ. Zool. 16: 113.

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38. , F. D. Wood and E. McNeil 1935. Univ. Calif. Publ. Zool. 41: 23.

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39. Lester, H. M. O. 1938. West Africa Med. J. 10: 2.

40. Meleney, H. E. 1925. Amer. J. Pathol. 1: 147.

41. Meyer, H. and M. X. de Oliveira 1948. Parasitol. 39: 91.

42. Most, H. and P. H. Lavietes 1947. Medicine 26: 221.

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45. and G. de Freitas 1944. Mem. Inst. Osivaldo Cruz 41: 303.

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51. Packchanian, A. 1939. Publ. Health Rep. 54: 1547.

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596 The Blood Flagellates

54. Pawlowsky, E. 1931. Centralbl. /. Bakt., Orig. 123: H.

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Med. 29: 453.

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XIII

Malaria

Introduction

The malarial parasites of man

The life-cycle of malarial parasites
Exo-erythrocytic phase
Erythrocytic phase

General features

Plasmodinm vivax

Plasmodium falciparum

Plasmodium malariae

Plasmodium ovale
Mosquito phase

Transfer of malarial parasites

The human malarias

Incubation periods

Prodromal symptoms

The paroxysm

Some characteristic effects of the

malarias
Duration of clinical attacks
Duration of infections
Relapses
Black water fever
Laboratory diagnosis
Chemotherapy
Control
Literature cited

INTRODUCTION

X^OR MANY CENTURIES malaria^ has been man's most important
protozoan disease. In spite of recent progress in malariology, the current
toll involves millions of cases annually, and malaria remains a serious
hindrance to economic and social development in various parts of the
world. In the eastern hemisphere, the history of malaria has included most
of Africa, southern and southeastern Asia, northern Australia, southern
Russia, England, and the European mainland bordering the Mediter-
ranean and Atlantic. Outbreaks have occurred as far north as Finland.
In the islands of the Pacific area, malaria extends southward from
Japan into the New Hebrides. In the western hemisphere malaria has
been prevalent from the central portions of South America to southern-
most Canada. Within this tremendous potential range, malaria has been
almost completely suppressed in a few regions and is gradually being

^ An encyclopedic review of malaria in all its phases, recently published under the
editorship of M. F. Boyd (13), will be invaluable to all who are interested in the
subject.

597

598 Malaria

brought under control in others. At the opposite extreme, there are still
areas in which perhaps 90 per cent of the population have malarial
infections every year. The history of malaria in North America (11, 44,
119) illustrates the results which may be expected from more or less
systematic efforts to control the disease.

The origin of malaria in North America is uncertain. Some authorities
suspect that the disease did not exist in the Am.ericas before their dis-
covery by Europeans. Others think that malaria was already endemic
when Europeans first reached America. At any rate, malaria has played
an important part in the history of North America for more than four
centuries. Introduction of slaves from Guinea into the West Indies was
begun about 1518 and the subsequently developed slave trade did much
to spread malaria, especially malignant tertian.

The early Spanish and French expeditions to the Gulf and south
Atlantic coasts probably brought malaria to North America, but some
time elapsed before the disease became important here. The settlers w^ho
reached Roanoke Island in 1585 apparently were not troubled by malaria.
However, those who came to Jamestown in 1607 had been recruited
mostly from the London area where malaria was then endemic. Within
four months more than 40 per cent of the settlers had died in what was
possibly an outbreak of malaria. By 1619, the slave trade also was begin-
ning to influence the malaria situation in Virginia. Along the Carolina
and Georgia coasts, malaria gradually increased with the establishment
of rice plantations, since the practice of flooding the fields provided
breeding grounds for anopheline mosquitoes. The cultivation of rice was
gradually extended southward. As a result, malaria flourished and the
prosperous coastal region soon became the most intensely malarial. The
disease also spread northward to New England, producing outbreaks in
Massachusetts in 1647, 1650, and 1668. Thus, a century before the out-
break of the American Revolution, malaria had become established
along the Atlantic coast from Massachusetts to Georgia. The Revolu-
tionary War introduced susceptible foreign troops into the malarial re-
gions along the coast and probably helped to spread the disease in the
southeastern area.

The close of the Revolution ended restrictions on migration. The
result was a westward movement of native easterners and immigrants.
Malaria accompanied the early migrants over the Appalachians and
beyond the Alleghenies to become endemic along the trails. Later
settlers passed through these malarial regions on their way westward
and helped to extend the disease into new territory. By 1850 most of the
United States — with the exception of the western plains and deserts,
northern Minnesota and Wisconsin, and the Appalachian and Rocky
Mountain highlands — was afflicted with malaria. The disease extended
from the Atlantic to the Pacific and from the Gulf of Mexico to the

Malaria 599

Canadian shores of Lake Erie. On the eastern coast, malaria was still
common in the Hudson River valley and along the shores of Long Island
Sound. Although it is uncertain when malaria reached the Pacific, the
early migrants evidently brought the disease overland to the lower
Columbia River valley and the Sacramento-San Joaquin valley in Cali-
fornia. An outbreak of chills and fever, possibly malaria, appeared among
the Indians of the Fort Vancouver region in 1829. Within three years,
about 90 per cent of the Indians had been exterminated. Another severe
epidemic, thought to have been malaria, attacked the California Indians
in 1833. The discovery of gold, which stimulated overland migration,
accelerated the introduction of new malarial strains.

In most of these malarial areas, epidemic outbreaks occurred only
during the warmer months of the year, but the situation in the southern
and southeastern states was more serious. The status of malaria in the
southern states is reflected in the fact that, from 1841 to 1847, 25.8 per
cent of the patients admitted to Charity Hospital in New Orleans were
suffering from malaria.^ For the United States as a whole, malaria seems
to have reached a peak about 1855, and the next few years showed a
gradual decline in incidence.

The Civil War interrupted this trend by introducing relatively sus-
ceptible Federal troops into malarial regions of the South. Movements
of the Confederate armies also contributed to the spread of malaria.
Information concerning malaria in the Confederate armies is scanty, but
it is known that the white Federal troops developed 1,163,184 cases of
malaria from May, 1861, to June, 1866.

Immediately after the Civil War, outbreaks of malaria increased in
some parts of the South, and it is likely that Federal troops also took
southern strains of the parasites home with them. Malaria broke out
in Connecticut, for example, and spread into Rhode Island and Mas-
sachusetts, the outbreak reaching a peak in 1881. In the South, increased
incidence of malaria was favored by unsettled conditions. Failure of
drainage systems extended the breeding grounds for mosquitoes, and
the partial failure of southern agriculture led to much undernourishment
with increased susceptibility to malaria. During the period, 1870-1900,
malaria was still important in many areas now more or less free from
the disease — Indiana, southern and northern Illinois, southeastern Kan-
sas, Ohio and Michigan along the shores of Lakes Erie and Ontario.
Cases were still fairly common in Philadelphia, on Manhattan Island,
and in Massachusetts.

Toward the close of the century, the incidence of malaria once more
began to drop. This trend has continued, and the recession has been
marked by the almost complete disappearance of malaria north of the

- By way of contrast, malaria accounted for only 0.58 per cent of admissions to the
same hospital for the period, 1933-1940.

600 Malaria

Ohio River and by its general restriction to the so-called "malarial belt."
This area extends eastward from the plains of Texas and Oklahoma and
includes the lower Mississippi valley and the Gulf Coast. Even within
this area, where malaria is mainly a rural disease, reduction in incidence
has been marked and continued reduction is to be expected with the
extension and improvement of control measures now in operation (1).
At present, however, endemic malaria in the southeastern states remains
a local problem of some importance as well as a potential threat to other
North American regions.

THE MALARIAL PARASITES OF MAN

Malarial parasites apparently were first seen by Meckel in 1847.
Their significance was not recognized until Laveran reported, in 1880,
that he was able to find them only in corpuscles of malaria patients. Five
years later, Marchiafava and Celli produced apparently the first cases
of experimental malaria by inoculating human volunteers with blood
containing the parasites.

Four species of Plasmodium are now generally recognized as parasites
of man: P. vivax, causing tertian (benign tertian) malaria; P. falciparum,
causing malignant tertian (subtertian, aestivo-autumnal) malaria; P. ma-
lariae, causing quartan malaria; and P. ovale, causing a comparatively
mild tertian type.

The relative incidence of these malarias varies in different parts of the
world. Benign tertian is primarily a disease of temperate and subtropical
areas and, while widely distributed throughout the tropics, it is ap-
parently uncommon in some tropical countries. Malignant tertian is pre-
dominantly tropical, although extending into temperate regions where
it is generally much less common than vivax malaria. Quartan malaria is
widely distributed but is usually a second-rate problem in comparison
with the dominant type (vivax or falciparum, malaria) in any given
region. However, there are exceptions, such as the Belgian Conge, in
which quartan malaria is especially prevalent. P. ovale produces a malaria
resembling but appreciably milder than benign tertian. Latent infections
tend to develop early and are less liable to relapse than in the other
malarias (103). P. ovale was observed by Craig (32) in American troops
returning from the Philippines, and was described later (33) as a variety
of P. vivax with a strong resemblance to P. malariae. The specific name,
P. ovale, was proposed by Stephens in 1922 after careful study of the
parasites (105). This species has since been investigated in additional
material (55, 77, 103, 108, 122). Strains have retained their characteristics
in passage through paretics (103, 122) and through mosquitoes (55, 103).
Examination of stained preparations convinced Craig (34) that P. ovale is
the same species which he had observed earlier. Infections have been re-
ported from certain parts of Africa (Belgian Congo, East Africa, Gold

Malaria 601

Coast, Nigeria, Sierra Leone, Uganda, West Africa), from Palestine,
western South America, and the Philippines. However, ovale malaria
seems to be rare wherever it has been found. The ecology of the Plas-
modhirn-Anopheles complex is not yet known well enough to explain
these differences in distribution, although the biothermal range of the
parasites may be a factor in some cases. P. vivax, for instance, apparently
does not develop in Anopheles quadrimaculatus at temperatures much
above 30° (111), whereas such temperatures seem to be satisfactory for
P. falciparum.

The evolutionary relationships of human malarial parasites and those
of apes are suggested by the apparent morphological identity of P. vivax,
P. falciparum, and P. malariae with P. schxuetzi Brumpt, P. reichenowi
Sluiter and Swellengrebel, and P. rodhaini Brumpt. In fact, it is debatable
whether these parasites of apes are specifically different from those of man.
Apparent physiological differentiation does indicate that P. vivax and
P. falciparum and their homologues in apes are distinct strains. P.
schxuetzi (85, 88, 89) and P. reichenoivi (6, 88) have failed to infect
artificially inoculated men, and P. falciparum has not infected chimpan-
zees under similar conditions (71). P. vivax, upon inoculation into
chimpanzees, occasionally produces a subpatent infection which persists
for several weeks without loss of virulence for man (85). The differentia-
tion of P. malariae and P. rodhaini seems to be less marked. Inoculation
of P. rodhaini into man has led to mild symptoms of quartan malaria and
the appearance of parasites showing the characteristics of P. malariae
(86). Strains of P. malariae also have proven infective for chimpanzees,
in which they retain their virulence for man (87).

THE LIFE-CYCLE OF MALARIAL
PARASITES

Exo-erythrocytic phase

For many years protozoologists were puzzled by the failure to
find malarial parasites early in an infection. The rather abrupt appear-
ance of parasites later on led to a suspicion, expressed clearly by Grassi
in 1900 and later by James (54), that sporozoites develop outside the
blood before invading erythrocytes. The confirmatory evidence is now
conclusive.

Experimental inoculations have indicated that parasites disappear
from the blood soon after introduction of sporozoites, are absent for
some time, and then suddenly reappear. Blood transfusions, within 7-30
minutes after inoculation of sporozoites, have transferred P. vivax and
P. falciparum to the recipients. Transfusions made after more than 30
minutes have given negative results (42). The blood does not become
infective again until about the eighth day with P. vivax (25, 42) and

602 Malaria

the sixth or seventh day with P. falciparum (23, 42). Likewise, P. cathe-
merium disappears from the blood of canaries within an hour after
inoculation and does not reappear until the end of the third day (118).
Coatney, cited by Sapero (90), has found also that large volumes of
blood from latent cases of vivax malaria fail to infect the recipients. The
latent phase thus resembles the prepatent period in that the blood con-
tains no demonstrable parasites. That the parasites are actually present
in the host is indicated by the subsequent relapse or primary attack.

The accumulation of morphological data, culminating in the ob-
servations of Shortt and his colleagues on primate malaria, gradually
brought to light this previously unknown exo-erythrocytic (E-E) phase

Fig. 13. 1. Exoerythrocytic schizonts (preerythrocytic phase) of Plas-
modium cynomolgi in hepatic cells of Macaca mulatta; schematic (after
Shortt). A. Stage recovered on fifth clay after inoculation by mosquitoes;
diameter of schizonts, lO-ll^tt. B. Vacuolated schizont recovered on the sev-
enth dav of the infection.

(Fig. 13.1). Non-pigmented E-E stages were perhaps first reported by
Raffaele (81), who found them in bone marrow endothelium of birds
infected with P. elongatiim and suggested (82) their origin from sporo-
zoites. Raffaele (83) later described non-pigmented P. vivax in human
bone-marrow five days after inoculation with sporozoites. From other
laboratories, E-E stages have been reported in P. gallitiaceum infections
(53, 57) and tissue cultures (48, 61), in tissue cultures of P. lophurae
(115), in canaries infected with P. cathemerium (121), in P. relictiim (31),
in P. mexicaniim of lizards (113), in monkeys infected with P. cynomolgi
(95, 97, 100), and from human liver early in a P. vivax infection (95, 99).
After inoculation of monkeys (Macaca mulatta) with sporozoites
of P. cynomolgi, E-E stages undergo growth and merogony in hepatic
parenchyma cells. By the fifth day the schizont approaches \\^ in di-

Malaria 603

ameter and at maturity, on the eighth or ninth day, measures 35-60[jl
(95, 100). Merogony results in about 1,000 merozoites, averaging I.Ijjl in
diameter (98). Some of these merozoites presumably enter red corpuscles
at the end of the prepatent period. The E-E cycle is continued in the
liver, where schizonts have been found in the fourth month of infection
and after a month of latency (97). Thus, for the first time in the history
of malaria, a complete description of the life-cycle became available for
a species parasitic in a mammal (96). Preliminary observations (95, 99)
have indicated that the E-E cycle of P. vivax closely resembles that of
P. cynomolgi.

The E-E stages of avian parasites have been observed mainly in
lymphoid-macrophage (reticulo-endothelial) cells. E-E stages of P. elonga-
tum develop primarily in wandering lymphoid-macrophage cells. Such
species as P. gaUinaceinn (53) and P. relictum (31) are found principally
in cajjillary endothelial cells, KiipfFer cells of the liver, and other fixed
cells lining sinuses of the bone marrow, lungs, and spleen. P. mexicanum
of lizards develops in both fixed and wandering cells (113). Two varieties
of E-E schizonts are found in P. relictum and P. gallinaceiini. Micro-
schizonts may produce about a thousand micromerozoites which are be-
lived to enter erythrocytes. Macroschizonts produce fewer and larger
macromerozoites which apparently invade lymphoid-macrophage cells
and continue the E-E cycle.

This E-E phase in malaria offers a logical explanation for prepatent
and postpatent periods, latency, and repopulation of the blood in re-
lapses. The direct invasion of tissue cells other than erythrocytes by the
inoculated sporozoites leads to a pre-erythrocytic cycle of growth and
merogony. This explains the failure to demonstrate parasites in the
blood during the prepatent period. However, the development of E-E
stages does not depend exclusively upon the introduction of sporozoites
in avian malaria. Inoculation of blood containing trophozoites of
P. galUnaceum, for example, may be followed by the appearance of E-E
stages in lymphoid-macrophage cells after 4-6 days (116). In fact, inocula-
tion of a single trophozoite into a chick has produced infections showing
E-E stages (39). Factors which eliminate erythrocytic stages, as the
primary attack passes into a latent phase in relapsing malarias, usually
do not eliminate the E-E stages. Accordingly, the E-E cycle continues
throughout latency and may persist for a long time, as indicated by
occasional relapse after a prolonged latency. It is more likely that
invasion of the blood is attempted periodically, only to fail under the
action of a stimulated malaricidal mechanism, than that the production
of "micromerozoites" destined for red corpuscles is completely suppressed
during latency. Sooner or later, however, the blood is repopulated in
relapsing malaria. The most logical explanation for the occurrence of
relapses is based upon immunological relationships (Chapter XIV).

604 Malaria

The erythrocytic phase

General features. The erythrocytic phase in a natural infection
normally is initiated by merozoites derived from E-E schizonts. Once
inside the red corpuscle, the young parasite usually develops a vacuole
which displaces the nucleus to the periphery, producing a "signet-ring"
stage. The ring, or young trophozoite, soon begins to grow. Binary fission
of ring stages has been suggested for P. vivax (3) and P. falciparum (50),
but this interpretation is not generally accepted. During growth, hemo-
globin is split into its protein component, which is used as food, and
hematin (76). As indicated by chemical and spectroscopic examination
(38, 47, 102), hematin is deposited in the retractile pigment granules of
erythrocytic stages. As estimated in infections of Macaca mulatta with
P. knowlesi, the hematin from about three-fourths of the corpuscular
hemoglobin is converted into pigment by the average parasite (74).

Nuclear division begins toward the end of the growth period. The
result is a multinucleate schizont, which undergoes merogony. The
resulting merozoites, with some residual cytoplasm containing the pig-
ment, are released into the blood stream. The pigment and other residual
material are ingested by phagocytes. Hence, the presence of pigment in
such cells indicates a malarial infection with a current or recently termi-
nated erythrocytic phase. The liberated merozoites which do not undergo
phagocytosis enter fresh red corpuscles, or sometimes reticulocytes, and
repeat the cycle of growth and merogony. The time required ranges from
about 24 to 72 hours in different species, with some variation among
strains of a single species. Length of the cycle in the St. Elizabeth strain
of P. vivax has averaged 43.4 hours; in the New Hebrides strain, 45.7
hours; and in the Baltimore strain, 41.5 hours (123).

During growth of the parasite, the corpuscle may undergo changes
which vary with the species of Plasmodium. Invaded corpuscles may
become enlarged, be distorted, become paler than the normal corpuscle,
undergo changes in reaction to the usual blood stains, or may show little
or no effect. Invaded corpuscles also tend to clump together in certain
malarial infections, such as P. knowlesi in monkeys (60). At the begin-
ning of the patent period, each corpuscle invaded by P. knowlesi becomes
coated with a thin self-adherent precipitate. As a result, such corpuscles
stick together. Since this coating substance is selectively ingested by
phagocytes, parasitized cells are rapidly ingested at this stage. As the
parasite-density increases, a fluffy precipitate forms, binding both invaded
and normal corpuscles into large masses. The blood now becomes thick
and sludge-like. Resistance of the larger masses causes the blood to flow
more slowly through the capillaries. Some of the smaller clumps, con-
taining both normal and invaded corpuscles, are ingested at this stage.
Later on, many of the larger clumps are broken up against the forks of

Malaria 605

arterioles into fragments small enough for phagocytosis. This leads to
substantial destruction of red corpuscles, parasitized and normal alike.

After several erythrocytic cycles of merogony, two types of gametocytes
normally appear in the peripheral blood, macrogametocytes usually being
more abundant than microgametocytes. Gametocytes may be expected,
in a primary attack, some time after a definite fever develops. In experi-
mental vivax malaria gametocytes are often present on the fifth day of
the patent period, usually persist during the clinical attack, and may still
be present for some time after the fever disappears. Infection of mos-
quitoes may be possible even after the blood contains less than 10 game-
tocytes/mm^. In laboratory-induced falciparum infections, gametocytes
are observed about the tenth day of the patent period and sometimes not
until after the primary attack subsides. A gametocyte density of 60/mm'^
is believed to be the minimum for infection of mosquitoes, and results
have usually been negative with less than lOO/mm^ (15, 26). In natural
infections with P. falciparum, gametocyte densities ranging from l/mm"^
to 90/mm-^ have proven infective for mosquitoes (125).

The factors responsible for differentiation of gametocytes are unknown.
Strains of P. vixiax vary in the numbers of gametocytes usually produced,
and the ability to produce gametocytes may decline during transmission
by blood inoculation exclusively (5). In addition, the ability to produce
gametocytes may be lost in an unnatural host. For instance, a strain of
P. elongatum, isolated from a sparrow and maintained in canaries and
ducks, stopped producing gametocytes at the fomteenth canary and the
fifteenth duck transfer. A return to sparrows failed to reverse the change
(72). Any strain inidergoing such a change under natural conditions
would necessarily perish at the end of its current infection. However, the
mere production of gametocytes in the vertebrate host does not insure
perpetuation of a strain. Mature gametocytes have a rather short life
in the vertebrate, perhaps only a day or so in the case of P. vivax (7),
and both types must be ingested by a suitable mosquito if the life-cycle is
to be completed.

The various stages in the erythrocytic cycle — rings, growth stages,
mature schizonts, stages of merogony, and gametocytes — differ morpho-
logically from species to species and furnish the major criteria for differ-
entiation of malarial parasites (36, 118a).

Erythrocytic phase in P. vivax (Fig. 13.2). The earliest stage in the
red corpuscle is a discoid form with a small nucleus. After development
of the usual vacuole, the ring measures about 2;^ in diameter and generally
contains a single chromatin inass, although sometimes two. A corpuscle
usually contains only one ring, occasionally two or three. Growing para-
sites appear as larger rings, and later on, as irregular amoeboid forms.
Refractile light brown pigment granules are deposited in the parasite
during growth. These inclusions show brownian movement in fresh

606 Malaria

Fig. 13. 2. Plasmodium vivax, semidiagrammatic: A. Uninvaded red cor-
puscle, in outline. B. Young ring. C. Marginal form. D. Older ring stage;
Schiiffner's dots indicated in cytoplasm of the corpuscle. E-G. Growth stages.
H-K. Binucleate, tetranucleate and multinucleate schizonts. L. Formation
of merozoites. M. Microgametocyte. N. Macrogametocyte.

preparations. With continued growth of the parasite, the corpuscle is
gradually enlarged to about 2-4 times the normal size and may be dis-
torted. Invaded corpuscles are usually decolorized and may show small
eosinophilic granules, Schiiffner's dots, possibly derived from the granules
of reticulocytes. The percentage of corpuscles showing Schiiffner's dots
may vary from patient to patient — for example, from 13.2 to 36.4 per
cent in specific cases (51). By the end of 36 hours, growth has practically
ceased and nuclear division is under way. At this point, the schizont
almost fills the enlarged corpuscle. After about 46 hours or less, depend-

Malaria 607

ing upon the strain, 12-24 daughter nuclei are present. After merogony,
rupture of the corpuscle liberates the merozoites, and the survivors enter
fresh corpuscles.

After a time, mature gametocytes appear in the peripheral blood. The
cytoplasm of the larger (8-10[j,) macrogametocyte usually stains a fairly
deep blue (Wright's stain); that of the smaller (7-8[ji,) microgametocyte,
a pale blue. The nucleus of the former is comparatively small and stains
a rather deep red, or sometimes bluish-red. The larger nucleus of the
microgametocyte is stained light red or pink. Numerous small brownish
pigment granules are distributed throughout the cytoplasm of the micro-
gametocyte. The larger and fewer pigment granules of the macrogameto-
cyte are often concentrated in the peripheral cytoplasm. Both types of
gametocytes practically fill the enlarged corpuscle at maturity.

Erythrocytic phase in P. falcipariwi (Fig. 13.3). Merogony is usually

Fig. 13. 3. Plasmodium falciparum, seniidiagrammatic: A. Young ring.
B. Ring ^v'ith two masses of chromatin. C. Double invasion of a corpuscle
in which Maurer's dots are indicated. D. Corpuscle with four young para-
sites. E. Older ring; corpuscle shows Maurer's dots. F. Two growth stages
in a corpuscle. G-I. Binucleate and multinucleate schizonts. J. Merozoites.
K. Undifferentiated gametocyte. L, M. Microgametocytes. N. Macrogameto-
cyte.

608 Malaria

completed in the visceral capillaries. Hence, the young rings, formed
shortly after merozoites invade fresh corpuscles, are normally the youngest
stages seen in blood smears. A characteristic feature is the fairly common
appearance of several, sometimes as many as six, small rings in one
corpuscle. The young ring usually measures not more than one-sixth the
diameter of the corpuscle. A thin film of cytoplasm encloses the vacuole
and the nuclear material is often seen as a small granule apparently
projecting from the outer surface of the ring. Occasionally, the chromatin
mass is rod-shaped, or there may be two or more small granules instead
of one. Early growth stages develop a thicker cytoplasmic layer, become
somewhat irregular in outline, and deposit dark brow-n or black pigment
granules. These larger and somewhat irregular rings, less amoeboid than
in P. v'wax, are probably comparable to half-grown forms of the latter.

The invaded corpuscle does not become enlarged, does not show
Schiiffner's dots, and may stain a little more intensely than the normal
corpuscle. Relatively coarse irregular eosinophilic granules i^Mmirer's
dots) are seen rather rarely, but small basophilic granules (dots of
Stephens and Christopher) may be expected somewhat more frequently.

In severe cases, particularly those with an unfavorable prognosis, all
stages of development may be found in blood smears. Ordinarily, how-
ever, grow'th stages remain in the peripheral circulation for about 24
hours and then drop out in the capillaries of the spleen, bone marrow,
and other internal organs where merogony is completed. Late stages
of growth and merogony have been observed also in dermal tissue smears
from children (79). This characteristic lagging of the older stages in
visceral capillaries is possibly the result of an acquired adhesiveness of
the invaded corpuscles, which tend to stick together and to the capillary
endothelium.

In the later stages the vacuole disappears and the cytoplasm appears
denser. This compact stage, shortly before nuclear division begins, is
not much larger than the largest rings seen in the peripheral blood. The
mature schizont, in which the pigment may occupy almost a third of the
cytoplasm, usually measures not more than two-thirds the diameter of
the corpuscle. Merogony produces 8-24 merozoites which measure 1.0[x or
less.

The early development of gametocytes occurs typically in the visceral
capillaries. As it reaches the peripheral circulation, the mature gameto-
cyte varies in form, even within a single strain (58, 59). Most commonly,
both types of gametocytes are sausage-shaped rather than crescentic. As
a rule, the two show fairly distinct differences, but there may be some
intergradation between gametocytes which are not quite mature. The
mature microgametocyte usually shows a fairly large, lightly stained
nucleus. The smaller nucleus of the macrogametocyte stains a little more
deeply. The cytoplasm of the macrogametocyte stains a rather deep blue;

Malaria 609

that of the microgametocyte, usually pale blue or lavender (Wright's
stain). The golden-brown pigment of the microgametocyte is usually
arranged rather loosely around or near the nucleus. The darker and
sometimes greenish-black pigment of the macrogametocyte usually forms
a compact aggregate partly or completely surrounding the nucleus. When
the gametocytes appear in the peripheral blood, the ring stages usually

Fig. 13. 4. Plasynodium malariae, semidiaqrammatic: A. Young ring. B-G.
Stages in growth. H-K. Binucleate and multinucleate schizonts. L. Mero-
zoites. M. Macrogametocyte. N. Microgametocyte.

decrease in number and it is not uncommon to find gametocytes as al-
most the only stages in blood smears. Mature gametocytes are believed to
live for only a few days in the blood (114). In some patients gametocytes
may disappear completely after a tune, and new ones are seen only after a
few more cycles of merogony (19).

Erythrocytic phase in P. malariae (Fig. 13. 4). This species differs from
P. vivax and P. falciparum in its longer asexual cycle and in the smaller
number of merozoites (usually 6-12) produced in merogony. The young

610 Malaria

rings, measuring from one-fourth to one-third the diameter of the
corpuscle, are similar to those of P. vivax. Although the cytoplasm and
chromatin are slightly coarser and the cytoplasm may stain a little more
intensely than in P. vivax, it is difficult or impossible to distinguish the
two at this stage. After a few hours of growth, the vacuole disappears and
the cytoplasm becomes compact. P. malariae rarely shows pseudopodia.

Fig. 13. 5. Plasmodium ovale: A. Normal red corpuscle. B, C. Young
parasites. D, E. Stages in growth; stippling of corpuscles indicated. F-I. Bi-
nucleate and multinucleate schizonts. J. Merozoites. K. Micrngametocyte.
L. Macrogametocyte. Semidiagrammatic.

Pigment is deposited early and is appreciably more abundant than in
the other parasites of man. Even half-grown parasites may show as many
as 30-50 dark or almost jet-black granules. Older growth stages may form
bands, stretching more or less completely across the corpuscle. Such bands
are usually considered a diagnostic feature because they are so much more
common in P. malariae than in the other species. In addition to the
bands, growth stages are often seen as compact masses without pseudo-
podia. The mature schizont approximates the diameter of the corpuscle.

Malaria 611

The invaded corpuscle is not enlarged and usually retains its normal
shape, color, and staining reaction.

The cytoplasm of the mature microgametocyte stains a light blue to
pale lavender (Wright's stain); that of the macrogametocyte, a fairly
deep blue. The nuclear material of the microgametocyte stains a light
pink and may occupy half the diameter of the parasite; that of the
macrogametocyte is more compact and is usually stained a bright red or
purple. The dark brown, or greenish brown, pigment granules are usually
scattered through the cytoplasm in the microgametocyte; more or less
restricted to the peripheral cytoplasm in the macrogametocyte. Each
gametocyte almost fills the unenlarged corpuscle.

Erythrocytic phase in P. ovale (Fig. 13.5). The small ring stages (108)
are usually coarser and stain more deeply than those of P. falciparum.
Both young and later growth stages resemble those of P. malariae in their
compact form, but are larger and contain slightly lighter, finer, and less
abimdant pigment granules. The corpuscle is somewhat enlarged and is
sometimes oval, occasionally rounded at one end and tapering at the
other, or often irregular or ragged in outline. Schriffner's dots are often
distinct and numerous, may be present even with early growth stages,
and may stain more intensely than in P. vivax infections. The mature
schizont is usually rather rounded and measures about three-fourths the
diameter of the corpuscle. Merogony often produces 8-10, but sometimes
as many as 14 merozoites.

The gametocytes are similar to those of P. vivax and P. malariae and
are usually not foimd in oval corpuscles. Enlargement of the corpuscle
and the presence of Schiiffner's dots distinguish the gametocytes of P.
ovale from those of P. malariae. Since they fill only about three-fourths of
the corpuscle, they are distinctly smaller than the gametocytes of P. vivax.

The mosquito phase'' (Fig. 13.6)

Although gametocytes establish infections only in the natural
hosts, maturation will take place even in vitro. Maturation of the micro-
gametocyte (exflagellation) involves nuclear division and budding to
form uniflagellate microgametes, the number of which is small — 4-8 in
P. vivax (84), 4-8 in P. jalciparum and 2-5 in P. malariae (124), accord-
ing to various reports. Exflagellation in vitro can usually be seen within
5-20 minutes after withdrawal of blood from a patient. The macrogame-
tocyte merely ruptures the enclosing corpuscle and then rounds up. The
possible occurrence of meiosis at this stage is suggested by observations
of MacDougall (62a). Fertilization is accomplished when a microgamete
penetrates the rounded macrogamete.

The zygote soon becomes an active ookinete which passes through

^Recent descriptions of the mosquito phase have been published for P. malariae
(65) and for P. vivax and P. jalciparum (64).

612 Malaria

the epithelium and rounds up beneath the outer layer of the mid-gut
within a period of 24-48 hours. Here the zygote begins to grow, apparently
enclosed in a thin "oocyst" membrane. Several to many oocysts may
develop in a single mosquito, usually without any appreciable effect on
the host. The rate of growth varies with the species of Plasmodium and
Anopheles and among different oocysts in the same mosquito, and is
influenced also by the external environment. Oocysts of P. vivax may
reach a diameter of 50[ji, or more in 1-2 weeks under favorable conditions.
Oocysts of P. falcipanitn reach a comparable size in two weeks or so,

/• '" \

■^::.::j

i;,'

Fig. 13. 6. Development of a malarial parasite in the mosquito; dia-
grammatic: A. Microgametogenesis, or "exflagellation.'" B. Macrogamete.
C. Ookinete. D. Zygote encysted on the wall of the stomach. E. Multinu-
cleate oocyst some time ijefore the formation of sporozoites. F. A sporo-
zoite.

while those of P. malariae grow a little more slowly (69). During growth,
rapid nuclear division occurs and sporogony finally produces thousands
of sporozoites. Eventual rupture of the "oocyst" releases the sporozoites
into the tissue spaces and some of them reach the mouth-parts of the
mosquito.

The time required for completion of the mosquito phase varies con-
siderably with environmental conditions. Under comparable laboratory
conditions, P. vivax and P. falcipanwi have required less than three
weeks and P. malariae about four (70), but these periods are shorter in
more favorable environments.

The sporozoites of P. vivax (25) and presumably the other species

Malaria 613

are able to penetrate the tissues of man after inoculation by a mosquito.
Within a short time, perhaps half an hour (42), they have entered
tissue cells to initiate the pre-erythrocytic phase.

TRANSFER OF MALARIAL
PARASITES

The relation of mosquitoes to malaria apparently was first suspected
by Lancisi, who stated in 1717 that marshes cause malaria through the
transformation of minute worms into mosquitoes which infuse a poisoned
liquid into the wounds they inflict. In 1883, Krieg and King again sug-
gested that malaria might be spread by mosquitoes, and this same opinion
was held by Laveran, Manson, Pfeiffer, and others. The theory was con-
firmed for bird malaria by Ross in 1898, and later in the same year, by
Grassi and his colleagues for P. falciparum. By the end of 1899, Grassi and
his associates had demonstrated similar cycles in P. malariae and P. vivax
and had transferred malaria to man from infected mosquitoes.

Aside from the dubious possibility that apes may serve as reservoirs, the
source of mosquito infection is a human reservoir with both types of
gametocytes. Young children are often the major source in tropical coun-
tries. For example, in Central African areas where malaria is endemic,
adults usually show only trophozoites of P. falciparum, or rarely a few
gametocytes of this species, whereas the gametocytes of all three major
species may be expected in children (92).

The ability to transmit human malaria is limited to anophelines. Of
these mosquitoes, only the "domesticated" types are usually important
because they are most likely to become infected. Once they have acquired
P. falciparum, mosquitoes may remain highly infective for about 10 days
after sporozoites appear but are no longer infective to man after 40 days
(27). The period of infectivity is somewhat longer for P. vivax (24). If
malaria is to be maintained in a human population, suitable mosquitoes
must be present in at least a minimal density. Hence, climatic conditions,
which affect both mosquito breeding and development of the parasites
in mosquitoes, exert an important influence on transfer of malaria.

Seasonal variations in incidence are more or less noticeable in malarial
regions. In general, vivax malaria is most common in the early spring
and through mid-summer in temperate regions. Initial attacks of malig-
nant tertian rarely occur before early summer and are usually to be ex-
pected in late summer and early autumn. Quartan malaria is more likely
to reach its peak in late autumn and early winter. The rainy and dry
seasons in the tropics are obviously major influences on seasonal inci-
dence. In temperate climates, however, changes in temperature may be
more important. For example, the biothermal range for P. vivax in
Anopheles quadrimaculatus is about 15-30°, with an optimum near 28°.
At temperatures above 30° development is inhibited, and the parasites

614 Malaria

are usually eliminated after 24 hours at 37.5°. P. vivax is more resistant
to low temperatures and arrested oocysts may pass the winter in mos-
quitoes and complete their development in the following spring (111).

In any season, a combination of circumstances may lead to a severe
outbreak of malaria, as opposed to the more common endemic condition.
Favorable climatic changes, permitting a marked increase in the anoph-
eline density, may produce such a result in a susceptible population
containing enough gametocyte carriers. An unusually wet season may
serve the purpose in an area normally too dry for dangerously heavy
mosquito densities. An unusually dry period might exert the same effect
by converting rapidly flowing streams into isolated pools suitable for
mosquito breeding. Importation of a prolific vector into new malarial
territory offering little hindrance to breeding may be followed by a severe
outbreak of malaria. Human activities, such as the migration of gameto-
cyte carriers into anopheline territory, also may start an outbreak in a
population relatively free from malaria.

Congenital transfer of malaria has been reported occasionally under
conditions which eliminate other possibilities, but there are no adequate
data for estimating the frequency of such transfer. Mechanical transfer
by inoculation of blood is a routine measine in malarial therapy of
syphilis and has occurred occasionally in blood transfusions. Storage of
blood in a blood bank for a week is not a complete safeguard against
the transfer of parasites in transfusions (94). Erythrocytic stages of P.
vivax may be stored, at —70°, in citrated or defibrinated blood for at
least five months without eliminating infectivity upon inoculation (91).
Mechanical transfer also may be accomplished by drug addicts through
common use of a hypodermic needle (75).

THE HUMAN MALARIAS

The incubation period

The number of sporozoites introduced is probably the most im-
portant influence on length of the incubation period (9, 16). The min-
imum for establishment of human infections is unknown, although
inoculation of single trophozoites has produced infections with P. know-
lesi in Macaca mulatta (30) and with P. cathemeriiim in canaries (104).
Relative susceptibility of the individual ranks next in importance. In
addition, the incubation period may vary with the strain of malarial
parasites. Climatic conditions also may have some significance, since
incubation periods in falciparum malaria may be relatively short from
October through December and relatively long during the winter months
(14).

The usual incubation periods are 14-18 days, with a common range of
^-35 days, for P, pivax; 18-21 days for P. ynaJariae; and 9-12 days for P.

Malaria 615

falciparum. In unusual cases symptoms may appear after much longer
periods. For example, mosquito inoculation of P. vivax has produced a
primary attack after 10 months (21). Likewise, under natural conditions,
autumnal mosquito inoculations sometimes do not lead to primary attacks
of vivax malaria until the following spring (101). A more unusual case,
involving an apparent "incubation" period of 19 years, has been re-
ported for P. maJariae (78). Such cases may be considered latent infections
and presumably involve an unusually prolonged pre-erythrocytic phase.
Somewhat similar to these latent infections, occasional cases show very
mild symptoms; unless treated, these may develop into typical cases.

Mixed infections may introduce complications. In experimental mix-
tures of P. vivax and P. falciparum (19), the latter was the first to populate
the blood and the early symptoms were those of falciparum malaria. P.
vivax later increased in number, while the population of P. falciparum
decreased rapidly, and a typical attack of benign tertian occurred next.

Prodromal symptoms

In a typical primary attack of vivax malaria, mild symptoms ap-
pear a day or so before the patent period. These include nausea, loss of
appetite, constipation, apathy, and sometimes insomnia. The mouth often
feels dry and the tongue may be thickly coated. Headache, muscular
pains, and aches in the joints soon develop, and there may be sensations
of chilliness. Comparable early symptoms may appear in quartan malaria,
but usually not before parasites are detectable. Such prodromal symptoms
are sometimes seen in falciparum cases but are often so insignificant
that the attack shows a sudden onset, particularly in partially resistant
individuals.

The paroxysm

Prodromal symptoms are followed by a series of paroxysms, the
length of the series varying with the patient and the type of malaria. A
clinical reaction may occur in naturally induced vivax malaria when the
parasite density approximates lO/mm^ (8), whereas recognizable symp-
toms may accompany the first appearance of parasites in the peripheral
blood in falciparum malaria (23). The complete paroxysm includes the
rigor (cold stage, chill), the fever stage, and the sweating stage.

The rigor usually begins with a chilly sensation, in the hands and feet
at first and more general later on. Acute shivering may follow, with
cyanosis of the lips and fingers tips. Rapid pulse and respiration, and
sometimes severe headaches, may be expected. Nausea and vomiting also
are fairly common. A rigor rarely initiates the first paroxysm in vivax
malaria, may not occur until after several days of intermittent fever, and
seldom precedes a peak temperature of less than 102° F. Appearance of
a rigor in the first paroxysm suggests previous experience with malaria

616 Malaria

or else a relapse. As the infection progresses, duration of the vivax rigor
may increase from 5-10 minutes to 1-3 hours. In quartan malaria, rigors
usually begin with temperatures of less than 100° F., although a tempera-
ture of 103-104° sometimes follows the first one. However, a rigor is not
always present in the quartan paroxysm. The paroxysm of malignant
tertian is often initiated by a sensation of chilliness, and in perhaps less
than a third of the cases, by a definite rigor. The factors inducing the
rigor are not definitely known. The appearance of specific toxins has not
been demonstrated, and similar symptoms can be induced by intra-

105-1

103

101

no r-

97-

mQlignani f erf fan -
guar fan
f erf /an

DAYS

Fig. 13. 7. Diagrammatic comparison of temjierature curves in malignant
tertian, quartan, and tertian malarias.

venous injection of foreign proteins or denatured normal serum. The
merozoites and residual protoplasm released at merogony presumably
could serve as such foreign proteins.

The fever stage overlaps the rigor. The temperature begins to rise
well before the end of the chill, or even near its beginning. As a result,
the patient soon feels hot instead of cold. Although duration of the fever
is variable, most of the surviving merozoites have penetrated corpuscles
before the fever disappears. In the vivax paroxysm, the fever may last
for 3-6 hours and the temperature curve (Fig. 13.7) generally shows an
abrupt rise, a sharp peak and a fairly rapid decline. A progressive de-
crease in the temperature peaks may be expected toward the end of a

Malaria 617

clinical attack. In infants and young children with vivax malaria the
fever is commonly continuous or remittent, without showing the perio-
dicity characteristic of the adult case. The fever stage in quartan malaria
is similar to that in tertian but the quartan temperature curves typically
show a steeper rise and fall. In malignant tertian a fever of 12-24 hours
is not uncommon. The temperature curves usually show fairly broad
peaks, sometimes broken by partial remission of the fever. In general,
the paroxysms are less clearly defined than in benign tertian and quartan
malaria and temperatures may remain above normal for as long as two
days or so.

The sweating stage of the tertian paroxysm sets in after the tempera-
ture has started to drop and may last 2-4 hours. The patient usually im-
proves rapidly and feels fairly comfortable within a few hours. The
sweating stage in quartan malaria is similar, but fails to bring such rapid
improvement. A subnormal temperature may persist for a day or two.
Sweating is usually less noticeable in malignant tertian, but the stage is
accompanied by subnormal temperatures as in the other malarias.

A tertian periodicity, with paroxysms on alternate days, is characteristic
of uncomplicated infections with P. vivax, P. ovale, and P. falciparum.
A quartan periodicity, with paroxysms at intervals of about 72 hours,
occurs in P. malariae infections. The exact periodicity may vary within
a species, however, and average intervals of 43.4, 45.7 and 41.5 hours
have been noted for three strains of P. vivax (123). Periodicity is influ-
enced also by the occurrence of double or multiple infections. A double
infection, for instance, may include strains undergoing merogony on
alternate days and producing quotidian paroxysms. In experimental
tertian malaria, quotidian paroxysms may occur even after a single in-
oculation (10). Such a course may change abruptly into a tertian one, or
a new cycle may develop in a tertian course to produce quotidian
paroxysms. Similarly, a double quartan course may become quotidian,
or a quotidian periodicity may revert to a quartan series. Naturally in-
fected patients, in contrast to those with induced malaria, show fewer of
these irregularities in quartan and tertian malaria. In malignant tertian,
on the other hand, changes and irregularities are common, and tempera-
ture curves sometimes suggest the lack of any basic organization. A simple
tertian course in falciparutn malaria may even indicate some degree of
resistance. The origin of these irregularities is uncertain. The appearance
of a new cycle in a tertian or quartan course might be attributed to fresh
invasion from an exo-erythrocytic reservoir, but the change from a quo-
tidian to a simple tertian or quartan series is another problem.

Some characteristic effects of the malarias

Anemia is inevitable in clinical attacks and normal red cell counts
are not to be expected except perhaps during the early erythrocytic

618 Malaria

phase. A marked anemia may occur within a few days, especially in
malignant tertian in which the parasitemia has reached 925,999/mm3 in
extreme cases (109). There is sometimes a temporary increase in leuco-
cytes during a paroxysm but such an increase, if it occurs at all, is fol-
lowed by a reduction. After some days in an uncomplicated infection
there is usually a leucopenia (3,500-4,500 leucocytes/mm^, or sometimes
less). The accumulation of pigment in leucocytes, mostly the large mono-
nuclears, is characteristic. Ingested pigment is to be expected also in the
lymphoid-macrophage cells in the viscera.

Enlargement of the spleen is another characteristic effect, so much so
that the "splenic index" has been used to advantage in malaria surveys.
Enlargement of the spleen in vivax malaria usually is not evident in
white adults until after a week or so of the patent period (110), but the
splenic response is more rapid in infants and young children. Spleno-
megaly is much less noticeable in quartan than in tertian malaria, usually
develops rather slowly in whites, and may be absent in negro patients.
Jaundice is fairly common and may be marked in some malignant tertian
cases. The condition may be expected in acute vivax malaria with a red
cell count dropping below 2,000,000 during the first week or ten days,
but is seen less frequently in slowly developing cases.

In contrast to the other types, falciparum malaria may be considered
potentially lethal, although some patients seem tolerant to fairly heavy
infections and may show comparatively mild attacks. In the simpler
falciparum cases, no particular organ system is extensively involved.
Even without localization, however, the parasites may multiply rapidly
enough to overwhelm the patient unless the infection is checked.

Localized, or pernicious, malignant tertian occurs primarily in the
tropics and in areas where the disease is highly endemic. Pernicious cases
are generally severe and their development is favored by malnutrition,
fatigue, heat prostration, drug addiction, and the like. The cerebral
varieties involve localization in the nervous system. Effects include delir-
ium, convulsions, failure of muscular coordination, amnesia, difficulties
in speech, partial paralysis, indications of meningitis, or simulation of
acute intoxication. The clumping of invaded corpuscles may lead to
thrombus formation in cerebral capillaries, sometimes with resulting coma
or death. The visceral (or algid) types of pernicious malaria involve
localization in the digestive and circulatory systems primarily. The sur-
face of the body feels cold. Symptoms may suggest acute appendicitis,
bacterial dysentery, cholera, gastritis, peritonitis, or typhoid fever. Cir-
culatory involvement often leads to angina-like pains and symptoms of
thrombosis, with indications of heart failure. However, fatal cases may be
the result of vascular collapse more often than of cardiac failure (67).
The adrenal glands may be invaded, sometimes with degenerative changes

Malaria 619

in the cortex, and adrenal insufficiency has been considered a possible
factor in fatal pernicious malaria. Involvement of the respiratory system
may lead to indications of bronchitis or pneumonia. Effects on the uro-
genital system may suggest nephritis, orchitis or oophoritis, and haemo-
globinuria or haematuria also may develop.

As complications of pregnancy, the malarias may be blamed for a
considerable amount of fetal, neonatal, and maternal mortality. Even
benign tertian is important in this respect and malignant tertian is par-
ticularly dangerous, both to the mother and to the fetus. The later the
falciparum infection occurs in pregnancy, the less is the chance of carry-
ing the fetus to term, and if the child is born alive it sometimes lives
for only a few days.

Duration of clinical attacks

Duration of the vivax attack varies with the strain of parasites,
with resistance of the individual, and apparently with the season of the
year. Under comparable experimental conditions, July-September cases
have shown longer clinical courses than January-March cases (22). Clin-
ical symptoms have mostly disappeared in experimental tertian when the
parasite density drops to about 100/mm^. In quartan malaria, duration
of clinical attacks has averaged 170 days in naturally inoculated whites
and 76 days in negroes. After artificial inoculation, the corresponding
averages were 81 and 53 days (9). Duration of attacks in experimental
falcipariwi malaria has averaged about II days, with a maximum of 36
(14). As in benign tertian, the length of the attack may vary with the
season, being relatively long during the fall and shorter during the winter
months.

Duration of infections

The duration of untreated infections is uncertain, in view of the
occasional occurrence of prolonged latency. Even in induced malaria it
is difficult to determine the end-point because failure to find the parasites
does not guaiantee the absence of a latent infection. In uncomplicated
vivax malaria, the attacks usually become less and less severe and eventu-
ally cease. However, the infection sometimes persists for at least two years
after the primary attack. In falciparum malaria, there are grounds for
believing that infections usually last no more than six months (12, 56).
P. malariae shows greater persistence and latent infections may last for
five years or more after the primary attack (106).

Relapses

Although the tendency of P. ovale infections to relapse is com-
paratively slight (68, 103), relapses are characteristic of the other human

620 Malaria

malarias. Recrudescences, which occur shortly after recovery from a
primary attack, are sometimes distinguished from relapses following a
fairly long period of latency.

The greatest tendency to relapse is noted in tertian malaria. In many
experimental infections (18), relapses have occurred after most primary
attacks interrupted by small doses of quinine, and after half of the spon-
taneously terminated primary attacks. The tendency to relapse varies
with the strain of P. vivax (18). Seasonal factors also may be significant,
since July-September cases have shown a greater tendency to relapse than
the January-March group (22). The pattern of relapse also varies with
the strain (29). The course of tertian malaria often involves a series of
"recrudescences" and then a period of latency, which may last 6-12 months
before the next relapse occurs. In stubborn cases, this sequence may be
repeated for several years after the primary attack. The St. Elizabeth
strain (United States) usually does not show marked recrudescence, but a
prolonged latent period and eventually a relapse are characteristic. The
Chesson strain (New Guinea) usually shows fairly regular renewals of
activity without prolonged latency. Relapses of the St. Elizabeth strain
seem to coincide approximately Avith the mosquito season in the southern
states. The Chesson strain is native to a region in which mosquitoes are
reasonably available throughout the year.

In jalciparum malaria, renewed activity shortly after the primary at-
tack is generally to be expected, but relapses after long latency are much
less common than in benign tertian. The tendency to produce "short-
term" relapses may vary with the strain, and the incidence of relapses
has ranged from 8.3 per cent (11) to 80.6 per cent (56) in experimental
infections.

Although infections with P. malariae sometimes last a long time and
relapses occur after apparently long periods of latency, little is known
about the pattern and incidence of relapses.

Malariologists now believe that relapses involve two different phases
in the life-cycle. A reactivation (recrudescence) occurring shortly after
the primary attack depends upon renewed multiplication of erythrocytic
stages not yet eliminated from the blood. The relapse following a long
period of latency involves a persistent exo-erythrocytic infection which
eventually supplies the merozoites for repopulation of the blood.

Blackwater fever

Blackwater fever occurs most frequently in individuals coming
from malaria-free areas into a region where malaria is highly endemic.
The specific cause is unknown. When it occurs, blackwater fever follows
prolonged cases of malaria which have been imperfectly treated, and
there is good evidence that P. jalcipariwi is always, or nearly always, the
only species involved. Inadequate dosage with quinine is not essential to

Malaria 621

development of the condition, since blackwater fever lias followed treat-
ment with other drugs such as atebrin. The onset is often marked by
rigors, bilious vomiting, jaundice, black urine, and general prostration.
The characteristic feature is intravascular hemolysis, followed by passage
of hemoglobin in the urine. The pathological effects are essentially those
of severe chronic malaria, with the complication of sudden and extensive
hemolysis. The only known preventive is adequate treatment of patients
in areas where blackwater fever is known. An extensive treatise on black-
water fever has been published by Stephens (107).

Laboratory diagnosis of malaria

Final diagnosis depends upon the detection of parasites in ma-
terial from the patient.^ Although certain serological techniques (Chapter
XIV) seem to be useful, they are not yet adequate substitutes for direct
demonstration of the parasites. Smears of bone marrow, obtained by
sternal puncture, have been used for diagnosis of chronic malaria, but
blood films are the preparations usually examined.

Both thick and thin films are often prepared for examination. Most
routine descriptions of the parasites are based upon thin-film prepara-
tions. The thick film, in which a large drop of blood is spread over a
small area of the slide, is the more efficient, both in saving time and in
insuring detection of the parasites. Since the thick-film techniques — the
rapid method of Field (45), the method of Barber and Komp (2), and
others — destroy the corpuscles, the technician must depend upon careful
microscopy and morphology of the parasites. With either type of films,
it may be necessary to examine slides prepared at successive intervals if
parasites are not detected at first. In any case, it is not sound practice to
base a negative report upon examination of thin films alone if malaria
is suspected.

Chemotherapy

Although malariologists seem to agree that P. falciparum should
be eliminated promptly, opinions have differed concerning the treatment
of quartan and benign tertian cases. Advocates of the "short-term" treat-
ment have disapproved attempts to eradicate P. vivax and P. malariae
during primary attacks, preferring clinical prophylaxis and suppressive
treatment during residence in malarial territory. Such recommendations
are based upon the assumption that individuals with sub-clinical infec-
tions will gradually develop an effective immunity, whereas prompt elim-
ination of the parasites will leave the individual susceptible to reinfection.
In presenting objections to the short-term treatment, Craig (35) has
stressed the shortcomings of active immunization in India, where the

* Comprehensive discussions of laboratory diagnosis have been published by Craig
(36) and Wilcox (118a).

622 Malaria

native population has had many generations in which to practice "pre-
munition" relatively undisturbed by chemotherapy.

From the practical standpoint, malarial therapy faces two problems
(90): suppression of the erythrocytic phase in both clinical prophylaxis
and clinical cure of primary attacks and relapses; and the elimination of
exo-erythrocytic stages. Solution of the second problem is much the more
difficult, but there is no true cure until exo-erythrocytic stages have been
eradicated. Suppressive therapy brings relief to the patient, and by elim-
inating erythrocytic stages for the moment, temporarily eliminates a
source of infection for mosquitoes. However, effective suppressive treat-
ment is often followed by relapse, especially in vivax malaria. In terms
of modern concepts, such a suppressant has been ineffective against exo-
erythrocytic stages.

Quinine, the traditional malaricidal drug, is a good suppressant, al-
though its activity may vary with the strain of parasites and especially
so in falciparum, malaria. Atebrin (atabrine, mepacrine, quinacrine) is
active against erythrocytic stages of all species, and is valuable also in
clinical prophylaxis. Both atebrin and quinine cause morphological
changes in trophozoites of P. vivax, as seen in blood films a few hours
after treatment (65a), but neither has any marked action on exo-erythro-
cytic stages. Chloroquine, which is well tolerated even by infants and is
an effective suppressant for vivax, falciparum, and quartan malaria (4,
40), also has little effect on exo-erythrocytic stages. Pentaquine, although
a poor suppressant, shows apparent activity against E-E stages (62).
Plasmochin (pamaquine, plasmoquine) is just a fairly satisfactory sup-
pressant for tertian and quartan malaria, but seems to be active against
E-E stages (62). However, the effects of plasmochin may vary with the
strain, in view of its failure to prevent relapse in mosquito-induced in-
fections with the Chesson strain of P. vivax (37). In fairly heavy dosage,
plasmochin is said to be an effective prophylactic against P. vivax and P.
falciparum (93), but the toxicity of the drug would seem to limit its
usefulness for this purpose. Paludrine (proguanil, chloroguanide), which
seems to be a fairly good suppressant for vivax and falciparum malaria
and is particularly active against pre-erythrocytic forms of P. falciparum
(63, 66), has an interesting delayed effect on both species. Gametocytes
mature in the treated patient and syngamy occurs after ingestion of
gametocytes by the mosquito. However, the resulting oocysts fail to ma-
ture in the vector (43, 63). Camoquin, another new drug, seems to be
about as good a suppressant as chloroquine and has given excellent re-
sults when administered in a single dose for moderate to heavy infections
with P. vivax and P. falciparum (49). The advantages of effective treat-
ment with a single oral dose are obvious.

The ideal malaricidal drug Avould be one harmless enough for use in
infants, active enough for the prompt suppression of acute infections,

Malaria 623

and effective enough against exo-erythrocytic stages to insure complete
prophylaxis and true cure. The search for such a drug is still in progress.^
At present, the closest approach to the desired effects has been obtained
with combinations of drugs. For example, the combination of quinine
and pentaquine produces a low relapse rate in tertian malaria (73, 112).
Likewise, quinine and plasmochin, as well as quinine and paludrine,
have real value in clearing up relapsing cases (73). Such combinations
as paludrine and atebrin, paludrine and chloroquine, and atebrin and
chloroquine also have been used in malignant tertian.

Control

Mass treatment and prophylaxis, with an ideal drug administered
to a docile or thoroughly cooperative population, probably could elimi-
nate malaria from a given area without distvirbing the local mosquitoes.
Since the perfect drug is not yet available and the human factor is rather
unpredictable, the most effective method for completely controlling ma-
laria involves the reduction of anophelines to such a low density that the
disease cannot be maintained in a given area. Successful results within
the shortest possible time would require a combination of mosquito
control and suppressive chemotherapy.

Long range measures, such as drainage of marshy areas and the stock-
ing of natural and artificial lakes with fish which eat mosquito larvae,
are effective deterrents to the breeding of mosquitoes. Treatment of stag-
nant pools and marshes with larvicides can be very effective where local
conditions permit such measures. Adequate screening of houses tends to
prevent contact of mosquitoes with man. In addition, some of the newer
insecticides promise striking results in the direct attack on adult mos-
quitoes. For instance, residual DDT spray has been tested in several dis-
tricts of Bombay Province with a population of about 1,600,000. After
one year of spraying human and animal shelters at intervals of 6-8 weeks,
the apparent incidence of malaria was reduced by 40-70 per cent in dif-
ferent areas (117). Practical tests in several tropical towns have shown
that malaria can be controlled to a satisfactory degree by combining the
use of DDT with suppressive chloroquine therapy (41). With the sys-
tematic application of available methods based upon sound knowledge
of anopheline ecology, the practical elimination of human malaria now
seems to be a distinct possibility. Attainment of this goal is retarded
mainly by economic factors.

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624 Malaria

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74. Morrison, D. B. and H. A. Jeskey 1948. ./. Nat. Mai. Soc. 7: 259.

75. Most, H. 1940. Amer. J. Trop. Med. 20: 551.

76. Moulder, J. W. and E. A. Evans 1946. /. Biol. Chem. 164: 145.

77. Miihlens, P. 1934. Arch. Schijjs-u. Tropenhyg. 38: 369.

78. 1937. Muench. Med. ]Voch. 84: 5.

79. Peel. E. and L. van Hoof 1948. Ann. Soc. Beige Med. Trop. 28: 273.

80. Porter, R. J. and C. G. Huff 1940. Amer. J. Trop. Med. 20: 869.

81. Raffaele, G. 1934. Rix>. di Malarial. 13: 331.

82. 1936. Riv. di Malarial. 15: 309.

83. 1937. Riv. di Malarial. 16: 413.

84. 1939. Riv. di Malarial. 18: 141.

85. Rodhain, J. 1939. C. R. Soc. Biol. 132: 69.

86. 1940. C. R. Soc. Biol. 133: 276.

87. 1948. Amer. J. Trop. Med. 28: 629.

88. and G. Muyelle 1938. C. R. Soc. Biol. 127: 1467.

89. and 1939. C. R. Soc. Biol. 131: 114.

90. Sapero, J. J. 1947. Amer. J. Trop. Med. 27: 271.

91. Saunders, G. M., D. W. Talmadge and V. Scott 1948. /. Lab. Clin. Med. 33: 1579.

92. Schwetz, J. 1949. Trans. Roy. Soc. Trop. Med. & Hyg. 42: 403.

93. Shannon, J. A. 1948. Proc. -fth Intern. Congr. Trop. Med. & Malaria 1: 714.

94. Sharnoff, J. G., J. Geiger and I. Selzer 1945. Ainer. J. Clin. Pathol. 15: 494.

95. Shortt, H. E. 1948. Proc. -ith Intern. Congr. Trop. Med. & Malaria 1: 607.

96. 1948. Trails. Roy. Soc. Trop. Med. & Hyg. 42: 227.

97. and P. C. C. Garnhara 1948. Brit. Med. J., 1225.

98. and 1948. Trans. Roy. Soc. Trop. Med. & Hyg. 41: 785.

99. — , and G. Covell 1948. Brit. Med. J., 547.

100. , and B. Malamos 1948. Brit. Med. J., 192.

101. Shute, P. G. 1939. /. Trap. Med. & Hyg. 42: 201.

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104. Stauber, L. A. 1939. /. Parasitol. 25: 95.

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626 Malaria

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Amer. J. Trop. Med. 28: 303.

XIV

Immunity and Resistance

Natural resistance

Acquired resistance
Active immunization

Leishmania

Trypanosoma

Babesia

Plasmodium

Coccidia
Passive immunization

Trypanosoma

Plasmodium

Factors involved in acquired resistance

Antibodies

Defensive mechanisms in trypanosome

infections
Defensive mechanisms in malaria

Serological diagnosis of infection
Agglutinin tests
Precipitin tests
Complement-fixation tests
Skin tests
Adhesion tests

Serological differentiation of species

Literature cited

NATURAL RESISTANCE

usT AS THERE ARE natural infections with Protozoa, so there
appear to be natural immunities to protozoan parasites, immunities
which probably should be attributed to biological incompatibility. A
natural immunity may be absolute, or it may be a relative immunity
which can be overcome by massive inoculation or by debilitating factors.

The degree of natural resistance to a given parasite commonly varies
with the host and may vary widely even within a single genus, as dem-
onstrated in Peromyscus (128). In highly susceptible species of Peromyscus,
infection with Trypanosoma hrucei is usually fatal within a week. In
another group of species, the infection is subacute and survival of the
mice averages about three months. In a third group, the infection runs
a chronic course and most of the mice apparently recover.

Within a single species, racial differences in natural resistance may
exist, although little is known about this aspect of immunity against
Protozoa. Differences between the apparent resistance of Europeans and
that of natives to tropical diseases have often been emphasized as ex-
amples of racial differences in immunity. These differences also have

627

628 Immunity and Resistance

been attributed to selection. Thus, native children may acquire endemic
diseases at an early age, with resulting death of the weakest. Conse-
quently, the survivors in each generation represent a selected group with
a resistance greater than that of the average incoming foreigner. So far
as malaria is concerned, some authorities believe that differences in the
immune status of racial groups depend primarily upon frequency of
infection. Whether this explanation accounts for the relatively higfi re-
sistance of American Negroes, perhaps even young children (32), to
Plasmodium vivax has been questioned. However, if there actually is such
a racial difference in resistance, it apparently does not extend to Pacific
strains of P. vivax since the susceptibility of American Negroes to Pacific
vivax malaria does not differ significantly from that of American whites
(38). Nevertheless, an apparently valid racial difference in susceptibility
to Plasmodium, knowlesi has been reported by Milam and Coggeshall
(115). Experimentally infected Negroes showed appreciably longer in-
cubation periods than whites and their blood remained infective to mon-
keys for a shorter time.

Individual variations in natural resistance also have been reported,
particularly in the induction of therapeutic infections with malarial para-
sites. If the possibility of previous experience with the parasites can be
eliminated, refractory individuals presumably exhibit an effective degree
of natural resistance.

The factors influencing occurrence and degree of natural resistance
are mostly unknown, since only a few cases have been investigated from
this standpoint. In some instances, body fluids of the host play an impor-
tant part in the fate of incoming parasites. Thus, coccidian oocysts pass
unchanged through the digestive tract of a naturally immune animal,
whereas hatching is apparently facilitated by the digestive fluids in a
susceptible animal (4). Occasionally, resistance may depend upon para-
siticidal properties of the body fluids. For example, after inoculation into
various cold-blooded vertebrates, Trypanosoma evansi fails to appear in
the blood of such animals as the eel, the serum of which destroys the
flagellates in vitro (104). An analogous factor presumably is responsible
for resistance of chickens to Plasmodium cathemerium. Immersion of
sporozoites in hen's blood for 30 minutes or more greatly reduces their
infectivity for canaries (21), which are normally susceptible to this
parasite.

For individual organisms, age is an important factor in resistance. In
general, young animals are more easily infected and usually show more
severe symptoms than older animals. Trypanosoma lewisi, for instance,
is frequently lethal in young rats but normally produces mild and self-
terminating infections in adult hosts (60, 64). T. cruzi also produces lethal
infections in young rats and comparatively mild infections in mature ani-
mals (100). Comparable differences have been noted even in chick em-

Immunity and Resistance 629

bryos of different ages. Embryos are highly susceptible to infection with
T. evansi at 8-14 days of incubation, but are quite resistant at 15-17 days
(23). Susceptibility to invasion by a particular route also may vary with
age of the host. Oral inoculation of rats with T. cruzi is usually possible
up to 12 days of age but becomes increasingly more difficult in older
animals (102).

Diet, and particularly the vitamin supply, may influence individual
resistance to infection. A well balanced diet is a predisposing factor in
human resistance to amoebiasis (2). Conversely, birds on a generally
deficient diet suffier abnormally severe attacks of malaria, with greater
tendency to relapse than in control animals (36). Certain high-protein
diets decrease the severity of flagellosis in rats (133), amoebic infections
in mice (134) and dogs (73), and balantodiosis in rats (150). Such effects
of proteins have been attributed to modification of the intestinal flora,
producing an environment unfavorable to Protozoa. In other cases, par-
ticular constituents of the diet, such as vitamins, may exert an important
influence.

Vitamin K, as a dietary supplement, protects chicks against Eimeria
tenella, reducing mortality from about 70 to 10 per cent (10). Likewise,
supplementary riboflavin (13), thiamine, or a combination of thiamine
and pyridoxine (14) decreases the intensity of coccidiosis in rats. A pro-
tective influence of ascorbic acid has been reported for Trypanosoma
brucei infections in guinea pigs (130). A low-biotin diet favors abnor-
mally high parasite densities in chickens and ducks infected with Plas-
modium lophiirae and in ducks infected with P. cathemerium (189, 190).
A biotin deficiency also prolongs and intensifies Trypanosoma lewisi in-
fections in rats, and even a moderate deficiency may lead to death (39,
40). Lack of folic acid increases the severity of P. lophurae infections in
chickens (152). A pantothenic acid deficiency, in rats infected with T.
lewisi, results in unusually high parasite densities, continued multiplica-
tion of the flagellates beyond the usual period, and death of the host in
extreme cases (20). Even an inorganic supplement — e.g., copper added to
the diet of rats infected with T. equiperdum (129) — may increase re-
sistance of the host.

In contrast to such instances in which an adequate supply of a vitamin
enhances resistance of the host or a deficiency lowers resistance, there are
cases in which development of the parasite is stimulated by a particular
vitamin in favorable concentrations. Such a relationship of vitamins to
coccidiosis of rats is indicated in a series of papers from Becker's labora-
tory. Preliminary observations (18) showed that dietary factors are di-
rectly related to the intensity of infections with Eirneria nieschulzi.
Addition of yeast to the diet stimulated production of oocysts to a maxi-
mum, while certain other supplements were somewhat less stimulatory.
Subsequent experiments showed that the yield of oocysts is increased by

630 Immunity and Resistance

supplementary pyridoxine (14) or pantothenate (19). Comparable find-
ings have been reported for malarial parasites. A riboflavin deficiency, in
chickens infected with Plasmodium lophurae, reduces the parasitemia to
less than one-fifth that in birds on a high-riboflavin diet (151). Panto-
thenic acid shows a similar influence on P. gallinaceum. Chickens with
a pantothenate deficiency develop much less severe trophozoite-induced
infections than controls on a normal diet. Oral dosage with analogues of
pantothenic acid produces much the same eflect as pantothenate defi-
ciency, the most active analogue (pantoyltauramido-4-chlorobenzene)
being at least four times as active as quinine (34).

The relative resistance of a particular host is influenced also by viru-
lence of the parasite, which may vary within a species. Such differences
in virulence are apparent in strains of avian malarial parasites (135), in
Plasmodium cynomolgi, P. inui, and P. knoxvlesi of monkeys (160), and
in malarial parasites of man (25, 82). Some strains of P. vivax have such
low virulence that they are of no value in malarial therapy. Differences
in virulence may be correlated with rates of reproduction. The relatively
virulent Madagascar strain of P. vivax averages 17-18 merozoites at merog-
ony; the less virulent Dutch strain, only 12-13. Strains of Entamoeba
histolytica also may vary in virulence, as indicated by their effects on
kittens (112), and strains retain their general characteristics in cultures
(114). Experimental reduction of virulence may be possible. For example,
a human passage strain of P. knoxvlesi, a species normally lethal to cer-
tain monkeys, has produced a mild chronic infection in these animals
(93). Likewise, virulence may be increased experimentally. A typical
strain of Trypanosoma gambiense, after seven passages through young
rats, caused death of adult rats in 4-7 days. Survival of adults infected with
the original strain ranged from 25 to 95 days (145). In similar fashion,
two strains of E. histolytica showed increased virulence for kittens after
seven passages through dogs. Upon return of the strains to cultures, how-
ever, virulence dropped to approximately the original levels after several
months (113).

ACQUIRED RESISTANCE

Resistance may be acquired actively, as a result of infection or
vaccination, or passively by transfer of antibodies from an actively im-
munized animal. The resistance which is acquired actively in certain
protozoan infections is a resistance to superinfection — "premunition" of
Ed. Sergent — and is dependent to a considerable extent upon persistence
of a latent infection, as in malaria. However, this resistance may last for
some time after apparent elimination of the parasites. In other cases, such
as Trypanosoma leioisi in the rat and coccidiosis in mammals, the para-
sites are finally eliminated so that a so-called "sterile" immunity is
developed.

Immunity and Resistance 631

Active immunization

Active immunization: Leishmania. Vaccination against oriental
sore was practiced empirically long before the causative organism was
discovered, and the practical value of this procedure has been confirmed
(22). In experimental immunization, results have varied with the host.
Monkeys are more readily immunized than dogs, and like man, often be-
come quite resistant to reinfection with L. tropica. Mice, on the other
hand, acquire practically no immunity. Prophylaxis with killed vaccines
has been generally unsuccessful, although such vaccines may have some
therapeutic value.

Laboratory animals are sometimes immune after recovery from infec-
tion with L. donovani (123), and it is generally believed that recovery
also leaves man resistant to reinfection. However, no effective method of
vaccination has been developed.

Active immunization: Trypanosoma. Development of acquired immu-
nity to a trypanosome was first demonstrated in rats recovering from in-
fections with T. lewisi (94). Development of such an immunity is limited
to rats more than 25 days old (64), and fails to occur even in adult rats
after hypophysectomy (59). The pathogenic trypanosomes are usually
lethal to laboratory animals, but sheep and goats sometimes recover from
chronic infections with a resulting immunity which lasts for several years
(65). Likewise, rats may recover spontaneously from infections with T.
cruzi and remain resistant to reinfection for at least five weeks (58).

Experimental immunization has followed several methods: inoculation
with a living, attenuated strain; inoculation with a virulent strain, fol-
lowed by adequate chemotherapy; and inoculation with killed trypano-
somes. Living attenuated vaccines were first used in attempts to immunize
cattle to T. brucei (99). Although the procedure apparently was successful
with some animals, this interpretation has been questioned on the basis
that cattle sometimes recover spontaneously from infection with this
trypanosome. However, rats have been immunized to T. lewisi with at-
tenuated cultures non-infective even in massive doses (127). Ehrlich and
Shiga (72) showed that, after inoculation with virulent trypanosomes and
subsequent chemotherapy, mice remain resistant to reinfection for sev-
eral weeks. These observations have been confirmed in other laboratories,
and similar results have been obtained with rats, rabbits, and guinea
pigs. Obviously, the practical value of this method is dubious. Most at-
tempts to use killed vaccines have been unsuccessful. However, such vac-
cines have immunized adult rats against T. lewisi (57, 126), although
vaccinated nurslings may succumb as readily as controls of the same age
(57). A few positive results have been reported for pathogenic trypano-
somes (35, 138, 148). Rats have been immunized against T. equinum,
to the extent that vaccinated animals always outlived the controls after

632 Immunity and Resistance

inoculation with a virulent strain, but absolute immunity was rarely
produced (138).

Active immunization: Babesia. Smith and Kilbourne (162) noted that
cattle surviving an attack of Texas fever possessed an immunity asso-
ciated with a persisting low-grade infection. Such infections, with con-
comitant immunity, may last as long as 12 years (149). Resistance of the
host is not absolute, since relapses may follow environmental or other
disturbances to the equilibrium.

Active immunization: Plasmodium. In areas where malaria is endemic,
many natives have the disease as children and the survivors seem to de-
velop a resistance to malaria. This resistance is believed to accompany
low-grade infections and to disappear gradually after the infections are
terminated.

Active immunization to P. vivax has been produced repeatedly under
experimental conditions (30, 31, 92, 198). Recovery from attacks is accom-
panied by an immunity which often prevents clinical attacks upon rein-
oculation with the homologous strain (27). However, there is little or no
protection against other strains (heterologous strains). Infection with
homologous trophozoites is frequently inhibited, although the introduc-
tion of homologous sporozoites may lead to a subclinical infection (26).
The exact duration of immunity to P. vivax is unknown. However, im-
munity may persist for six or seven years, as indicated by light infections
with mild symptoms or none at all following reinoculation with the
homologous strain (29). A comparable immunity, developed against P.
falciparum, often aborts a second clinical attack, although some multipli-
cation of the parasites may follow reinoculation (33). Inoculation of a
heterologous strain may induce a new clinical attack almost as severe as
the first but an increased tolerance is sometimes indicated by a shorter
attack and a lower parasitemia (28). In the case of P. ovale, the immu-
nity is usually more effective against heterologous strains than it is in
P. falciparum or P. vivax (161).

The phenomenon of relapse, although apparently less common in P.
ovale infections (109), is characteristic of other human malarias. Relapses
in falciparum malaria are usually of the "short-term" type, occurring
within a few weeks after apparent recovery from the original attack. Re-
lapses in vivax malaria, and especially in quartan malaria, are often
"long-term." Cases of the former many relapse after 6-12 months, and
quartan after even longer periods. The occurrence of relapse is sometimes
attributed to factors which lower resistance of the host — adverse climatic
changes, prolonged fatigue, surgical shock, pregnancy, inadequate diet,
and the like. However, an adequate immunological explanation for this
recurrence of symptoms, after a specific immunity presumably has been
developed, is highly desirable. Certain experimental data are suggestive.
For example, a relapse in monkeys infected with P. knowlesi is preceded

Immunity and Resistance

633

by a decrease in litre of the protective antibody, while a rise in titre
occurs after recovery from the relapse (47). Furthermore, superinfection
with the homologous strain (St. Elizabeth strain of P. vivax) is possible
after prolonged periods of latency (49). This indicates a gradual relaxa-
tion of the defensive mechanism during latency. On such grounds, it has
been suggested (172) that antigenic stimulation during primary attacks
with P. vivax and P. malariae usually induces a temporary low-grade im-
munity. This immunity wears off after the disappearance of erythrocytic
stages. The result is a relapse. Relapses bring further antigenic stimula-
tion, producing an immunity which may eventually become potent
enough to eliminate the parasites.

Homologous immunities, similar to those in the human malarias, are
developed against parasites of monkeys (160) and of birds (171). In
avian and simian, as well as in human malaria, the immunity is believed
to be primarily a resistance to superinfection in animals carrying a low-
grade infection with the homologous strain. Such an immunity may
persist for some time, in diminishing degree, after elimination of the
infection. The immunity of canaries to P. cathemerium decreases gradu-
ally from the first to the sixth month after cure and is no longer detect-
able after eight months (80). Man also develops an apparently sterile
immunity to P. knowlesi (115). A similar temporary immunity to P.
knowlesi has been obtained by chemotherapeutic elimination of latent
infections in monkeys (43, 107) and also against P. vivax in man by
dosage with pentaquine (48, 197a). This residual immunity against P.
vivax varies in intensity with the number of relapses rather than dura-
tion of the infection, and is lost rather rapidly after elimination of the
parasites.

In a few instances, resistance to Plasmodium has been induced with
killed vaccines. Resistance of canaries to P. cathemerium has been in-
creased by vaccination with formalin-killed parasites (79), and similar
vaccines have immunized ducks to P. cathemerium and P. lophnrae (187).
Striking results have been obtained in rhesus monkeys vaccinated with
killed P. knowlesi, emulsified in paraffin oil containing killed Mycobac-
terium tuberculosis (76). Although P. knowlesi is usually lethal, inocula-
tion of the vaccinated animals resulted in mild infections of short duration.
However, vaccination of man against P. vivax has produced no significant
protection (87).

Different stages in the life-cycle may vary in their susceptibility to
antibodies. Apparently normal pre-erythrocytic stages, but few or no
erythrocytic stages, have appeared after heavy inoculation of immunized
chickens with sporozoites of P. gallinaceum (90). In addition, chickens
vaccinated with inactivated sporozoites of P. gallinaceum are partially
immune to sporozoites but not to erythrocytic stages (143).

Active immujiization: Coccidia. Andrews (3), with his observations

634 Immunity and Resistance

that dogs and cats remain immune at least seven months after recovery
from infections with Isospora, was one of the first to demonstrate im-
munization against Coccidia. Similar results have been obtained with
other mammals (9) and with chickens (191, 192) by feeding Coccidia in
small doses. The severity of the infection is correlated with the degree
of acquired immunity, and very light infections may induce no appreci-
able resistance. Immunity against Coccidia is a sterile immunity which
prevents development of the homologous parasites. Thus, sporozoites of
Eimeria tenella may invade intestinal cells of immune chickens but soon
disintegrate (192).

Vaccination with non-viable parasites has been unsuccessful in chick-
ens (191), rabbits (9), and rats (12). These unpromising results, and the
ineffectiveness of antiserum prophylaxis, led Becker to suggest that im-
munity to coccidiosis cannot be explained on the basis of a generalized
response of the host's tissues. Instead, resistance may involve a tissue
immunity which spreads from centers of infection over the remaining
epithelial layer (12).

Passive immunization

Passive immunization: Trypanosorna. In certain host-parasite com-
binations, antiserum from a recovered animal, or from one with a chronic
or subacute infection, is protective when inoculated simultaneously with
pathogenic trypanosomes (e.g., T. briicei, T. crirJ, T. equiperdiun). Se-
rum prophylaxis against T. cnizi, although not preventing infection, does
induce a mild type of trypanosomiasis in rats (58). Passive immunization
likewise is effective against T. leioisi (167) and T. duttoni (170). Lacteal
transfer of antibodies, from actively or passively immunized females to
nurslings, has been demonstrated with T. lewisi (55, 56) and T. cruzi
(102) in rats. During the first 24 hours, a newborn rat can receive enough
antibodies in milk to protect it against an inoculum of 1,000,000 T.
lewisi. The results with T. cruzi are usually an abortive infection and
survival of the nursling. Placental transfer of antibodies appears to be
insignificant in these cases, as indicated in the exchange of litters between
immunized and normal females.

Serum therapy has been more or less beneficial in some instances. Upon
treatment with antiserum, goats infected with T. congolense have de-
veloped mild infections with recovery after three months, while controls
have died (144). Beneficial effects of antiserum have been obtained also
with T. equinum in mice (176). Such treatment of T. cruzi infections in
rats induces an incomplete crisis in the blood, but a relapse occurs soon
after the last injection of serum (58).

Passive immunization: Plasmodiutn. Although the results are of uncer-
tain practical value, favorable effects of antiserum in human malaria
have been reported occasionally (95, 106, 163). Sera from monkeys with

Immunity and Resistance 635

chronic P. inui or P. knowlesi infections also are beneficial to monkeys
with the homologous infection (46, 47). Likewise, serum therapy has pro-
tected canaries against P. cathemerhim (86) and P. circumflexum (110)
and chickens against P. lophurae when adequate dosage was continued
over a long enough period (180).

In certain parts of Africa, the comparative incidence of malaria in
infants and in older children has suggested to some workers that passive
immunization may be important in man. Cases of malaria are relatively
rare in young infants but become more and more common toward the end
of the first year. On this basis, it has seemed possible that resistant mothers
transmit to their infants an immunity which is rather effective during the
first few months after birth and then gradually disappears.

FACTORS INVOLVED IN ACQUIRED
RESISTANCE

Antibodies

The development of an acquired immunity may involve both a
specific intensification of the host's normal defensive reactions and the
appearance of defensive factors not present in the normal animal. The
mechanism of resistance may include an increased phagocytic activity,
specific for the homologous parasite, as well as the production of specific
antibodies affecting the parasite directly. Substances which induce such
reactions upon parenteral introduction into an animal are known as
antigens. In general, an antigen may be considered a protein which, if it
is to show antigenic properties in a particular animal, must be chemically
foreign to that animal. Since Protozoa, like other microorganisms, con-
tain more than a single type of antigen, any strain should be considered
an antigenic complex rather than a pure antigen. There is probably a
certain amount of overlapping among related Protozoa. One or more
similar, or possibly identical, antigens (group antigens) may occur in
several strains or in several species. Other antigens (species-specific or
strain-specific) are limited to a single species or a single strain. Among
bacteria, strain-specificity may depend upon certain non-protein sub-
stances (Jmpteyies) which modify the antigenicity of proteins. Such sub-
stances have not yet been investigated extensively in Protozoa. However,
possibly specific polysaccharides have been reported from leptomonad
stages of Leishmania tropica (153) and also from Trypanosoma cruzi,
Leishmania brasiliensis, L. donovani, Endotrypanum schaudinni, Lepto-
monas culicidarurn, and L. oncopelti (119a). It is interesting that the
polysaccharide fractions from Leptomonas gave negative precipitin tests
with antisera for the other flagellates, while T. cruzi showed fairly strong
cross-reactions with anti-Leishmania sera. It has been suggested that the
lipoid fraction, rather than the carbohydrate fraction, is related to the
antigenic peculiarities of trypanosomes (97a).

636 Immunity and Resistance

Introduction of an antigen into an animal induces the appearance of
antibodies which react specifically with that particular antigen (homol-
ogous antigen). Introduction of an antigenic complex (e.g., Protozoa,
bacteria) induces the appearance of various antibodies corresponding to
the different antigens of the complex. Some of these antibodies will react
only with the particular microorganism involved. Others, induced by
group antigens, will react also with related microorganisms which con-
tain such antigens. Antigen-antibody reactions of the latter type are often
termed group reactions. Such group reactions may form the basis of a
cross-immunity, in which an animal immunized to one strain of parasites
shows a detectable immunity to a related strain. Antibodies, which are
associated with the globulin fraction of the serum proteins, are evidently
proteins. On the basis of their reactions with antigens, they are usually
termed agglutinins, precipitins, lysins, and opsonins, although the uni-
tarian theory holds that a single antibody produces the various reactions
under appropriate conditions. True antitoxins, comparable to those in-
duced by bacterial exotoxins, have not been demonstrated in animals
infected with Protozoa. The complement-fixation reaction, involving
"complement-fixing" antibodies, is discussed below.

Under suitable conditions, a particular antibody and its homologous
antigen will react in a characteristic fashion. A precipitin reaction in-
volves the "precipitation" of a non-cellular antigen by a specific precipitin
in the presence of an electrolyte. An agglutinin reaction involves agglu-
tination of a cellular antigen (bacteria. Protozoa, etc.) by a specific
agglutinin under similar conditions. In agglutination of Trypanosoma
equiperdum, for example, the flagellates form clumps visible macroscop-
ically (138). Under the microscope, the flagellates appear in characteristic
rosettes, since the bodies stick together more readily than the motile
flagella. Lysis, which also involves a cellular antigen, may bring about
disintegration of Protozoa. In lysis of Bodo caiidatus, motility is first re-
duced and then the flagellates round up, become transparent and finally
disintegrate (140). A lysin, unlike precipitins and agglutinins, acts on the
homologous antigen only in combination with complement. A heat-labile
complex of substances, complement (or alexin) is found in normal serum
as well as in serum from immunized animals. An antiserum containing
a lysin loses its lytic activity if it is heated (e.g., for 15 minutes at 56° C).
Reactivation is produced by adding a suitable amount of normal serum
and thus restoring complement. The opsonic effect, also dependent upon
complement, is expressed as an increased phagocytic activity against the
homologous antigen. More than one of these various antibodies are to be
expected in animals infected with a given parasite. Rabbits infected with
Trypanosoma cruzi develop precipitins, agglutinins, lysins, and comple-
ment-fixing antibodies (154). Likewise, monkeys infected with Plasmo-
dium knowlesi (69) develop agglutinins, complement-fixing antibodies

Immunity and Resistance 637

and so-called protective antibodies (probably opsonins). Precipitins (169)
and agglutinins (69, 110) also have been reported in other malarial in-
fections.

Defensive mechanisms in trypanosome infections

While it is possible to observe various antigen-antibody reactions
in vitro, the composite action on the parasite — the effect of the defensive
mechanism as a whole — can be comprehended only by the study of para-
site populations during infections. This method has been followed by
Taliaferro and his associates (171, 173) who have studied the development
of resistance in various host-parasite combinations. Growth of popula-
tions has been traced by counting the flagellates in blood samples at
intervals throughout the infections. Rate of reproduction has been esti-
mated by computing the coefficient of variation in length of the flagel-

day s

Fig. 14. 1. An acute lethal infection: Trypanosoma rhodesiense in
a mouse (after Taliaferro and Taliaferro). A relatively constant coef-
ficient of variation (C. V.) indicates a uniform rate of reproduction.

638

Immunity and Resistance

lates at appropriate intervals. This procedure is based upon the premise
that in a rapidly dividing population, there is greater variation in length
and a larger coefficient of variation than in an adult population of fully
grown flagellates. The rate of reproduction may be estimated also by
determining the percentage of dividing flagellates (175). Any destruction
of parasites may be estimated on the basis of significant decrease in
parasite density. Such methods have been applied to the analysis of
acute lethal infections, relapsing lethal infections, and infections with
non-pathogenic species.

Acute lethal infections (Taliaferro's "continuous fatal" type) are the
simplest type, showing merely an incubation period and then a sharp

thousands/mm^

200

days

Fig. 14. 2. A relapsing lethal infection: Trypanosoma rhodesiense in a
guinea pig (after Taliaferro and Taliaferro).

increase in the parasite population. In mice infected with T. rhodesiense
(178), the flagellates are detectable about four days after inoculation,
and they continue to multiply until the host dies on the seventh or
eighth day (Fig. 14. 1.). The coefficient of variation remains fairly constant,
indicating a uniform fission-rate, and there is no significant break in the
growth-curve. The obvious conclusion is that the mouse develops no
appreciable resistance to T. rhodesiense.

Relapsing lethal infections (Taliaferro's "intermittent fatal" type) are
produced in a number of host-parasite combinations — T. brucei, T.
gambiense, and T. rhodesiense in guinea pigs, rabbits, and rats; T. eqiii-
num and T. evansi in rats; T. rhodesiense in man and the cat. With
T. rhodesiense in the guinea pig (178), there are irregular increases and
decreases in parasite density (Fig. 14. 2.). The decreases are referred to as
crises; the subsequent increases in parasite population, as relapses. Very

Immunity and Resistance

639

few flagellates are present during the first three weeks. A week or so later,
the population reaches a moderate density and then undergoes a crisis
in which most of the flagellates are destroyed. During the succeeding
chronic phase the survivors, as the relapse strain, multiply at the normal
rate in the presence of a trypanolysin to which they seem to be no longer
susceptible. The resulting increase in trypanosomes produces the first
relapse, which is followed by a second crisis. This crisis presumably
involves a new lysin, the appearance of which is induced by the anti-
genically modified relapse strain. The action of the second lysin on

300

thousands/mm-^

200

100

\ — ' "-1

'*"'■' *"* * divding forms

days

Fig. 14. 3. A non-lethal infection: Trypanosoma leiuisi in a rat (after
Taliaferro and Pavlinova). Early cessation of reproduction is indicated by
marked decreases in the coefficient of variation (C. V.) and the percentage of
dividing forms.

the relapse strain is comparable to that of the first on the original, or
passage strain. The typical infection shows only a few crises and relapses,
and death of the host usually occurs during the second month. Since
fission-rate remains practically constant, as indicated by the coefficient of
variation, the acquired immunity is expressed primarily through the
action of lysins which produce the crises.

Trypanosoma lewisi in the rat (178) produces a non-lethal infection.
After a short incubation period, the parasite density rises rapidly (Fig.
14. 3), usually without killing the adult rat. During the second week, the
trypanosomes begin to decrease in number. This decrease may be quite
rapid in some rats (178), somewhat more gradual in others (Fig. 14. 3).

640 Immunity and Resistance

In either case, reproduction of the flagellates (as indicated by the coeffi-
cient of variation and the percentage of divising forms) is rapid at first
but shows a sharp decline after the first few days and has practically
ceased within two weeks. Throughout the rest of the infection, there
seems to be no further reproduction, although the persisting flagellates
remain infective for normal rats (1 7 la).

This inhibition of reproduction is attributed to the appearance of an
antibody, ablastin, which prevents fission without destroying the trypano-

ihousands/mm-^

600-

500-

400-

Effecf of Ab/asfin

300-

200-

30-

'.20-

100-

c.v.

600

500.

400

300

thousands/mm^

CONTROL

^ —

'^i

§/

30-

•■■' ^/\

20-

d:/

/ "■■

\ 10-

..--

day s

Fig. 14. 4. The effect of ablastin on development of Trypanosoma lewisi in
tfie rat. The experimental animal ^\'as inoculated with T. lewisi suspended in
serinii containing ablastin; the control, Avith a comparable inoculum of trypano-
somes from the same source but suspended in normal serum.

somes. Ablastin serum, transferred to non-immune rats (Fig. 14. 4) pro-
duces the same effect (167). The importance of ablastin in defence against
reinfection has been questioned by Augustine (5), who observed dividing
flagellates in immune rats after massive intraperitoneal inoculation with
T. lewisi. If these dividing flagellates were not present in the inocula ot
200-900 million T. lewisi, or if there was a significant increase in dividing
forms after inoculation, it would appear that ablastin is relatively
inactive against reinfection by this route. On the other hand, the data
of Becker and Lysenko (16) are in accord with the view that ablastin
and trypanolysin are separate antibodies. At any rate, ablastin (or the

Immunity and Resistance 641

ablastin effect) appears after about four days in a primary infection and
the titre increases almost explosively toward the end of the first week
(50). The ability to produce ablastin is well marked in animals of 25
days or older. Young rats apparently produce little or no ablastin and
often die with T. lewisi infections (60). Production of ablastin also may
depend upon the diet of the host. A pantothenate deficiency almost
doubles the period of multiplication in T. lewisi infections (17, 20), and
a biotin deficiency likewise delays the production of ablastin as well as
trypanolysin (40). Irradiation of rats with X-rays (120) and dosage with
sodium salicylate (15) also delay the ablastin effect.

The first crisis is caused by a lysin, which upon passive transfer to rats
with early infections, induces a crisis within a few hours (51). The second
crisis, according to different suggestions, depends primarily upon the
action of a new lysin (167), mainly upon phagocytosis (137), or perhaps
upon both factors.

The relative importance of cellular mechanisms in resistance to trypa-
nosomiasis is uncertain, but the possible significance of phagocytes in
general has been considered by various workers. Phagocytosis has been
reported in vitro and in vivo. Ingested flagellates are sometimes seen in
circulating leucocytes, chiefly the large mononuclears, and also in fixed
tissue phagocytes. Furthermore, rabbits which survive infections with
T. brucei show an increase in percentage and in total number of mono-
cytes just before the first crisis. Rats and non-resistant rabbits show no
such increase (81). An absolute monocytosis also occurs about the time
of the first crisis in rats which survive T. lewisi infections, but not in
those which are to die (64). In addition to any possible importance in
phagocytosis, the lymphoid-macrophage cells have been considered as
a source of ablastin — for example, by Regendanz and Kikuth (137) who
noted that splenectomy usually delayed the ablastin effect for several
days and was sometimes followed by death. Others (175) have failed to
detect any marked effect of splenectomy on the production of ablastin.
A possible relation of lymphoid-macrophage cells to formation of lysins
also has been considered. Denison (63), for instance, has traced the effects
of blockade with trypan blue upon the production of T. cruzi lysin in
rats. In hanging-drop preparations, antiserum from infected normals
produced lysis much more rapidly than that from blockaded animals, and
the antibody titre was higher in the former serum.

Defensive mechanisms in malaria

Early investigations on bird malaria (reviews: 168, 171) showed
that the incubation period — in canaries infected with Plasmodium
cathemerium, for instance — is followed by an acute stage in which 30-50
per cent of the corpuscles are invaded. If the bird survives, the acute
phase is terminated by a crisis which eliminates most of the parasites

642 Immunity and Resistance

(Fig. 14. 5). There follows a chronic phase of a week or more, during
which a few parasites can be detected in blood smears. The chronic phase
gradually fades into a latent stage, during which the parasites cannot
be found in the blood. Subsequently, relapses may occur. Each relapse,
checked by another crisis, is followed by a new latent period.

Taliaferro and his associates concluded that the bird acquires no
resistance during the incubation period and early acute phase. The
first crisis was attributed to a stimulated malaricidal mechanism, which
from an early stage of the infection, was already destroying the majority

arasites/

100 n

40-

30-

20-

0,!!

Si!

•*^ji

<b: i

10-

:3: :

<o.' !

1 ;
; 1

'* ^^••••«

day

Fig. 14. 5. A typical malarial infection in a canary (after Taliaferro).

of merozoites — approximately 67 per cent at each merogony in specific
cases (166). Accumulation of carbon dioxide in the blood, in unfavorable
concentration, also has been suggested as a factor contributing to the
crisis (139). It was believed that the high malaricidal rate prevented
repopulation of the blood during latency, although reproduction con-
tinued at the original rate. A relapse was assumed to involve temporary
relaxation of the malaricidal mechanism. This concept of the malarial
infection in birds was modified slightly by Boyd (24) in observations on
P. cathemerium in canaries. During the first day, division-rate was high
(16.3 and 16.0 merozoites at merogony in two of the birds). During the
next couple of days, while the parasite density was increasing enormously,

Immunity and Resistance 643

the rate of reproduction dropped 25-50 per cent before the first crisis.
After the crisis, the division-rate rose again and became fairly constant,
but usually failed to reach the original level (Fig. 14. 6). The malaricidal
rate also varied. During the first day, 50-60 per cent of the parasites were
destroyed after each merogony. Three or four days later, the rate reached
90 per cent or higher and then remained at about this level. In similar
fashion, the malaricidal rate in P. knowlesi infections in monkeys in-

8-
7
6-
5

parasites/lOO red cells

m/s
20

%
90

70-1

days

Fig. 14. 6. Plasmodium cathemerium in a canary (after G. H. Boyd).
The rate of reproduction is indicated by the average nuinber of merozoites
produced by each schizont (m/s). The death rate is expressed as percentage
of parasites destroyed in each merogonic cycle.

creases from about zero to approximately 90 per cent in acute infections
(183a).

The primary factor in the malaricidal mechanism is phagocytosis;
the lymphoid-macrophage, or "reticulo-endothelial," cells play the domi-
nant role. Although it was once believed that phagocytes are mainly
scavengers in malaria, the evidence indicates that normal parasites are
ingested (174, 188). Indirect evidence also has been obtained by splenec-
tomy and by blockade of the tissue phagocytes. The latter procedure
involves the injection of material Avhich is ingested by phagocytes and
subsequently interferes with phagocytosis of parasites. Splenectomy in
monkeys with chronic or latent infections is often followed by relapse,

644 Immunity and Resistance

very severe with some species of Plasmodium. In other cases, a natural
tolerance may be eliminated by splenectomy. Much the same effects have
been produced by blockading techniques.

In natural resistance — that is, in the normal animal — phagocytosis ap-
pears to be non-specific. Circulating phagocytes seem to take little part
in destruction of the parasites and the macrophages show only sluggish
phagocytosis. In the early stages of infection, the phagocytes possibly
ingest only moribund parasites (78, 84).

As the infection progresses, some degree of immunity is developed.
Previous experience of monkeys with malaria, involving activation of
lymphoid-macrophage cells, apparently facilitates the development of
immunity against a new strain (118). Cellular responses in the spleen
of monkeys (177) include both multiplication of lymphocytes in the
splenic nodules, followed by their migration into the red pulp, and later
multiplication of lymphocytes and their transformation into macrophages
in the pulp. The result is a marked increase in the number of phagocytes.
The reproduction of macrophages as such apparently occurs only to a
minor extent. As would be expected, such agents as X-rays in heavy
dosage (185) and nitrogen mustard (183), which destroy lymphocytes,
retard the development of immunity.

In addition to the increase in number of phagocytes, the phagocytosis
of homologous parasites is specifically stimulated. This response suggests
the influence of an opsonin (47, 199). It is now widely believed that the
appearance of such opsonins, or "protective antibodies," is characteristic
of malarial immunity. In inonkeys, corpuscles invaded by P. knoivlesi
become coated with a precipitate which is selectively ingested by phago-
cytes (98a.) This phenomenon implies stimulation of phagocytes rather
than a harmful action of opsonins on the parasites. Phagocytosis occurs
principally in regions where the blood flows relatively slowly and comes
into close contact with the phagocytes. Such regions are represented
particularly by the liver, bone marrow, and spleen. Acquired immunity
to malaria, in the phraseology of Taliaferro, is thus expressed primarily
as an intensified and specific phagocytosis in such strategically located
organs as the spleen. The sluggish and non-specific phagocytosis of the
normal animal usually can not prevent establishment of an infection,
but the development of immunity increases phagocytosis to such an
extent that the infection is brought more or less under control.

The malaricidal mechanism, particularly after development of a potent
immunity, probably plays a significant part in the net results of chemo-
therapy. This conclusion is indicated, for example, by the results of
splenectomy in chickens infected with P. gaUinaceum (184). Direct inter-
ference with the lymphoid-macrophage cells throws more than the usual
load on a malaricidal drug in the elimination of infections. A similar
situation may exist in therapy following a low-grade immunological re-

Immunity and Resistance 645

sponse to a mild infection or in the early chemotherapeutic suppression
of primary attacks. Conversely, artificial stimulation of the defensive
mechanism in conjunction with administration of malaricidal drugs may
intensify effects on the parasites. As reported by Garcia (77a), injections
of tetanus toxoid following atebrin or chloroquine therapy have reduced
the relapse rate with P. vivax from an average of 90 to 9.5 per cent and
that with P. falciparum from 90 per cent to zero.

SEROLOGICAL DIAGNOSIS OF
INFECTION

Diagnosis of a protozoan infection is usually based upon detection
of the parasites in body fluids or other materials. This method works well
enough when the parasites are present in reasonably large numbers. In
early infections and in chronic and latent stages, however, the usual
laboratory examination becomes time consuming and often fruitless.
Such difficulties have stimulated attempts to apply antigen-antibody
reactions to diagnosis of protozoan infections. These tests are based upon
the principle that the infected animal will sooner or later develop anti-
bodies which react specifically with the causative organism.

In the usual procedure, a suitable antigen — a suspension of the para-
sites for an agglutinin test, or an extract for a precipitin or a complement-
fixation test — is prepared from the suspected organisms. Serum from the
infected animal is then tested with this antigen. A positive reaction, after
elimination of possible group reactions, indicates that the host has
developed antibodies against the particular parasite. A positive test, with
corroboratory clinical evidence, may thus be considered presumptive
evidence of infection.

Agglutinin tests

A suspension of the parasite in physiological salt solution is mixed
with dilute serum from the suspected host. Agglutination becomes in-
creasingly significant as the dilution of the serum is increased. Agglutina-
tion at a low dilution may represent merely a group reaction. Since
group agglutinins are usually present in concentrations lower than those
of antibodies specific for the homologous parasite, they are gradually
eliminated with increasing dilution of the serum. A positive test at high
titre thus indicates that the host has produced specific agglutinins for
the test antigen.

The results obtained with kala azar have been contradictory. Some
tests have been fairly successful, while others have not been clear cut.
Caronia (41) demonstrated agglutinins in children infected with L. dono-
vani, but concluded that the titre was too low for diagnostic purposes.
More recently, however. Row (141) has obtained well marked agglutina-
tion of flagellates from cultures. Agglutination of trypanosomes was first

646 Immunity and Resistance

demonstrated with sera from rats infected with T. leiuisi (103). Practical
application has been fairly successful in the diagnosis of dourine in
horses. Although the diagnostic value of the reaction has not been de-
termined, erythrocytic stages of avian malarial parasites (110) and P.
knowlesi of monkeys (79) are agglutinated by homologus antisera.

Precipitin tests

The test antigen is usually prepared as an extract of the suspected
parasite. Various dilutions of the antigen, in physiological salt solution,
are then tested with serum from the host. Group reactions may be
eliminated by increasing the dilution of the antigen. Consequently, a
reaction with the antigen in high dilution has the same general signifi-
cance as agglutination with high dilution of the test serum. In addition
to demonstrating specific antibodies in the blood of the host, the test
also may detect antigens of the parasite in body fluids of the host, as
in Trypanosoma equiperdum infections of laboratory animals (131). In
this case, a known antiserum is tested with material from the host, serving
as the test antigen.

The precipitin reaction has been applied to diagnosis of dourine in
horses, and has been tried also in diagnosis of human trypanosomiasis
(119, 158, 159). Muniz (119) has found the test reliable for active cases
of Chagas' disease, although much less sensitive than the complement-
fixation reaction in chronic cases. Precipitin tests have proved positive
for well developed Entamoeba histolytica infections in cats, although
negative for early infections and dying animals (194). Good results have
been reported also for malaria (142, 169). Group reactions, common to
sera from patients with P. falciparum and P. vivax, have been noted.
However, more intense reactions are obtained with the homologous
antigen (142).

Complement-fixation tests

Specific complement-fixation depends upon the fact that an anti-
gen and its homologous complement-fixing antibody will "fix," or com-
bine with complement. If either the antigen or the homologous antibody
is absent, complement is not fixed. The results are read in terms of an
indicator, the so-called hemolytic system. In carrying out such a test,
measured quantities of the test antigen, the test serum (heated to in-
activate the complement), and complement (in normal guinea pig serum)
are added, in physiological salt solution, to a serological test tube. After
incubation, a suspension of red corpuscles and an appropriate amount
of inactivated serum containing homologous hemolysin are added to the
tube. The red corpuscles and the hemolytic serum constitute the "hemo-
lytic system." The complete inixture is incubated and later examined
for effects on the red corpuscles. A settling out of the corpuscles without

Immunity and Resistance 647

hemolysis indicates that complement was fixed in the test reaction, since
there was none available for the hemolytic reaction. Absence of hemolysis
thus indicates that the test serum contains antibodies homologous for
the test antigen. On the other hand, the occurrence of hemolysis, indi-
cating that complement was not fixed in the test reaction and hence was
free to combine with the red corpuscles and hemolysin, demonstrates
that the test serum does not contain the homologous antibodies. In the
usual procedure, the test is checked with various control tubes containing
no test antigen, no test serum, neither test antigen nor test serum, or
only red corpuscles, as well as with complete systems containing known
positive and negative test sera.

Complement-fixation has sometimes shown good correlation with other
methods for diagnosis of leishmaniasis. Using L. donovani antigen pre-
pared from spleens of infected hamsters, Hindle, Hou, and Patton (89)
obtained good results with sera from kala-azar patients. Comparable and
more significant results have been reported for antigens prepared from
cultures (6, 61, 77, 124). The test is positive in early cases and seems to
be highly specific (77). The usual procedure also has been reversed by
using antiserum from immunized rabbits to detect Leishmania antigens
in human blood (121). Complement-fixation has been useful in the
diagnosis of dourine because tests are positive at an early stage, and in
spite of group reactions with Trypanosoma evansi, seem to be reliable.
Complement-fixation also has been used extensively in the diagnosis of
Chagas' disease (122). With antigens prepared from cultures of T. criizi,
the test is dependable and apparently is not complicated by cross-reactions
with Wassermann sera (96). A polysaccharide fraction prepared from
T. cruzi also has proven effective as a test antigen (119a).

Izar (91) and Scalas (146) apparently were the first to report success
with complement-fixation in amoebiasis. Subsequently, the results of
Craig (52, 53, 54) and later workers, with antigens prepared from cultures
of E. histolytica, indicated the practical value of this test in mild intestinal
amoebiasis. The important handicap to wider application seems to have
been the difficulty of preparing effective test antigens. Establishment of
E. histolytica in cultures with one strain of bacteria (136) and the current
availability of commercially prepared antigen should eliminate certain
variables caused by uncontrolled bacterial flora. However, one modifica-
tion of the test, carried out with commercially produced materials, seems
to be useful for diagnosis of hepatic but not intestinal amoebiasis (90a).

Application of complement-fixation to diagnosis of malaria was not
successful at first because sensitivity of the tests was too low. More re-
cently, reasonably good results have been obtained with antigens prepared
from parasitized human or monkey blood (44, 45, 62, 67, 71, 83, 98, 111,
164, 165) and from chicken blood containing P. gallinaceum (83, 98).
Such tests also will detect malarial antigens, in the blood of the host.

648 Immunity and Resistance

which fix complement in the presence of antiserum (70). Although com-
plement-fixation with P. knowlesi or P. gallinaceum antigen in human
malaria is a group reaction and false reactions are sometimes obtained
with syphilitic sera (7, 83), its practical value as a supplementary method
in diagnosing mild infections with P. vivax seems to have been demon-
strated (68). However, the test seems to have no value in latent vivax
malaria. An interesting outgrowth of these investigations is the demon-
stration that P. gallinaceum antigen is effective in complement-fixation
tests for Haemoproteus columbae in pigeons (197). Perhaps the relation-
ship between Plasmodium and Haemoproteus is closer than is generally
believed.

Tests with human sera from known and suspected cases of toxoplasmo-
sis have indicated that complement-fixation may be useful in diagnosis
of active toxoplasmosis (196). Complement-fixation tests may be positive
also in animals infected with Coccidia (8, 42) but the diagnostic value
is uncertain.

In addition to specific complement-fixation, in which the reaction is
dependent upon the presence of a particular antigen and its homologous
antibodies, non-specific complement-fixation tests have been used exten-
sively in serological diagnosis. In these tests, the test "antigen" bears no
apparent relation to the parasite causing the infection under consider-
ation. The best known example is the Wassermann reaction, in which
the test "antigen" is extracted from normal ox heart. A comparable
non-specific test has been tried in diagnosis of kala azar (155, 156), the
"antigen" being prepared from a human strain of Mycobacterium tuber-
culosis. Although occasional false positives have been obtained in pulmo-
nary tuberculosis, this test for kala azar seems to be fairly reliable.

Skin tests

Diagnostic skin tests depend upon a cutaneous inflammatory re-
action induced by an antigen, introduced either by intradermal injection
or by the scratch method, into an animal containing homologous anti-
bodies. A positive reaction in man usually involves both immediate and
delayed reactions. In lower animals which react at all, an inflammatory
reaction usually develops the day after inoculation. The minute reddened
area of a negative reaction is readily distinguished from a positive test.
Skin reactions to Leishmania have been obtained in rabbits immunized
to L. donovani and L. tropica (193) and also in human cases of dermal
leishmaniasis (117, 153). Positive tests have been reported also in rabbits
immunized to Trypanosoma cruzi (154), in human amoebiasis (147), and
in guinea pigs for several weeks after recovery from coccidiosis (88).
Preliminary experience with an intradermal test for human malaria has
been promising. With antigen prepared from Plasmodium gallinaceum,
the test compares favorably with examination of blood films (108).

Immunity and Resistance 649

Adhesion tests

Adhesion, or "adhesin," tests, in diagnosis of trypanosomiasis, in-
volve mixing citrated blood from the host with a suspension of the
suspected parasites. A positive test, in which red corpuscles and some-
times blood platelets stick to the flagellates, indicates that the blood con-
tains antibodies specific for the trypanosomes. The reaction apparently
depends upon the presence of complement (195). Adhesion tests have
been applied to diagnosis of trypanosomiasis in man and other animals
(37, 66, 132, 179, 195) as well as infections with Leishmania tropica (116).

SEROLOGICAL DIFFERENTIATION
OF SPECIES

In the differentiation of species by means of serological reactions,
microorganisms are tested with known antisera. A positive reaction, such
as agglutination, indicates that the antiserum contains antibodies homol-
ogous for the test organism. This establishes the identity of the strain,
provided group reactions have been eliminated. The specificity of an
agglutin test can be increased by preliminary absorption of the anti-
serum with appropriate heterologous antigens so as to eliminate some or
most of the group antibodies. Even the quantitative interpretation of
group reactions may throw some light on degrees of taxonomic relation-
ship (85, 186). Serological tests have some value in differentiating morpho-
logically similar organisms, but the data must be interpreted cautiously
because the tests are so sensitive. For instance, agglutinin tests have
distinguished between strains of trypanosomes derived from one original
stock but maintained in different host species (138).

Agglutinin tests have given good results in differentiating types of
Leishmania. Bandi (11), who was interested in the status of "Leishmania
canis," found that either L. canis or L. donovani agglutinin would react
with either strain in titres up to 160. Neither agglutinin was active
against L. tropica in titres above 70. L. canis and L. donovani thus
seemed to be serologically identical, while both were distinct from
L. tropica. More recently, agglutinin tests have indicated that L. dono-
vani, L. tropica, and L. brasiliensis are serologically distinct (74, 97, 125),
although group reactions may be expected with low dilutions of aggluti-
nating sera. Group agglutination is eliminated in higher dilutions, while
homologous agglutination may still be detectable at titres of 2,560-2,580
(157). Adler and Theodor (1) used agglutinin tests in identifying an
invertebrate host of L. tropica with their demonstration that "Herpeto-
monas papatasii," an intestinal flagellate of sandflies, is serologically
identical with L. tropica.

Specific lysins also have been tried in the identification of Trypano-
somidae. By inoculating flagellates into culture media to which known

650 Immunity and Resistance

lytic antisera had been added, da Fonseca (75) was able to distinguish
Leishmania brasiliensis from L. tropica. Growth of L. brasiliensis was
inhibited by anti-brasiliejisis lysin but not by Rnti-tropica lysin. The re-
sults were the reverse with cultures of L. tropica. Neither antiserum pre-
vented growth of L. donovani in cultures. There are also indications that
species of Trypanosoma may be differentiated by lysis in vitro (105), al-
though there may be difficulties with group reactions.

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Index

Numbers in italics refer to pages on which figures occur. In the text, names of
authors are cited mostly by numbers referring to papers listed at the end of each
chapter. Accordingly, the names of relatively few authors are listed separately in the
index.

Ablastin, 640
Acanthamoeba, 239

castellanii, 236
Acantbochiasma, 217
Acanthocystidina, 207
Acanthocystis, 209

lubella, 208
Acanthofca, 130
Acantbometia, 217

pel/ucida, 213
Acanthometion (see: Acanthometra)
Acanthoma, 217
Acanthosphaera, 218
Acantbospoia, 282

repe/inf, 281
Acanthosporidae, 282
AcantbostomeUa, 392
Acclimatization, 509
Acephalina, 287
Achromatic figures,

extranuclear, 66

intranuclear, 67
Aciculum, 16
Acineta, 420

cornuta, 417

livadfana, 417
Acinetactis, 202

arnaudofB, 20^
Acinetidae, 420
Acinetides, 420

varians, 417
Acinetopsfs, 420

eJegans, 417
Acnidosporidea, 323
Acrasina, 228
Acrasfs, 228
Actfnelfus, 217
Actfnobolina, 339

vorax, 340
Actinobolinidae, 339
Actinocephalidae, 282
AcdnocephaJus, 282

parvus, 281
Actinocoma, 226

ramosa, 226

Actinolophus, 209

peduncuJatus, 209
Actinonionas, 171, 202

mirabiJis, 203
Actinomyxida, 317
Actinophrydina, 206
Actinophrys, 206

pontfca, 207

so7, 207
Actinopodea, 202
Actinosphaerium, 206

eichorni, 207
Actipylina, 216
Acutispora, 285
Adelea, 296

ovata, oocyst, 295
syzygy, 291
Adeleida, 296
Adeleidae, 296
Adeleina, 296
AdeJina, 296

deronis, 295
syzygy, 291
Adler, S., 649
African coast fever, 306
African sleeping sickness, 583

chemotherapy, 587

control, 588

Gambian type, 585

laboratory diagnosis, 587

reservoirs, 588

Rhodesian type, 587

transmission, 585
AgareJIa, 317

graci/is, 315
Aggregata, 298

eberthi, 298
mitosis, 66
Aggregatidae, 298
Aikinetocystidae, 287
AiJcinetocystis, 287

singu/aris, 272
AlbatrossieJ/a, 393
AJbertisieJ/a, 290
Allantocystidae, 287

654

Index 655

AI7antocystis, 287

dasyhe/ei, 288
Al/antosoina, 420
intestinaJis, 418
AUogTomia, 265
du/ardini, 264
laticol/are, 253
Allogromiidae, 264
AJJoiozona, 342

trfzona, 341
Amaurochaete, 233
Amicronucleate ciliates, 49
Amoeba, 239
dubia, 238

gullet-like structures, 28, 29
internal electrolyte concentration, 471
locomotion, 489
proteus, 238
Amoebiasis, 555

causative organism, 555
chemotherapy, 560
complement-fixation tests, 647
control, 562

laboratory diagnosis, 568
primary, 559
Amoebida, 233
Amoebidae, 237
Amoeboaphe/idium, 221
Amphacanthus, 395
Amphibotre/Ja, 339
enigmatica, 340
Amphibotrellidae, 339
Amphichrj'sis, 125
Amphidiniopsis, 145
kofoidi, 138, 146
Amphidinium, 143

dentatum, 142
Amphileptidae, 341
Amphi/eptus, 342
cJaparedei, 540
AmphiJonche, 217
Amphimonadidae, iSo
Amphimonas, 180
cyc/opum, 179
globosa, 179
Amphisie/ia, 399
Arnpfiitrema, 265
stenostoma, 264
wrightianum, 264
AmphizoneJ/a, 249

vioJacea, 243
Amphore/Ja, 393
AmphoreJJopsis, 393
AmphorocephaJus, 282
Amphoroides, 282

ca/verti, 281
Amphosome, 33
AmpJecteJ/a, 393
AmpIecteJ/opsis, 393
AmpuJ/acuIa, 342

ampuJ/a, 341
AmyJoodfnium, 150

oceJJatum, 141
Anarma, 420
brevis, 418
Ancistrel/a, 374

Ancistrocoma, 371

dissiwiUs, 372
Ancistrocomidae, 371
Ancistrospira, 374
Ancistrum, 374

pernix, 373
Ancyrophora, 282

uncinata, 281
Ancystropodium, 398
AndreuJa, 371
Andrews, J. M., 535, 633
Angeiocystis, 298
Anisocoiiiides, 371
Aniso/obus, 285
Anisonema, 168

acinus, 167
Anophr)s, 364

saJmicida, 365
AnopJophrya, 379

gamniari, 377
Anoplophryidae, 379
Anthophysis, 127
Anthorhynchus, 282

sophiae, 281
Antibiotics, effects, 486
Antigenic types

cytoplasmic inheritance, 524
inheritance, Paramecium, 517
Anurosporidinm, 328

pe/seneeri, 328
ApheJidiopsis, 221
Aphehdium, 221
Apiococcus, 152
Apodinium, 150
ApoJocystis, 289

minuta, 272, 288
Apostomina, 374
ArachnuJa, 224

impatiens, 223
Arcel/a, 249

vulgaris, 243
Arcellidae, 249
Archidiscus, 218
Arcyria, 233

cinerea, 232
Aristerostoma, 364
Arthrochrysis, 127
Arthropyxis, 127
Artodiscus, 265

saJtans, 26^
Ascog/ena, 166

vaginicola, 165
Askenasia, 345
volvox, 344
Aspidisca, 398
polystyh, 397
turrita, 397
Aspidiscidae, 398
AssuJina, 250

semi/unum, 248
Astasia, 166
comma, 165
dangeardii, 165
longa, 165
torta, 165
Asteropbora, 282

656 Index

Astomina, 376
Astramoeba, 240

Stella, 2^8
AstTocystella, 290
Astrodisculus, 209

radians, 208
Astropbrya, 422
Astiorhiza arenaria, 255
AsMozoon, 408
phiioime, ^o^
Astylozoonidae, 407
Atelodinium, 150
Atopodinium, 403

fibulatum, 402
Augustine, D. L., 640
Aulacantha, 219
Aufosphaera, 219
Autogamy, 80

genetic significance, 518, 519
Paramecium, 84
Axoneme, 12
Axopodia, 11
Axostyles

resorption in fission, 58
structure, 14

Babesia, 308

bigemina, 307, 308
Babesiida, 306
Babesiidae, 309
BaciUidium, 322

argoisi, 320
Badhamia, 233

n?agna, 233
Baikalodendion, 422

augustatum, 419
Baika/ophria, 422

acanthogammari, 419
Ba/anonema, 364

diibium, 363
Balantidiidae, 381
Balantidioides, 388

miiscicoJa, 387
Balantidiosis, 563
Balantidium, 581
coil, 563, ^6^
praenucleatum, 382
Ba/antiophorus, 370
Ball. G. H., 528
Ba//adyna, 398
parvu/a, 399
BdUadynopsis, 398
Ballodora, 409
Barbetia, 221
Barbu/anympha, 193

ut alula, 192
Barrouxia, 301

schneideri, 299
Basal granule, 18
Bathysipbon bumilis, 2ec
Beale, G. H., 524
Beccaricystis, 290

ioriai, 288
Becker, E. R., 629, 640
Beers, C. D., 72, 97

Belar, K., 63, 206
Beloides, 282
Bertramia, 328
Bicoeca, 126, 175
Biiocuiine//a globula, 2^6
Biomyxa, 225
mefdaria, 22^
vagans, 224
Biotypes, 506
Bipedinomonas, 152
Bishop, A., 547
Bizone, 364
Blackhead in poultrv

Histomonas mekagridis, 173
Tikhomonas gallinaium, 190
Blackwater fever, 620
Blastodiniidae, 150
Blastodinium, 150
spinulosum, 149
Bkphamma, 388
hyalinum, 389
lateritia, 389
Bkpbamconus, 342

cervicab's, 341
Blepharocoridae, 353
Blepharocorys, 353
curvigula, ^^^
equi, 354
Blepharoplast, 12
BkphaTopTosthium, 342

pireum, ^^1
Bkpharosphaera, 342

intestinalis, ^^1
Blepbarozoum, 342
Bodeiia, 265
Bodo, 180

caudatus, ly, lyg
Bodonidae, 180
Body form,

maintenance of, 5
variations, 3
Boeck, W. C., 548
Boell, E. J.. 91
Borgert, A., 128
Botlniapsis, 282
Bo\'eria, 374

teredini, 373
Boyd, G. H., 642
Boyd, M. F., 597
Boyers, L. M., 559
BTachiomonas, 152

westiana, J54
Brahmachari, U. N., 580, 581
Brancbioecefes, 353
Biandtklla, 393
Biesslaua, 355
skaiia, ^^6
Br}'ometopus, 385
Bryopbiya, 355
Bryopbyllum, 342
carinatum, 340
Bucbneiklla, 379

criodrib, 377
Budding
external, 61
internal (Suctorea), 61

Index 657

Bu/bocepha/us, 287

elongatus, 284
BuUanympha, 187
BundJeia, 342

postciUata, 341
Burk, M., 230
Bmsaopsis, 395
Bursaria, 383

truncate//a, 582
excystment, 78
Bursaridium, 383

pseudobuisaria, 382
Bursariidae, 385
BurseUa, 347
Biitschli, O., 95, 106
Biitschlia, 342

nana, 341
Biitschliella, 379

nasuta, 377
Biitschliidae, 342
Buxtonella, 349

su/cata, 350

Caduceia, 187

buguioni, 188
Cacnonjorpha, 385

niedusuJa, 386
Calkins, G. N., 2, 95, 96, 97, 106, 109, 111
Callimastigidae, 1 84
Callimastix, 1 84

cqui, 183
Ca/onympha, 188
Calonympliidae, 187
CaloscoJex, 391;
CaJospira, 376
CaJyptotiicha, 370
CaJyptrosphaera, 130
Camerina e/egans, 253
Campascus, 249

triqueter, 248
Campbell, A. S., 391
Caziiptonema, 206
Cannophi/us (see: Dicfyocha)
Cannosphaera. 219
CantharieJJa, 393
Carbohydrate metabolism, 466

hexoses, dissimilation, 467

monosaccharides, utilization {table), 466

synthesis, 470
Carchesium, 411

po/ypinum, ^11
Carcinoecetes, 284

hesperus, 275, 281
Carcinogenic substances,

effects on growth, 486
Cardiostoma, 364
Carteria, 152

coccifera, 154
Caryospora, 301
Caryotricha, 398
Caryotropha, 300

mesnili, 291, 298
Caryotrophidae, 300
Castanidium, 219

sol, skeleton, 219
Catenoid "colonies," 7

Cau2ico/a, 410
valvata, ,^08
CauUeTyella, 282

pipfentis, 280
Cell Theory, relation to Protozoa, 3
CeUonieUa, 133

pa/ensis, 132
Celloniellidae, 133
Cenolarcus, 218
Cenosphaera, 218

macropora, 216
Centrophore/Ja, 342

lasciolata, 340
Centro pyxis, 249

aculeata, 2^^, 247
Cepedea, 337

pun/abensis, ^^^
Cepedella, 371
Cephalina, 282
CephaJoidophora, 284

coimniinis, 281
Cephaloidophoridae, 284
Cepha/omonas, 155
Cepha/othamion, 127
Cepha/otrichium, 391
Ceratiidae, 146
Ceratfomyxa, 233
Ceraffum, 146

hirundineJ/a, 147
theca in fission, 57
Ceratomyxa, 316

shasta, 313, ^1^
Ceratomyxidae, 316
Ceratospora, 290
Cercobodo, 180
Cercomonas, 180
Certesi'a, 398

quadrinuc/eata, 397
Chadefaud, M., 160, 168
Cbaenea, 347

limicoh, 347

teres, 347
Chaetospira, 398
Chagas, C, 589
Chagas' disease

acute form, 592

causative organism, 589

chronic form, 595

complement-fixation tests, 647

control, 594

distribution, 589

laboratory diagnosis, 594

precipitin tests, 646

reservoirs, 590

transmission, 590
ChagaseJJa, 296

hartmanni, 295
ChaHengeron, 219

armafum, skeleton, 219
Chalubinskia, 145

tatrfca, 146
Chaos, 240
Characioch/oris, 1 52
Charon, 353

equi, 354
Chasmatostoma, 364

658 Index

Chatton, E., 92, 371, 380, 398
Chattonelh, 170

suhsala, 169
Chattonidiidae, 383
Chattonidium, 383

setense, ^82
Cheissin, E., 379, 380
Cheissinia, 374

baicalensis, 373
Chcnioautotroph, 433
Chen, Y. T., 168
Chilodochona, 412

quenncTStedti, ^13
Chilodochonidae, 412
Chilodonella, 342

caudafa, 343

cucu//us, 343
Chi7odonfopsis, 348

muscoruni, 349
Chilomastix, 182

intestinaUs, 181

magna, 181

mesniH, 547, 548
Chilomonas, 137
ChiJophrj'a, 347
ChitoanastTum, 218
Ch]arn}dob/epharis, 153
Chlamydobotivs (see: PjTobotrys)
ChJamydodon, 342

tn'quefrus. 343
Chlamvdodontidae, 342
Chlamydomonadidae, 152
ChJaniydornonas, 1 52

unibonata, 1^^
Cblamydomyxa, 224
ChJaniydophrys (see: Pamphagus)
Ch/orobrachfs, 152
Chforoceras, 152
ChJorodesmus, 126
ChJorogonium, 1 52
Chhiomeson, 133

parva, 134
Chloromonadida, ii8, 168
Chloromyxidae, 316
ChJoTomyxum, 316

tri/iigum, 315
Ch/orophysema, 152
Ch/orosaccus, 134
Choanocystel/a, 290
Choanocystis, 212

JepiduJa, 211
Choanocystofdes, 290

costaricensis, 289
Choanoflagellates, 175
Choanophrya, 423
Chondropus, 210
Chonotrichida, 333, 411
Chromatoid bodies, 38
Chromatophores, 33

pigments, 35
Chromidia, 39
Chromidina, 376

elegans, 375
Chromidinidae, 380
ChromuJina, 125

annu/ata, 119

ChromuJina (Cont.) :

conmiutata, 119
Chromulinidae, 125
Chroomonas, 136

baJtica, 1^6

vectinis, 1^6
Chiysamoeba, 131
Chrysapsis, 125

fencstrafa, 119
Chrysarachnion, 130

insidians, 131
Chrj'sidiastrum, 131

catenafum, 131
Chrysocapsa, 132
Chrysocapsidae, 132
Chrjsocapsina, 132
Chrysochromuh'na, 127

parva, 127
Chrysococcocystis, 126
Chrysococcus, 126

unibonatiis, 120
Chrysocrinus, 132
Chrysodendron, 127
Chrysog/ena, 126
Chrysonionadida

chromatophores, 121

colonial organization, 122

cortical specializations, 119

diagnostic features, 117

encystment, 123

feeding habits, 118

life-c\clcs, 123

suborders, 124
ChrysosphaereJ/a, 126

Jongispina, 123
Chytriodinfum, 150
Cienkowskya, 209

merescfiJcowsJcyi, 208
Cilia, 18

derivatives of, 19
Ciliatea, 333

food requirements, 448
Ciliophora, 332
Ci/iophr\'s, 202

marina, 203
CfJiospina, 391
Cinetochi/um, 364
Circoponis, 219
Cirri, 19
C/adomonas, 180

fruticu/osa, 6
CJadonema, 127

pauperum, 122
CJadotricha, 398
Clathielh toieli, 211
Clathwsoius, 230
C/athrostoma, 354

vimfnaJe, 354
Clathrostomidae, 353
CJathniJina, 212

elegans, 210, 211
Cleveland, L. R., 63, 171
Cleve/andeJJa, 383

elongata, 384
Clevelandellidae, 383
Ch'macocycJis, 391

Index 659

Climacostomum, 388

virens, 389
CJypeoIina, 249

marginata, 247
Cnidosporidea, 511
Coatney, G. R., 602
Coccidia, 290
life-cycles, 291
oocysts, 293, 294
sporoblasts, 294
subdivisions, 295
Coccidiosis, man, 565
Coccodinium, 150

duboscqi, 149
Coccolithina, 130
Coccoliths, 130
CoccoJithus, 130

waHichi, 129
Coccomonas, 155
Coccomyxa, 316
morovf, 315
slavinae, 320
Coccomyxidae, 316
Coccospora, 322
Coccosporidae, 322
CochJiatoxum, 396
CochJiodinium, 143
Jebourae, 138
puJcheUum, 142
CochJiophiJus, 371

depressus, 372
Cochbopodium, 249

granulatum, 246
Codonarfa, 391
Codonella, 391

rapa, 392
Codonellidae, 391
Codonellopsis, 391

longa, 392
Codonellopsidae, 391
Codonobotr)'S, 127
Codonocladium umbeJ/atum, 174
Codonodendron, 126

ocenatum, 122
Codonopsis, 391
Codonosigopsis, 175

sociaJis, ij^
Codosiga, 175
botrytis, 174
elegans, ij
Codosigidae, 175
Coelacantha, 219
Coelodendrum, 219
CoeJomonas, 170
Coelorhynchus, 282

heros, 281
Coelosporidium, 328

periplanetae, 327, 328
CoementeJ/a, 219
Coenonia, 228
Cohen, B. M., 92
Cohnilembidae, 364
CohniJembus, 364
punctatus, 363
CoJacium, 166

vesiculosum, 34, 165

Colacfum (Cont.) :
vesiculosum (Cont.):
budding, 62, 72
Colepidae, 342
CoJepismatophiJa, 286

watsonae, 28^
Cokps, 345

amphacanthus, 343
hirtus, 343
CoIIineJJa, 349
gundii, 350
CoJ/odictyon, 152
tricibatum, 153
CoIIosphaera, 218
CoJJozoum, 218
Colonies, organization, 6
Colpidium, 367
coJpoda, 363

chromatin elimination, 70
Colpoda, 355
steinii, 354
Colpodidae, 354
CoJponema, 180

Joxodes, 179
Cometodendron, 421

digftatum, ^18
Cometoides, 282
Commensalism, 527

evolutionary goal of parasites, 528
Concboceras, 219
Conchophthiriidae, 371
Conebophtbirius, 371
anodontae, 372

mitosis, micronucleus, 67
ConcbopbyJJum, 374
Concretion-vacuole, 32
CondyJostoma, 385
arenarfum, ^8^
vorax, 384
Condylostomidae, 383
Conidiophryidae, 355
Conidiophrys, 355

pib'suctor, 356
Conjugation

cytoplasmic lag in inheritance, 512
factors inducing, 90
genetic effects, 512

micronuclear behavior, 513, 514
intervarietal crosses, 93
macro- and microconjugants, 89
mating reactions, 85
mating types, 92
nuclear behavior, 86
pairing, 85

stimulatory effects, 97
survival after, 97
variations within a species, 90
Contractile tube, Haptophrya, 32
Contractile vacuoles

hydrostatic regulation, 470
vacuolar cycle, 31, 472
vacuolar pores, 32
Copeland, H. F., 2
Copemetopus, 385
Copromastix, 1 82
prowazeJci, 181

660 Index

Coria, N. A., 449
Corliss, J. O., 362, 367
Cornuspira phnoibis, 256
Corone, 156

bohemica, 1^6
Coronympha, 188

clevehndi, 189
Cortex, differentiations of, 8
Coitiniscus, 218
Corycella, 282

armata, 272
Corycia, 249

flava, 246
Coiytbion, 250
Costa, 1 5

behavior in fission, 58
Costia, 182

necatrix, 181
Cothurnia, 410

acuta, ^08

canthocamptf, ^08
Cougomdella, 322

magna, 320
Coulston, F., 303
Coxliella, 391

fasciata, 392
Coxliellidae, 391
Craig. C. F., 557, 568, 569, 647
Cranotherfdium, 353
Cratere/Ja, 392

urceoJata, 392
Craterocjstis, 290

papua, 288
Crebfcoma, 371

carinata, 372
Cresta, 16

Crfbostomum bradyf, 255
Crfbran'a, 233
CricundeJJa, 393
Cristigera, 370

setosa, 569
Crithidia, 177

euryophthaJmf, ijS
CrobyJura, 347
Cromyodrymus, 218
Cryptobia, 178

helicis, 15, 179
Cryptobiidae, 178
Cryptochib'dium, 364

echini, 365
Cryptochrysidae, 135
Cryptochr\'sis, 1 36

atJantica, 1^6

commutata, 136
Cni'ptodifHugia, 249

conipressa, 246
Cryptoglena, 166

pigra, 165
Cryptomonadida

diagnostic features, 117, 134

families, 135

food requirements, 445
Cn,'ptomonadidae, 137
Cryptomonas, 137

simiUs, 136
Cryptopharvnx, 342

Cryptopharynx (Cont.) :

setigcrus. 543
Cryptosporidiidae, 300
Cryptosporidium, 300

panuni, 298
Cryptozoite, 303
Crystals, cytoplasmic

Amoeba, 39

Paramecium, 39
Ctedoctema, 370

acanthocrj'pta, 369
Ctenostomina, 401
Cucurbite/Ia, 249

mespfliformis, 247
Culture media (tabJe)

CbiJomonas paramecium, 430

Tetrah}'mena pyTifoimis. 430
Cunhaia, 395

Cyanide-stable respiration, 456
Cyanomonas, 137
Cyathodiniidae, 355
Cyathodinium, 355

late fission, 60

pirifornie, 356
Cyathomonas, 137
CycJammina cance/Jata, 255
CycJidium, 370

giaucoma, 369
Cyc/ochaeta, 410

domerguei, 407
Cyc/ogramma, 348
Cyclonexis, 127

annularis, 123
CycJonympha (see: Teratonympha)
CycJopbrya, 423

magna, 421
C}'cJoposthifdae, 395, 396
Cycioposthium, 396

bipaJrnatum, 596
CycJospora, 301

caryoJytica, 299
Cyc/otrfcbium, 345

g'gas, 344
CymatocycJis, 392
Cyphoderfa, 250

tiochus, 248
Cyrto/ophosis, 364
Cyrtopbora, 126
Cystidina, 249
Cvstidium, 218
Cvstobia, 290
Cystoccphalus, 287

aJgerianus, 284
Cystodinedria, 149
Cystodinium, 149

iners, 148
Cystospora, 230
Cysts

protective
structure, 74
viability, 75

reproductive, 75
Cytochdus, 218
Cytogamy, 85

genetic significance, 520

Index 661

Cytoplasmic inclusions (see: Food reserves,
Mitochondria, Osmiophilic inclusions,
Vacuome, Volutin)
Cytoplasmic inheritance, 521

antigenic types, 524

killer trait, Paramecium, 521

mating tj'pes, 523
Cytostome, 28
Cyttarocyclidae, 392
CyttarocycJis, 392

acutifoimis, 392

Dacty/ochiamys, 339

pisciformis, 340
Dactylophoridae, 285
DactyJophorus, 285

robustus, 283
Dact)Jophrya, 420
Dacty/osaccus, 265
Dacty/osoma, 309, 310

jahni, 309

ranarum, 309
DadayieUa, 393
DaJh'ngeria, 180
Dasytricha, 357

ruminantium, 358
DatuTeUa, 393
Dauermodifikationen, 510
Deflandre, C, 13, 130
DeJtopyJum, 367

ihabdoides, 365
De/totrichonympha, 195
De Morgan, W., 376
Dendrocometes, 421

paradoxus, 414, 418
Dendrocometidae, 420
DendrOHionas, 127
Dendrophrya erecta, 2^^
Dendrorhynchus, 285
Dendrosoma, 422

radians, 419
Dendrosomidae, 421
Dendrosomides, 422

truncata, 419
Dentostomina bermudiana, 256
Derepy.vis, 126

amphora, 120
Desmarella, 175

moniliformis, 174
Desmothoracina, 211
Deutomerite, 273
Deutsch/andia, 130
Devescovina, 187

arta, 186

vestita, 186
Devescovinidae, 187
Dewey, C. V., 450
Dexiotrichides, 364
DiaphoTodon, 249, 265

mobiJe, 2^8
Diceras, 353

bfcornis, 351
DichiJum, 364
Dictyocha, 130

specuJum, 128
Dfctyocysfa, 392

Dictyocysta (Cont.) :

mira, 392
Dictyocystidae, 392
Dictyophimus, 218

graciJfpes, skeleton, 217
Dictyostelium, 228

discofdeum, 227

mucoroides, 227
Dfdesmfs, 342

ovah's, 341
Didiniidae, 345
Didinium, 345

nasutum, 344

excystment, 77
Didymium, 233

annu/atum, 233

meJanospermum, 233
Didymophyes, 285
Didymophyidae, 285
Dientamoeba, 241

fragiiis, 553
Difflugfa, 249

corona, 9

pyriformis, 247
Difflugiidae, 249
Digestion, 460

carbohydrates, 463

disaccharides, utilization (taWe), 464

food vacuoles, 461

lipids, 464

polysaccharides, utilization (table), 463

proteins, 462
Dileptus, 353

anser, 352

trichocvsts, 27
Diller, W. F., 84, 508, 513, 519
Dimastigamoeba, 234

simplex, 235
Dimastigamoebidae, 234
Dimoipha, 171, 202

mutans, 203
mitosis, 64
DimorpheJIa, 202

elegans, 203
Dfnamoeba, 240

mirabi/is, 239
Dinastridfum, 149
Dinema, 168
Dinenympha, 184

fimbriata, 183
Dinobryon, 127

stokesff, 120

utriculus, 120
Dinocapsina, 147
Dinococcina, 149
Dinoflagellida

diagnostic features, 118

epicone, 1 39

girdle, 138

holozoic habits, 139

hypocone, 139

life-cycles, 140

pigments, 139

pusules, 139

suborders, 141

sulcus, 138

662 Index

Dinoflagellida (Cont):

theca, 139
Dfnomonas, 180

tubeicuhta, 179
Dinophysidae, 147
Dinophysis, 147

diegensis, 147
Dinopodiella, 149

phaseolus, 148
Dinosphaera, 145

palusf rfs, 146
Diophrys, 398

appendiculatus, 399
Diplocolpus, 217
Diploconus, 217
Diplocystidae, 287
Diphcystis, 287
DipJodinium, 395

monocanthum, 395
DipJogromia, 265

brunneri, 263
Dip7omita, 180

socia/is, 179
DipJophrs'S, 265

archeri, 264
Dip/opJasfron, 395
Diplopsalis, 145

Jentfcu/afa, 144
Dip/osiga, 175
Diplosigopsis, 175

entzil, J 74
DipJostauron, 152
Diporfodon, 132
Dfscomorpha, 403

pectinata, 402
Discomorphidae, 403
Discophrya, 423

Jonga, 421
Discophryidae, 423
Discoibynchus, 282
Discosoma, 421
Discosphaera, 130
Disematostoma, 364

biitschJfi, 365
Dissodinium, 149
Disfephanus (see: Dicfyocha)
Distigma, 166

senni'i, 165
Disfigmopsfs, 166

grassei, 165
Dftoxum, 396

fiminudeum, 395
Dobell, C. C, 3, 548, 553
DobeUia, 296
Dobelliidae, 296
Doflein, F., 107
DogieleUa, 379
DoJichodiniuni, 147

Jineatum, 147
Dorisiella, 301

scolepedis, 299
Dorofaspis, 218

heteropora, 213
Drepanomonas, 362

denfafa, 361

Drugs

acquired resistance, 509

effects on metabolism, 485
DruppuJa, 218
DuboscqeUa, 150
Duboscqia, 322

kgeri, 320, 321
Dujardin, F., 105
Dunalielh, 152
Dysniorphococcus, 155

variabi Jis, 1 55
Dysterfa, 345

navicuJa, 344
Dysteriidae, 345

Echinocystis, 289
Echinomera, 285
Echinophrya, 423

honida, ^21
Echfnospora, 301

labbei, 299
Ehrenberg, C. G., 104
Eimeria, 301

stiedae, 299
Eimeriida, 297
Eimeriidae, 300
E/aeorhanis, 209
ElastcT, 212

greefi, 211
Eleutheropyxis, 131
Elliott, A. M., 441
ElJobiophrya, 410

donacis, ^oj
life-cycle, 406
Ellobiopsidae, 150
EUobiopsis, 150
EJytrop/astron, 395

bubab', 395
Embadomonas (see: Retortomonas)
EncheJydfum, 353

ampbora, 352
Enchehodon, 347

e/egans, 347
Encbe/yomorpba, 339

vermicularis, 340
Enche/ys, 347

gasterosteus, 347
Encystment, 75

factors inducing, 76

precystic changes, 75
Endamoeba, 241

granosa, 240

simuJans, 240
Endamoebidae, 240
Endodiniiim, 150
EndoJfmax, 241

nana, 551, 552

termftfs, 240
Endomixis

genetic significance, 518

Paramecium aureb'a, 94
Endosome, 46
Endospbaera, 420

engeJmanni, ^18
Endotbiya media, 2^6

Index 663

Eneithecoma, 371

properans, 372
Engelmann, T. W., 95
Enop/astron, 395
Enriques, P., 96
Entamoeba, 241

buccaJis (see: gingiVaJis)

coU, 554, 556

gingivaJis, 545, 546

histolytica, 555, 556
invasion of tissues, 557

invadens, 36, 240
Enterocystis, 289
Enteromonas, 182
Entodiniomorphina, 393
Entodinium, 395

bfconcavum, 394
Entodiscus, 356

borea/is, 357
Entorhipidiidae, 355
Entorhi'pidium, 356

echini, 357

mu/timicronucJeatum, 357

tenue, 557
Entosfphon, 168

sulcatum, 167
Enfzia, 145
Eodinium, 395

poiygonale, 39-^
Epalcidae, 401
EpaJxis, 401

striata, 402
Ephelota, 423

gemniipara, 421
Ephelotidae, 423
EpicaneJJa, 392
EpicJintes, 398
Epicone, 139
Epfcranel/a, 395
Epicystfs, 126
Epidinium, 395

caudatum, 394
Epimecophrva, 364
Epimerite, 273
EpioTclla, 392
EpipJastron, 395
Epiplocvlidae, 392
Epip/ocy /is, 392
Epipy.vis, 126
EpirhabdoneJJa, 393
EpirhabdoseJ/a, 393
Epistylidae, 408
Epistyiis, 409

chr)'seinidis, 404

horizonta/is, telotroch, 404
Epitheca, 139
EremopJastron, 395

bovis, 395
Erythropsis

cornuta, 34

extrudens, J38
EschaneustyJa, 398
Espe/oia, 364

mucicoJa, 365
Eucamptocera, 348
Euchitonia, 218

Euchrysomonadina, 125
Euciliatia, 337
Eucomonympha, 195

inula, 194
Eucyrtidium, 218

cranioides, skeleton, 217
EudipJodinium, 395

maggii, 394
Eudorina, 157
Eug/ena, 166

giaciJis, 16^

apochlorotic strains, 164

oxyuris, 163

piscifoimis, 163

socia bib's, 163

tripten's, 163
Eug/enamorpha, i66
Euglenida

chromatophores, 160

diagnostic features, 118, 160

flagella, 162

food requirements, 447

life-cycles, 162

paramylum, 37, 162

subdivisions, 164

"trichocysts," 28
Eug/enocapsa, 167
Euglenoidina, 166
Euglypha, 250

alveolata, 9

aspera, 243
Euglyphidae, 250
Eugregarinida, 282
Euheterochlorina, 133
Eumycetozoina, 230

life-cycles, 231
Euphysetta, 219
Eup/otaspis, 398, 401

cionaeco/a, 397
Euplotes, 398

harpa, 399
Euplotidae, 398
Eup/otidium, 398

agitatum, 397
Eurycbiium, 364
Eurysporina, 316
Eutintinnus, 393

brandtf, 392
Eutreptia, 166

viddis, 165
Eutreptie/Ja, 166

marina, 163
Eutrichomastix (see: Monocercomonas)
Excystment

Bursaria truncate/Ja, y8

Didinium nasutum, 77

factors inducing, 79

mechanisms, 76
Exuvielh, 142

perforata, 1^1

Fabrea, 388

salina, 389
Faure-Fremiet, E., 333, 360, 410
Favella, 392
Favellidae, 392

664 Index

Fibrillar systems

ciliates, 20

functional significance, 24
Filopodia, 11
Fischerina helix, 2^6
Fission

ciliates, 58

continuity of basal granules, 56, 60

flagellates, 56

reorganization (ciliates), 59

resorption of organelles, 56

Sarcodina, 58

self-reproducing organelles, 56

stoi7iatogenesis (Tetrahymenidae), 60
FhbeUiih, 240

mira, 239
Flagella

behavior in fission, 58

structure, 12

types, 13
Flagellosis, 550

laboratorj' diagnosis, 568
Foai'na, 187

taenioJa, 188
Foettingeria, 376

actinarium, 375
Fo//icuJina, 385

acuieata, larva, 38^

vnidis, 384
Folliculinidae, 385
FollicuUnopsis, 385

producta, 384
Fonseca, F. da, 650
Fonsecaia, 287

Food requirements (see also: Vitamin require-
ments)

Chrysomonadida, 445

ciliates, 448

Cryptomonadida, 445

determination of, 431

Dinoflagellida, 445

Euglcnida, 447

minerals, 435

Phytomonadida, 446

Protomastigida, 447

Sarcodina, 448

Trichomonadida, 448

Trypanosomidae, 447
Food reserves

lipids, 38

polysaccharides, 37

proteins, 38
Foraminiferida

diagnostic features, 2 50

feeding habits, 252

life-cycles

alternation of generations, 257
duration of, 262
gametogenesis, 259

pseudopodia, 250

taxonomy, 262

tests, 254
FoTtiella, 153
Fowler, E. H., 524
Freitas, G. de, 590
Frenze/ina, 249

Fienzelina (Cont.):

leniformis, 2^j
Frontonia, 364

leucas, 363, ^6^

paiva, 365
FiontonieUa, 364
Frontoniidae, 364
Frye, W. W., 555
Fu/igo, 233

septica, 235
FurciUa. 152
Furgason, W. H., 364

Gamoc\stfs, 285
Ganymedes, 2 88

anapsides, 28S
Ganvmcdidae, 288
Gargariiis, 374

gargaritis, 373
Garnhani, P. C. G., 303
Gastrocirrhus, 398

steiitoreus, 400
GastrostyJa, 398

steinii. 399
Geiman, Q. M., 548
Geliella, 408

vagans, 404
Geneiorh)nchus, 282
Geph^ramoeba, 226

de/icatii/a, 226
Giaidia. 1S4

Janib/ia, 549, 550

mun's, 1S5
Gigantoch/oris, 153
Giganfomonas, 187

hercuJea, 72
Girdle, 138
Glaser, R. W., 449
Ghucowa, 367

scinti/Jans, 366
Glenodiniidae, 145
GJenodiniopsis, 145

steinii. 146
GJenodfnium, 145

cinctuin, 1^^
Gleochloiis, 134
Gleoc\stis stage, 7
Gleodiniidae, 148
GJeodfnium, 148

niontainim, 1^8
Gleowonas, 153
G/ossate//a, 410

tiiitinnabu/uni, ^o^
Giugea, 322

acerinac, 320
Glycogen, 58

distribution in fission (Stentor), 55
Golgi material (see: Osmiophilic inclusions)
Goniocoma, 371
Gonfum, 157

pectoraJe, 157

development of colony, 157

socfaJe, 5
Gonospora, 290

varfa, 289
Gonostomum, 398

Index 665

Gonyaulacidae, 145
Gonyauhx, 145

acateneUa, 9
Gonyostomutn, lyo

semen, 169
Gorgonosoma, 422

arbuscuh, 419
Gregarina, 285

rigida, syzygy, 2J^
Gregarinidae, 285
Gregarinidia, 271

life-cycles, 274

morphology, 271

spores, 276

dispersal of, 276, 278
Gregory, L. H., 96
Growth of Protozoa

cultures, tj'pes of, 473

"diauxie," 480

individual organisms, 475

light vs. darkness, 484

pH optimum, 480

pH relationships, 478 (table, 479)
Astasia /onga, 480, 481

populations, 474
death phases, 477
initial stationary phase, 475
lag phase, 475
logarithmic growth, 476
maximal density, 476
negative growth acceleration, 476
size of inoculum, 477
Gruberia, 388

calkinsi, 389
Gur/eya, 322

richardi, 320
GuttuJina, 228
GuttuJinopsis, 228
Guyenotfa, 319
Gymnodiniidae, 143
Gymnodinina, 143
Gymnodfnioides, 376

inJ:}'Stans, 375
Gymnodinium, 143

catenatum, chain, 141

dissimile, 142

dorsum, 138

racemosus, 158
Gymnostomina, 338
Gyrodinium, 143

dorsum, 13

me/o, 142

submarinum, 142
Gyromonas, 184

ambulans, 18^

Haematococcidae, 155
Haematococcus, 155

pluvfaJis, 155
Haemogregarina, 296

stepanowl, 297
Haemogregarinidae, 296
Haemogregarinina, 296
Haemoproteidae, 305
Haemoproteus, 306

columbae, 305

Haemosporidia, 301
Ha/opappus, 130
HaJterfa, 390

ge/eiana, 390
Halteriidae, 390
Hance, R. T., 508
Haplosporidia, 326
Haphspoiidium, 328

cauiieryf, 328

cernosvftovi, 327, 328

chftonfs, 328

heterocfrrf, 328
HapJozoon, 1 50

c/ymeneJJae, 149

dogieli, 149
Haptopbr}'a, 379

chain-formation, 8

contractile tube, 32

michiganensis, 377
Haptophryidae, 379
Harrison, J. A., 524
Hartmann, M., 108
HartmanneJJa, 240

kJitzkei. 239
HartmannuJa, 345

entzi, ^^^
Hastatella, 408

radians, 404
Hatt, P., 279
Hawking, F., 303
HedTiocystis, 212

peiiucida, 205, 210

retfcuJata, 211
He/eopera, 249

picta, 2^j

rosea, 243
Hehaktis, 132
HeJiapsis, 130

muta bill's, 131
HeJicoprorodon, 347

gigas, 546
Helicosporida, 322
HeUcospoTidium, 322

parasiticum, 323
Helicostotna, 370
HeJicostomeUa, 391
Heliobodo, 171

radians, 172
HeUochona, 412

sessilis, 413
HeUocbTysis, 132

erodians, 1 3 1

sphagnkoh. 131
Helioflagellida, 202
Heliozoida

diagnostic features, 203

feeding habits, 206

life-cycles, :c6

test, 204
HemicycJiostyJa, 398
Hemidinium, 145

nasutum. 144
Hemispeira, 374

asteriasi, 373
Hemispeiridae, ^5-1
Hemitrichia serpu/a, 233

666 Index

Hemixis, 94
Henneguya, jiy

magna, ^1^
Hentscbeha, 285

thaJassemae, 28^
Hepatozoidae, 296
Hepafozoon, 297
adiei, 295
cam's, 295
muris, 295
Herpetomonas, lyj

muscarum, ij6
Herpefophrya, 379
Hertwig, R., 95
Heteroautotroph, 435
Heterocapsina, 134
Heterochlorida

diagnostic features, 117, 133
encystment, 133
Heterocineta, 371

phoTonopsidis, 372
Hetemcinetopsis, 371
Heterocoma, 37^
Heterodiniidae, iaj
Hetewdinium, 147

scrippsi, 1^^
Heteiohgynion, 131

oedogonii, 1^1
Hetewmastix, 152
Heteronema, 168

acus, 166
Hetewphiys, 209
m}TJopoda, 209
Heterotrichina, 381
Hexaconus, 218
//exactmomyxon, 319
Hexamastix, 187

termopsfdfs, 186
Hcxainita, 184
gigas, 185
pitheci, 18^
Hexamitidac, 1 84
He.vamitus puJcher, 13
Hinshaw, H. C, 546
Hirmoc)-stis, 285
Hirschheld, H., 410
Histioba/antfum, 370

semisetatum, 369
Histiona, 126, 175
Histomonas, 171

meJeagrfdfs, 172, 173
Histiio, 399
Hoare, C. A., 297
Holdfast organelles, Astomina, 378
Hollande, A., 33, 164, 166
HoJocoma, 371
Holomastigotes, 193
Holomastigotidae, 193
7:foJomastigotoides, 193
chromosome cvcle, 63
hemi'gymnum, 193
HoJophr)'3, 347
coronata, 346
ob/onga, 346
Holophryidae, 345
Holophrj'oides, 342

Holosticba, 398
Icess/eri, 399
novitas, ^00
Holotrichida, 338
Homa/ogastra, 364

setosa, 363
Homa/ozoon, 353
Hopkins, D. L., 555
HopUtophr)-a, 379

secans, 378
Hoplitophryidae, 379
Hoplonyinpha, 193

natator, 192
Hoplonymphidae, 192
Host-specificity, 532
Hsiung, T.-S., 342
Huff, C. G., 303
Hutner, S. H., 445
Hya/obr\'on, 127
iauterbornif, 120
ramosiim, 6
Hyalocephalus, 166
Hyalodiscus, 226, 240

rubicundus, 225
Hya /ogo ni u m , 153

klebsii, 154
Hya/osphem'a, 249

cuneata, 2^6
HyaJospora, 285
Hyalosponna, 285

cambo/opsisae, 28^
Hydramoeba, 241
hydroxena, 240
Hydruridae, 133
Hydrurus, 133
Hyman, L. H., 3
Hymenomoiias, 130

roseo/a, 129
Hymenostomina, 362
Hyperamminoides elegans, 255
Hyperdevescovina, 187

mitrata, 188
Hypermastigida, 190
Hypnodinium, 149
sphaeTicum, 1^8
Hypnomonas, 153
Hypocoma, 374
parasitica, 373
Hypocomaga/ma, 371
Hypocomatidium, 371

sphaeiii, 372
HypocomeWa, 371
Hypocomidae, 374
Hypocomidium, 371
Hypocomina, 371
tegu/arum, 372
Hypocomoides, 371

my till, 372
Hypocone, 139
Hypotheca, 1 39
Hypotiichidium, 398

conicuin, ^00
Hypotrichina, 396
Hysterocfneta, 367

eisem'ae, 368
Hysterocinetidae, 367

Index 667

Ichthyophthiiius, 370

muhiEUis, 368
Icbthyospoiidium, 328
Idi'onynipha, 193
I/eonema, 347
Immunity (see: Resistance)
Infections, 535
Infraciliary network, 24
Infraciliature, 24
Inquiline, 528
Jnsfgnicoma, 371

venusta, 372
Intoshellina, 380

poJ/ansky, 380
Intoshellinidae, 379
Jntrasfy'ium, 411
lodamoeba, 241

biitschlif, 552
Jri'dia

diaphana, 251, 259

Jucida, 260

seiialis, 253, 259
Irradiation

radium, effects of, 488

spectra, 486

ultraviolet, effects of, 487

X-rays, effects of, 488
Isocomides, 371
Isospora, 301

belli, 565

bigemfna, 299

hominis, 564, 566
Isotn'cha, 357

bubaJi, 358

intestinaJis, 358
Isotrichidae, 357

Jahn, T. L., 113, 115, 451, 452
James, S. P., 303
Jarrina, 301

paludosa, 299
Jennings, H. S., 91, 92, 97, 507, 512
Joenia, 190
Joeinidae, 190
Joenina, 190
Joenopsis, 190
Johnson, G., 569
Jollos, v., 510
Joukowsky, D., 95

Kahl, 339, 362, 364, 401
KahJia, 398

costata, 400
Kala-azar, 577
Karyolysidae, 297
KaryoJysus, 297

7acertarum, 297
Karyomastigonts, 18

multiple, 18
Katharobes, 431
Kellersberger, E. R., 587
Kent, W. S., 105, 106
Kentrochona, 412

nebaJiae, 413
Kephyrion, 126

spiraJe, 120

Kephyriopsfs (see: PseudoJcephyrion)
Kerona, 398

polyporum, 400
Keronopsis, 399
KhawJcinea, 167
Kidder, G. W., 442, 450
Kidderia, 371
Kikuth, W., 641
Kimball, R. F., 93
Kinetoplast, 1 8
Kirby, H., 180, 548
KiibyeUa, 184
Klebsiella, 167

alligata, 161
Kline, A. P., 449
Klossia, 296
Khssiella, 296
Klossiellidae, 296
Kofoid, C. A., 3, 145, 391, 546
Kofoidei/a, 379
Kofoidia, igi

loiiculata, igi
Kofoidiidae, 190
Kofoidina, 285
Korschikoffia, 1 52
Krernastochrj'Sis, 126

pendens, 124
Krichenbauer, H., 164
Kudo, R. R., 110
Kupferberg, A. A., 569
Kybotfon, 131

LaackmannieJJa, 391
Laboratory diagnosis

leishmaniasis, 580

malaria, 621

Protozoa of intestine, 568

Protozoa of mouth, 568

Trichomonas vagina/is, 569

Trypanosomiasis, African, 587

Tr\panosomiasis, American, 594
Labyrinthomyxa, 221
LabyrinthuJa, 221

macrocystis, 220, 221

zopfi, 220, 221
Labyrinthulidae, 220
LachmanneJIa, 379
Lacrymaria, 347

olor, 347
Lagenoeca, 175

gJobuJosa, 174
Lagenophryidae, 409
Lagenophrys, 409

Jabiata, ^o^
Lagynion, 131

subovatum, 131
Lagynophrya, 347

simp/ex, 346
LamborneJJa, 364
Lanipoxanthiuni, 218
LankestereJ/a, 301

minima, 300
Lankesterellidae, 301
Lankesten'a, 287

culicis, 288
Laivulina, 370

668 Index

Lecanophrya, 423
Lecbriopyla, 360

mystax, 361
Lecquereusj'a, 249

spiralis, 243
Lecudina, 285

pe/Jucfda, 284
Lccudinidae, 285
Lecytbion, 285

fha/assemae, 283
Lecytbium, 265

granu/atum, 26^
Legendrea, 353

Joyezae, 352
LegereHa, 296
Legerellidae, 296
Legeria, 282
Leidyana, 285
Leishmania, 177

brasiJiensfs, 579

chagasi, 576

chamae/onis, 176

donovani, 575, 576

tropica, 576, 578
Leishmaniasis

agglutinin tests, 645

causative organisms, 574

chemotherapy, 581

complement-fixation tests, 647, 648

control, 581

laboratory diagnosis, 580

muco-cutaneous, 579

oriental sore, 578

transmission, 579

visceral, 575
Lembadion, 364

biiJJinum, 363
LembadioneJJa, 364
Leniboides (see: Paralembiis)
Lepiswatophih, 286
Lepochromu/ina, 126
LepocincJis, 167

maissoni, 165
Leptomonas, 177

ctenocephali, 17

patellae, 176
Leptomyxa, 224

reticulata, 224
Leptospironympha, 193
Leptotheca, 316

oblmacheri, 312, 313
Lernaeophrya, 422

capitata, 419
Leucocytozoon, 306

coccyziis, gametocytes, 305
Leucophra, 362, 367
Leucophrydium, 364
Leucophrys (see: Tetrahymena)
Leucoplasts, 33
Leucosin, 38
Leukapsis, 130
Leukopyxis, 131
Licea, 233
Lichnophora, 385

macfarlandi, 386
Lichnophoridae, 385

Lieberkiihnia, 265

wagneri, 263
Life-cycles

dimorphism, 73

general types, 71

meiosis in {table), 80

physiological, 94

polymorphism, 73

significance of, 73
Ligniera, 230
Lionotus, 342

hsciola, 340
Lipids, synthesis of, 470
Lipocystis, 282

polyspora, 280
Lipotropha, 282

macrospora, 280
Lissodiniidae, 145
Lithocircus, 218

annularis, skeleton, 217
LithocoUa, 209
Lithocystis, 290

brachycercus, 289
Lithoiopus, 218
Lithoptera, 218
Lobomonas, 153

rostra ta, 1^^
Locomotion, 489

amoeboid movement, 489

ciliates, 491

flagellates, 490
Lobopodia, 11
Lohmannie//a, 391

eJegans, 390
LophocephaJus, 287
Lophomonadidae, 190
Lophomonadina, 190
Lophomonas, 190

striata, 191
Lorica, 10

Tintinnina, 391
Loxocepha/us, 367

colpidiopsis, 365
Loxodes, 348

lostTum, 30

striatus, 347
Loxodidae, 348
LoxophyJJum, 342

lostiatum, ^^o
Ludio, 385
Luminella, 392
Lwoff, A., 371, 380, 449
Lycoga/a, 233
Lyramula, 130

McDonald, }. D., 563

MacDougall, M. S., 508

MachadoeJJa, 282
triatoinae, 278

Macromastix, 182
Japsa, 181

Macronucleus

behavior in fission, 68
"dispersed" type (Di/eptus), 49
elimination of chromatin, 69
functional significance, 48

Index 669

Macronucleus (Cont.) :

genetic significance, 520

morphology, 47
Macrospironympha, 193
Macrotrichomonas, 187

lighti, 188
Mahcophiys, 364
Malaria

blackwater fever, 620

causative organisms, 600

chemotherapy, 621

complement-fixation tests, 647

control, 623

distribution, 597

duration of attacks, 619

duration of infections, 619

effects in man, 617

fevers, 616

incubation periods, 614

laborator)' diagnosis, 621

North America, 598

paroxysms, 61;

periodicity of paroxysms, 617

pernicious, 618

prodromal symptoms, 615

relapses, 619, 632

rigors, 615

transmission, 613
Ma//eoch/oris, 153
Ma/Jeodendron, 134
Mallomonas, 126

dentata, 120
Margarita, 233
Marsupiogaster, 168

striata, i6y
Maryna, 360

socfaJfs, 358
Marynidae, 357
Massartfa, 143
Mastfgamoeba, 171
Mastigel/a, 171

poJymastfx, 172
Mastigina, 171

hyJae, 172, 173
Mastigonts

dissociation from nuclei, 18

organization, 18
Mating types

cytoplasmic inheritance, 523

EupJotes patella, 93

inheritance
autogamy, 519
conjugation, 514

Paramecium aureJia, 92

P. bursaria, 92

P. caudatum, 93
Maupas, E., 90, 91, 95
MaupaseJJa, 380

criodrili, 380
Maupasellidae, 380
MayoreJ/a, 240

conipes, 238
Medusetta, 219
Meiosis (table), 80

conjugant, 81

gametic, 80

Meiosis (Cont.) :

Myxosporida, 314

zygotic, 81
Meleney, H. E., 557
Membranelle, 19
Membranosorus, 230
Menoidium, 167

cultellus, 16^

obtusum, i6j
Menospora, 285

polyacantha, 284
Menosporidae, 285
Merocystis, 298
Merodfnium, 150
Merogregarina,

amaroucii, 2 78
Merose/enidium, 282

keilini, 280
Merotrichia, 170

capita ta, 169
Meseies, 390
MesniieHa, 379

muJtispicuIata, 378
Mesocena (see: Dictyocha)
Mesodinium, 345

acarus, 344
Meso/oenia, 190
Mesostigma, 152

vfride, 153
Metabolism

carbohydrate, 466

effects of drugs, 485

nitrogen, 465

Metacineta, 423
Metacoronympha, 188
Metacryptozoites, 303
Metac}cb's, 392
Metacystidae, 348
Metacystis, 348

cJongata, 349
Metadevescovina, 187

modica. 188
Metadinium, 395

medium, 395
Metafo/iiculina, 385
Metamera, 285

reynoJdsi, 283
Metaphrya, 379
Metaradiophrya, 379

asymmetrica, 377
MetasaccinobacuJus, 184
Metastwmbidium, 390
Metopidae, 385
Metopus, 385

mathiasi, 386
Metz, C. B., 516
Microfol/icuJina, 385

b'mnoriae, 384
Microgromia, 265

e/egantuJa, 263
Micro/oenia, 190

ratcb'ffei, 191
Miciometes, 265

paludosa, 263
Micronucleus

amicronucleate ciliates, 49

670 Index

Micronuclcus (Cont.) :

functional significance, 48
mitosis, 65
origin (ontogeny), 47
Aficroregma, 347
MicrorhopaJodina, 184

mu/tinucJeata, J 83
Microsporida, 319
life-cycles, 320
spores, 319
Miciothorax, 362

viridis, 361
Middleton, A. R., 507
MikrogJena, 126
MiJJiamina lata, 256
Minchin, E. A., 108
Mineral requirements, 435
Mitochondria

cytoplasmic distribution, 40
functional significance, 40
types, 40
Mitosis

achromatic figures, 66
eumitosis, 63
HoJoniastigotoides, 63
interphase chromatin, 62
micronuclcus, 65
nuclear membrane, 65
origin of chromosomes, 62
paramitosis, 63
Mitraspora, 316

cyprini, 315
Mixotricha, 195
Moewus, F., 82, 511
Mohr, J. L., 412
Monadodendron, 127

dfstans, 122
Monas, 126

vestita, 121
Monocercomonadidae, 187
Monocercomonas, 187
phyj/opnagae, 1 86
verrens, 186
Monocercomonoides, 184

pilleata, 14, 183
MonochiJum, 364
Monocystidae, 288
Afonocystis, 290

agilis, 288
Monod, J., 480
Monodfnium, 345
Monodontophrya, 380

Icijenskiji, 580
Monoductidae, 286
Monoductus, 286

Junafus, 284
Monomastigocystis, 212

brachypous, 210
Monopylina, 218
Monosfga, 175

angustata, 174
Mrazekia, 322

lumbricuU, 320
Mrazekiella, 379

intermedia, 378
Afrazekiidae, 322

Mugard, H., 362, 367, 370

Miiller, O. F., 94, 104

Mailer's vesicles (see: Sensory vacuoles)

AluJticiJia, 171

AfuJtifascicuJatum, 420

eJegans, 417
Muniz, J., 590, 646
Musgrave, W. E., 558
Mussel poisoning, GoynauJax, 536
Mutations, 508
Mycetozoida, 227
Mycterothri'x, 360

erJangeri, 358
Mylestoma, 403

anatinum, 402
Mylestomidae, 402
Myonemes, 25
Afyriophrjs, 210
Alyriospora, 298

trophoniae, 298
Myxidiidae, 316
Aiyxidium, 317

meJum, 315
Alyxobilatus, 317

asymrnetricus, 312
Myxobolidae, 317
AlyxoboJus, 317

osburni, 321
MyxochJoris, 133

sphagnicoJa, 1^^
Myxochrysidae, 132
Alyxochrysis, 132

paradoxa, 124
AfyxophyJJum, 374
Afyxopodia, 11
Afyxoproteus, 316

cornutus, 315
Myxosoma, jr)

olcobo/ienjis, 315
Myxosomatidae, 517
Myxosporida, 311

life-cycles, 312

spore formation, 314
Myxotheca, 265

NadineHa, 249

tenelJa, 247
NaegJeria, 254

gruberi, 235

tachypodia, 235
NageJielJa, 13^

natans, 152
Nageliellidae, 133
Nannophr}a, 347
NassuJa, 348

graciJis, 349
Nassulidae, 348
Nautococcus, 153

mammiJatus, 1^4
NebeJa, 250

vitraea, 249
Nematocystis, 290

anguiUuh, 272
Nematocysts, 28
Nematopsis, 287

Jegeri, 275, 286

Index 671

Neoactinomy.xon, 319

glohosum, 519
Nephrfdiophaga, 328
Nephrochloiis, 133

salina, 1^^
Nephroselmidae, 137
Nephioselmis, 137

olivacea, 137
Neuromotor apparatus, 21
Neuroneme system, 23
Neuschloss, S., 509
NeviUina coronata, 256
NicoUelh, 349

ctenodactyJi, 350
Nina, 285

gracilis, 283
Nitrogen metabolism, 465
Nitrogenous excretion, 470
Noble, E. R., 546
Noctiluca, 143

scintillans, 142
Noctilucidae, 143
Nosema, 322

eJongatum, 320

termitis, 320, 321
Nosematidae, 322
Notoso/enus, 168

apocamptus, 16 j
Nucleaiia, 226

cauJescens, 226
Nuclei

dimorphism, ciliates, 46

vesicular, 45, 46
Nutrition (see also: Food requirements), 429

autotrophic, 430 (table, 433)

chemoautotrophic, 433

heteroautotrophic, ^33

heterotrophic, 430

holozoic, 430

photoautotrophic, 433

saprozoic, 430
Nyctotherus, 388

cordiformfs, 387

development of macronucleus, 88

Icyphodes, 387

Ocellus, 37
Ochromonadiaae, 126
Ochromonas, 126

granularis, 119, 121

pinguis, iig

leptans, iig
Ochryostyhn, 126
Octomitus (see: Hexamita)
Octosporea, 322

bayeri, 320
OdontophoreJIa, 393
OiJcomonas, 126

termo, 121
Oligotrichina, 390
OJithodiscus, 133

Juteus, 134
Onychodromopsis, 398
Oocephalus, 287
Onychodromus, 398

grandis, 399

Oocysts, Coccidia, 292
Oodiniurn, 1 50
Oopyxis, 249
OpaJina, 337

obtrigonoidea, 335

ranarum, 335
Opalinidae, 336
Opalinopsis (see: Chromidina)
Opercu/aria, 409

ramosa, ^o^
Ophfuraespira, 376
Ophrydiidae, 410
Ophrydium, 410

g/ans, 405

Jemnae, 405
Ophr}'ocephaJus, 423
Ophryocystidae, 279
Ophryocystis, 282

mesniJi, 278

schneideii, 278
Ophryodendridae, 423

Ophryodendron, 423

vermiform bud, 421
Ophr}0gJena, 370

atra, 368
Ophryoglenidae, 367
Ophryoscolecidae, 393, 395

membranelles, arrangement, 394

skeletal plates, arrangement, 394
OphryoscoJex, 395

caudatus, 394
Opistbomitus (see: Oxymonas)
Opisthotricha, 399
Opisthotiichum, 395
OrcadeUa, 233
Orchitophrya, 379
Oriental sore, 578
OimoseUa, 393
Orosphaera, 218
Orthodon, 348

hamatus, 349
Osmiophilic inclusions, 43
Ostenfe/diel/a, 230
Ostracodfnium, 395

cJipeoIum, 395
Ovivora, 298

tha/assemae, 291, 298
gametes, 83
schizogony, 55
Oxidation-reduction potentials, 451

culture media, 452

internal, 452
Oxidations, 454

adenosine phosphate system, 458

catalase, 457

cytochrome pigments, 455

cytochrome system, 455
poisoning techniques, 455

diphosphothiamine enzymes, 457

fiavoprotein enzymes, 457

glutathione, 457

pantothenic acid enzymes, 458

peroxidase, 457

pyridine nucleotide enzymes, 456

pyridoxine enzymes, 4157

tricarboxylic acid cycle, 458

672 Index

OxnereUa, 209
Oxygen consumption

applications of data, 452

factors influencing, 453
Oxygen relationships

ecological distribution, 450

growth of cultures, 451

parasites, 450
Oxymonas, 184

di'morpha, 18 j
Oxyphysis, 147
OxyrrhiS, 143

marina, 141

tentacii/ifera, 1^1
Oxytiicha, 398

p/atystoma, 399
Oxytrichidae, 398

Packchanian, A., 528
Palatinella, 126
Palm, B. T., 230
Pa/marium, 385
Palmella stages, 7
Painphagus, 249
mutabilis, 246
Pandonna, 157
morum, 157
Parabasal apparatus
behavior in fission, 58
free-living flagellates, 16
structure, 16
ParaWepharisma, 388
bacteriophora, 3S9
Parachaenia (see: Ancistrocoma)
Paracineta, 420 (sometimes assigned to Podo-
phryidae, 423)
p/euromammae, 422
Parac/eveJandia, 383

brevis, 384
Paradesmose, 66
Paradileptus, 353

conicus, 352
Paradinium, 150
Paraeijg/ypha, 250

reticulata, 248
Paraeup/otes, 400
tortugensis, 400
Paraeuplotidae, 400
Parafave/Ia, 393
ParafoJhcu/lna, 385

hiiundo, 3S4
Parag/aucoma, 367
rostrata (?), 366
Paiaholosticha, 398

ovafa, 400
Parahypocoma, 374
Paraisotricha, 360

minuta, 358
Paraisotrichidae, 360
Paraisotrichopsfs, 342

composita, 341
Para/oenia, 187

giassii, 186
Para/embus, 370
Parameciidae, 360

Paramecium, 360
aure/ia, 359
endoniixis, 94
hemixis, 94
mating types, 92
bursaria, 359

mating types, 92
calkinsi, 359
caudatum, 359

mating types, 93
muJtimicronuc/eatum, 350
po/ycaryum, 359
tiichium, 359
woodruffi, 359
Paramylum, 37
ParanassuJa, 348

microstoma, 349
Paranyctotherus, 388

kirbyi, 387
Parapodophr)'a, 423

atypica, 422
ParapoJytoma, 153
Parasitism, 527

evolution of parasites, 530
host-specificity, 532
infections, 535
parasites of man

geographical distribution, 537
parasites of Protozoa, 535
taxonomic distribution, 533
transfer of parasites, 537
Paraspathidium, 353
Parastrombidium, 391
ParavorticeJJa, 410
clymenellae, ^o^
PaimuJina, 249
cyathus, 247
Parthenogenesis, 80
ParundeUa, 393
Paruro/eptus, 399
Pascher, A., 124, 152, 511
Pascherie//a, 156

tetras, 1 56
PaulineUa, 250

chroniatophora, 248
PaviUardia, 143

tentacuJifera, 142
Pavonina Habellifoimis, 255
Pearl, R., 512
Pearse, A. S., 115
PebiiUa, 385
Pedinella, 126
Pedinomonas, 152

minor, 153
Pedinopeia, 155
Pediostomum, 388
Pedogamy, 80

Heliozoida, 84
Pe/atotricha, 392

ampuJJa, 392
Pelatotrichidae, 392
Peiatractus, 348

constractus, 349
PeJecypohora, 574
Pellicle, 8
Pelodinium, 401

Index 673

Pelodinium (Cont.) :

renifonne, 402
Pe/oniyxa, 240

caro/inensfs, mitosis, 65

paJustris, 239
Pelta, 16
Pehomyxa, 230
PenardieUa, 355

unduhta, 352
Penfafrichon7onas. 190

hominis, 13, 548, 549
Pentatrichouiorioides, 190
Peranema. 168

perforatorium, 168

trichophoniin, 166
Peranemoidina, 167
Peranemopsis, 168

striata, 166
PerezeJ/a, 379
Pericaryon, 376
Peridiniidae, 145
Peridinina, 144
Peridinium, 145

kuhzynskii, 146
Periplast, 9
Peripylina, 218
Perispira, 353

ovum, 351
Peristome

peristomial organelles
EupJotes, 30
Oxytricha, 29
Paramecium, 28
StyJonychia, 29
Peritrichida, 333, 403

life-cycles, 405
Peritromidae, 385
Peiitiomus, 388

kahli, ^86
Perseia, 379

dogieJi, 377
Petalomonadoidina, 168
PetaJomonas, 168

dorsaJis, 167
Pfeiffeiinella, 301
Phacodinium, 388
Phacotidae, 155
Phacotus, 155
Phacus, 167

pJeuronectes, 165

pyrum, 163

quinquemarginatus, 16^

toita, 16-^
Pfiaeop/aca, 133
Phaeosphaera, 133
P/iaJacroma, 147
Phalansteriidae, 175
Pha/ansterium, 175

digitatum, 6
Phanerozoites, 303
Pharyngeal-basket, 31
Pharyngcal-rod apparatus, 31, 168

resorption in fission, 58
Phasco/odon, 342

vortice//a, 343
Phialoides, 282

Philastei, 570

digitiformis, 369
Philasteiides, 370

armata, 369
Philasteriidae, 370
Phorefophr}a, 376
Photoautotroph, 433
Photoreceptors (see: Ocellus, Stigma)
Pbractaspis, 218
Phryngaye//a, 249
Phtorophrya, 376
Phy//ocardium, 1 52
Phy/Jomitus, 180

amyJophagus, 179
PhyJ/omonas, 153
Physa/ophrya, 360
Physarum, 233

ieucopus, 233

po/ycephaJum, 232
Physematicum, 218
Physiological life-cycle, 94
Physomonas (see: Monas)
Phytodinediia, 149

procubans, 148
Phytodiniidae, 149
Ph)'todinium, 149
Phytomastigophorea, 117
Phytomonadida, 118, 150

food requirements, 446
Phytomonas, 177
Pigments

chromatophores, 55

Chrysomonadina, 3 5

cytoplasmic

lipoproteins, Stentoi, 35
protective, Eug/cna rubra, 35
toxic, B/epharisma, 35

Dinoflagellida, 35, 139

Euglenida, 35

Phytomonadida, 35
Pi/eocephaJus, 282
Pfnaciocystis, 210
Pinaciophora, 210

fluviatiJis, 208
Pipetta, 218
Pitelka, D. R., 168
Pithothorax, 347
PJacocista, 250

/ens, 249
PJacus, 348

socialis, 346
P?agfocampa, 348

Jongfs, 546

marina, 346
PJagfophrys, 249, 265

parvipunctata, 246
PlagiopyJa, 360

nasuta, 361
Plagiopylidae, 360
P/agiorhiza, 131
PJagiospira, 374
PJagiotoma, 388

Jumbrici, 386
Plagiotomidae, 388
Plakea stage, 1 57
Plasmodiida, 302

674 Index

Plasmodiida (Cont.) :

life-cycles, 303
Plasmodiidae, 306
Phsmodiophoia, 230
Plasmodiophorina, 228
Phsmodium, 306

circiimfle.xum, in mosquito, 302

cynomoigi, 602

eJongatum, 304

erythrocvtic phase, 604

exoerythrocytic phase, 303, 304, 601

fa/ciparuiii, 607

ga/h'naceum, 304

ma/ariae, 609

mosquito phase, 611

ovale, 610, 611

leUctinn, 304

tricarboxylic acid cycle, 469

vfvax, 605, 606
Plasmotomy, 56
PJatophrya, 348

spumacoJa, 346
PJatophrya (Suctorea), 422

rotundata, 421
P/a tychlorfs, 153
PJatychrysis, 127

pigra, 127
PJatycoJa, 410

Jongico//is, ^08
PlatydoTina, 157

caudata, 157

development of colony, 157
PJatymonas, 153

tetratheJe, 154
P/atynematum, 364

hyah'num, 363
Platysporina, 316
PJatytheca, 131
Pieodorina, 157
PJeurocoptes, 370
PJeurocystis, 290
P/euroinonas, 180

/acuJans, 179
PJeuronema, 370

setigerum, 369
Pleuronematidae, 370
PJeurotricha, 398

grandis, ^00
PJistophora, 322

intestina/is, 320
Poci/Jomonas, 152
PodactineJius, 218
Podocyathus, 423
Podolampidae, 145
Podophrya, 423

Exa, life-cycle, 415

parasitica, 422
Podophryidae, 423
Polar capsules, 311
Polyblepharidae, 1 52
PoJybJepharides, 1 52
Po/ydinium, 396

mysoreum, 396
Polykrikidae, 143
Po/ykriJcos. 143

schwartzf , 1 42

Polymastigida, 180
Polymastigidae, 184
PoJymastix, 184

phyJIophagae, 183
Po/ymorpha, 342

ampuJ/a, ^^1
Polypbiagma crihosum, 256
PoJypJastron, 395

muJtivesicuJatum, 395
Po/yrhabdina, 285

spion/s, 284
Poiysphondyb'um, 228
PoJyspira, 376
PoJytoma, 153
PoJytomena, 152

citii, 153
PompboJyxophrys, 210

punicea, 209
PontiguJasia, 249

incisa, 247
Pontosphaera, 130
Poiella, 142
Poroecus, 392
Porospora, 287

gigantea, 286
Porosporidae, 286

life-cycles, 278
PorostyJon, 132
Porpostoma, 370

notatum, 369
Porter, R. J., 303
Poteriochromonas, 127
Poteriodendron, 126, 175

petioJafum, 6
Pottsia, 420
Powell, W. N., 567
Preer, J. R., 522

Pringsheim, E. G., 114, 164, 166, 429
Prisma tospora, 282

evansi, 281
ProamphoreJJa, 393

PiohoscidieUa (see; AlicrorhopaJodina)
Proboieria, 374
ProJophomonas, 190

toco/pa, 191
PropJecteJ/a, 393
Proroccntrina, 142
Prorocentriim, 142
Prorodon, 348

parafarctus, 346

teres, 346
Prorodonopsis, 342

coli, 341
PiostelidieUa, 393
Proteomyxida, 220
Proteromonas, 180

Jacertae, 179
ProtoanopJophrya, 379
Protochrysis, 137

phaeophycearum, 137
Protociliatia, 334

geographical distribution, 337

life-cycles, 336

taxonomic relationships, 336
Protocrucia, 388

tuzeti, 389

Index 675

PTotocymatocyclis, 392
Protodini'fer (see: Protonocti/uca)
ProfomagaJhaesia, 285
Protomastigida, 173
Protomerite, 273
Protomonas, 221
Protonocti/uca, 143

tentacuJatum, 158
Protonoctilucidae, 143
Protoodinium, 150
Protoopalina, 336

intestinalis, 355

montana, 335
Protoopalinidae, 336
Protophna, 374
ProtophryogJena, 370
Protoradiophn'a, 379

fissispicuJata, 378
ProtorhabdoneJJa, 393
Protospongia, 175

haecJceJii, 174
Protrichomonas, 187
Protympanium, 218
Provasoli, L., 445
ProwazeJceJJa (see: Protcromonas)
Provvazekia (see: Bodo)
Prymnesiidae, 127
Prymnesium, 127

parvum, 127
Psammonvx vuJcanicus, 256
Pseudastrorhiza sihirica, 255
PscudobJepharisma, 388
Pseudobodo, 180

minima, 179
PseudochJamys, 249

pateJJa, 246
Pseudocyst, gregarines, 276
Pseudodevescovina, 187

unif?ageJ/ata, 188
Pseudodifflugia, 249

fu/va, 247
Pseudo/oJIicuIina, 385

aictica, 384
Pseudogemma, 420
PseudogJaucoma, 364
PseudoJcephyrion, 127

minutissimum, 120
PseiidoWossia, 298

Pseudomal/omonas (see: Ma?/omonas)
Pseudomincrothorax, 362

agibs, 361
Pseudoplasmodium (Acrasina), 227, 228
Pseudopodia, 11
Pseiidoprorodon, 348

emmae, 347
Pseudospora, 221

parasitica, 221, 222

rovignensis, 222

voJvocis, 222
Pseudosporidae, 221
Pseudosporopsjs, 221
Pseudostiombidium, 398
Pseudotricbomonas, 187

Jceib'ni, 1 86
Psetidotricbonympba, 195
Pseiidotrypanosoma, 190

Pseudotrypanosoma (Cont.) :

gigantea, 189
Psilotricba, 399
Pteridomonas, 171
Pteiomonas, 155

anguJosa, 155
Pterospora, 290
Ptychocyclidae, 393
Ptychocyclis, 393
Ptycbostomuni, 367

pygostoma, 368
Pusules, 139
Pycnothricidae, 348
Pycnotbrix, 349

n]onoc}'stoides, 350
Pyramidocbrysis, 126
Pyramidomonas, 152
Pyramimonas, 152

tetrarbyncbus, 153
Pyrenoids

fission, behavior in, 33, 58

functional significance, 33

morphology, 33
Pyrobotrys, 1 56

squarrosa, 1^6
Pyrotbeca, 322
Pyrsonympba, 184

minor, 18'^
Pyrsonymphidae, 184
Pyxicola, 410

entzi, ^08
Pyxidicu/a, 249

opercuJata, 246
Pyxidium, 409

cothurnoides, ^0^
Pyxinia, 282
Pyxinoides, 285

pugetensis, 283

Quadrula, 250
discoides, 2^8

RaabeHa, 371
Races, 506

characteristics, 506

induced changes, 508

spontaneous changes, 507
Raciborskia, 149
RaciborskieJJa, 152

urog/enoides, 1^^
Radiolarida, 212

central capsules, 212

colonial types, 214

life-cycles, 214

skeletons, 212

subdivisions, 216
Radiopbrya, 379

boplites, 378

Jum brief, 378
RaflFaele, G., 303, 602
Raffel, D., 508
Rainev's corpuscles, 325
Raphidiopbrys, 210

pallida, 208
Rapbidocystis, 210

infestans, 205

676 Index

Red tide, 137
Regendanz, P., 641
Reichenow, E., 110
Reichenowella, 388

nigricans, ^8j
Reichenowellidae, 388
Remanella, 348

margaritifera, 347
Reproduction, methods, 54
Resistance
acquired, 630
antibodies, 635
factors involved, 635
active immunization
Babesia, 632
Coccidia, 633
Leishmania, 631
Plasmodium, 632
Trypanosoma, 631
defensive mechanisms
malaria, 641
trypanosomes, 637
diet, influence of, 629
natural, 627

factors influencing, 628
individual variations, 628
racial differences, 627
passive immunization
Plasmodium, 634
Trypanosoma, 634
virulence of parasite, 630
vitamins, effects on, 629
Respiratory quotients, 453 (tabic, 454)
Responses to stimuli (see: Stimuli)
ReticuJaria, 233

Reticulopodia (see: myxopodia)
Retortomonadidae, 182
Retortomonas, 182
agiJis, 181
giyllotalpae, 181
intestinalis, 546, 547
Reuling, F., 567
Rhabdocystis, 290
Rhabdomonas, 167

incuTva, 165
RhabdoneJIa, 393

henseni, 392
Rhabdonellidae, 393
RhabdoneJIopsis, 393
Rhabdophn'a, 422
RhabdoseiJa, 393
Rhabdosphaera, 130
RhabdostyJa, 409

ovum, 404
Rhaphidomonas, 170
Rhinodiscii/us, 364
Rhipidodendron. 180
Rhizocarium, 379
Rhizochloridina, 133
Rhizoch/oris, 133

arachnoides, 135
Rhizochr\sidae, 130
Rhizochrysis, 130

planJctonica, 131
Rhizochrysodina, 1 30
Rhizomastigida, 171

Rhizomastix, 171

gracilis, iji, 173
RhizonubecuJa adherens, 255
Rhizopodea, 219
Rhizopodia (see: Myxopodia)
Rhodomonas, 137

baltica, 136

iacustris, 136
RhopaJonia, 285
Rhopaiophrya, 348
Rhynchefa, 423
Rhynchocystidae, 290
Rhynchocystis, 290

pilosa, 288
Rhynchogromia, 265

h'nearis, 263
Rhynchonympha, 193
Rhynchophrya, 423
Rhynchosaccus, 265
Rickeitia, 170

Saccammina fiagilis, 255
SaccinobacuJus, 184

doroaxostyJus, iS^
Salpingacantba, 393
Salpingella, 393

acuminata, 392
Sa/pingeJ/oides, 393
SaJpingoeca, 175

brunnea, 174
Sappinia, 228
Saprobes, 431
Saprodinium, 401

integrum, 402
Saphrophi/us, 364

putrinus, 365
Sarcocyst, 324
SaTcocystis, 324

lacertae, 325

miescheriana, 325

muns, 325

p/atydactyh, 325

tene/Ia, 325
Sarcodina, 202
Sarcosporidia, 324

molds, possible relation to, 326
Scaphidion, 345
Schaeffer, A. A., 226
SchaudinneJia, 290

henJeae, 272
Schaudinnellidae, 290
Schiller, J., 130, 160
Schizocystidae, 280
Schizocystis, 282

iegeri, 280
Schizogony, 56, 62
Schizogregarinida, 279
Schneideiia, 282
Schuhzella, 265
SchuJtzeliina, 380
Sciadophora, 282
Scopula, 403
ScourfieJdia, 153

compJanata, 154
Scyia, 285

Index 677

Scyphfdfa, 410

ameirui, ^05

physaTum, ^o^
Scvphidiidae, 410
Scytomonas, 168
Seguela, J., 398
Selection, effects of, 507
SeJenidium, 282

cauUeryi, 280
Seknochloiis, 153
Selenococcidiidae, 301
Se/enococcidium, 301

intermedium, 300
SeJenocystis, 282
Sensory bristles, 24
Sensory vacuoles, 32
Septicepha/us, 285
Serological reaction?

diagnosis of infections
adhesion tests, 649
agglutinin tests, 645
complement-fixation tests, 646
precipitin tests, 646
skin tests, 648

differentiation of species, 649
Sexual phenomena, varieties, 79
Sheath, flagellar, 12
Shells, 10
She//acJ:ia, 301

bolivaii, 300
Shortt, H. E., 303, 602
Siebold, C. T. E. v., 105
Siedleckia, 282
Silicoflagellina, 128
Silver-line system

ciliates, 21

flagellates, 2;
Sinodiniidae, 144
Sfnuolinea, 316

capsuJarfs, 315
SkadovstieIJa, 127
Skeletal plates

Colepidae, 342

Entodiniomorphlna, 393
Skeletons, Radiolarida, 11, 212
Slime-molds (see: Eumycetozoina)
SnydereJ/a, 188

tabogae, 1^
SoJenophrya, 420
Sonderia, 360

phar}'ngea, 361
SonderieJ/a, 360
Sonneborn, T. M., 92, 508, 512, 518, 520,

. 521, 523
Sorodiscus, 230
Sorosphaera, 230
Spasmostoma, 348

vi'ride, 346
Spathidiidae, 349
Spathidioides, 353

exsecata, 351
Spathfdium, 353

amphoriforme, 351
SpeJaeophrya, 423
Spermatozopsis, 1 52
Sphaeractinomyxon, 319

Sphaeractinomyxon (Cent.) :

gigas, 312, 319
SphaerelJopsis, 153
Sphaerocapsa, 218
Sphaerocystis, 286
Sphaerodinfuin, 146

limneticum, 146
Sphaeroeca, 175

volvox, ly^
Sphaeiomyxa, 317

baibiani, 315
Sphaerophrya, 423

magna, 42 1
Spbaeioibynchus, 287
Sphaerospora, 316
Sphaerosporidae, 316

poJymorpha, 315
Sphaerosporidae, 316
Sphaerosporina, 316
Sphaerotrichium, 391
Sphaerozoum, 218
SphenochJoris, 153
Sphenoderia, 250

lenta, 249
Sphenomonas, 168

teres, 167
Sphenophrya, 374

dosiniae, 373
Sphenophr)idae, 374
SpiriUina vivipaia, 259, 260
Spi'rocbona, 412

dcgans, 413

patella, 415
Spirochonidae, 412
Spiwcystis, 282
Spirodinium, 396

equi, 396
Spirog/ugea, 322

octospora, 320
Spirogonium, 153
Spirornonas, 180
Spiron}mpha, 193

portcri, 193
Spirophrya, 376

subparasitica, 375
Spirorhynchus, 385

verrucosa, 386
Spirostomidae, 388
Spirostomina, 388
SpiTOStomum, 388

teres, 389
Spirotrichida, 380
Spirotrichonympha, 193

bispira, transverse fission, 56

e/egans, 193
SpirotrichonympheHa, 193
Spfrotrichosoma, 193
Spirozona, 360

caudata, 358
Spirozonidae, 360
Spondylomoridae, 155
SpondyJomorum, 1 56

quaternarium, 156
Spongomonas, 180

uve/Ja, 179
Spongospora, 230

678 Index

Spores

Grcgarinidia, 277

Haplosporidia, 328

Microsporidia, 320

Myxosporidia, 315
Sporoblasts

Coccidia, 294

Microsporidia, 321

Myxosporidia, 313
Sporomyxa, 230

tenebrionis, 229
Sporozoa, 270
Sprince, H., 569
SquaJophrya, 420

maciostyh, ^18
Stabler, R. M., 546

Staborgan (see: Pharyngeal-rod apparatus)
Stalk-muscles, 25
Starch, stored, 38
StasziceUa, 146

dinobryonis, 1 46
Staurocyc/ia, 218
Stauro/oenina, 193

assimiUs, 192
Staurojoeninidae, 193
Stauro/oiiche micropora, 216
Staurophn'a, 422
Staurojphaera, 218
Steensfrupie/Ja, 393
StegochiJum, 364
Stein, S. N. F. v., 105
SteineJJa, 379
Steinia, 399
Steinina, 282

rotundata, 281
Stelidiella, 393
Stemonitfs, 233
Stem peJ/fa, 322

magna, 320
Sfenocodon, 127
Stenophora, 287

shyamaprasadi, 284
Stenophoridae, 287
Stenoseme/la, 391
Stentor, 388

auricuJatus, 389

felici, 389
Stentoridae, 388
Stephanocodon, 126
Stephanonympha, 188
Stephanoon, 157
Stephanopogon, 348

nuclei, 47
Stephanoporos, 132
Stephanosphaera, 155

phn'ialis, 155
Sterromonas (see: Monas)
Sticho/ormis, 218
Stichotricha, 399

nanJcingensis, 399
Stictospora, 282
Stigma, 37
Stimuli, responses to

electric current, 493

light, 493

temperature, 494

Stimuli, responses to (Cont.) :

typical reactions, 492
Stfpitococcus, 133

capense, 135
Stokesia, 364
StoJcesieJJa, 127

Jepteca, 9, 120
Stomatochone, 126
Stomatophora, 290

simplex, 289
Stomatophoridae, 290
Strains, 506
Strcblomastigidae, 182
Streb/omastix, 182

stn'x, 181
Streptomonas, 180

cordata, 179
Streptomycin

bleaching of EugJena, 486
Strobilidiidae, 390
Stiobilidium, 391

gyrans, 390
Strom bidium, 390
Strongy/idium, 399

maritimum, 400
StyJobrj'on, 127
Stylocephalidae, 287
Sty/ocephaJus, 287

giganteus, 284
StyJochona, 412

coronata, 413
Stylochonidae, 412
Sty/ocoma, 399
StyJocometes, 421
StyJocystis, 282
StyJodinium, 149

sphaera, 1^8
Stylonetbes, 399
Sty/onychia, 399
StyJophrya, 423

polymorpha, 421
Styhpyxis, 126
StyJosphaeridiiim, 153
Suctorea, 333, 413

ingestion, 415

larvae, ^16

life-cycles, 415

taxonomic relationships, 333, 420

tentacles, 414, 415
SuJcoarcus, 342

peUuciduJus, 341
Sulcus, 138
Swanson, B. K., 445
Symbiosis, 527, 528, 529
Symmetry, 3
Synactinomyxon, 319
Syncrypta, 126

volvox, 5
Syncryptidae, 126
Syncystis, 282
Syndinium, 150
Syngamy

anisogamy, 80

biochemical anisogamy, 82

haploid flagellates, 511

isogamy, 80

Index 679

Syngamy (Cont.) :

sex substances, ChJamydomonas, 82

uncertain cases, 81
Synochromonas, 127
Synophrya, 376

hypertrophica, ^j^
Synura, 127

uveJ/a, 123
Syracosphaera, 130

mediterranea, 120

puichra, 129
Syrfngopharynx, 371
Syzygy

Coccidia, 291

Gregarinidia, 274

Tachyb/aston, 420
Tachysoma, 399
Taeniocystfs, 282

niira, 272, 281
Taliaferro, W. H., 637, 638, 642, 644
Tanabe, M., 548
Tannreuther, G. W., 168
Te/omyxa, 322

glugeiformis, 320
Telomyxidae, 322
Telosporidea, 270
Telotroch, 406
Te/otrochidium, 409

henneguyf, 404

/ohannfnae, ^o^
Temperature

biothermal range, 482

coefficients (do values), 483

lethal, 482

thermal increments (/x values), 484
Teratonympha, 194, 195
Teratonymphidae, 195
Tests, 10, 241
Testacida, 241

ecology, 245

life-histories, 244

pseudopodia, 242

subdivisions, 245

tests, 241
TetrabJepharfs, 153
TetrachJoris, 152
Tetractinomyxidae, 319
Tetractinomyxon, 319
Tetradimorpha, 202

radiata, 203
Tetradinfum, 149

/avanfcum, 148
Tetrahymena, 362, 367

patu/a, 366

pyrfformis (see also: T. gelefi. Glaucoma
pyriformis), 365, 366
carcinogenic substances, effects, 486
culture media, 450
cytochrome system, 455
disaccharides utilized, 464
endopeptidases, 462
glycolysis, 469
lipids synthesized, 470
mineral requirements, 437
monosaccharides utilized, 466

Tetrahymena (Cont.):

pyriformis (Cont.) :

oxygen relationships, 451
pH relationships, 479
polysaccharides utilized, 463
respiratory quotients, 454
tricarboxylic acid cycle, 460, 469
ultraviolet irradiation, 487
vitamin requirements, 440

vorax, 366

form, effects of diet, 4
Tetrahymenidae, 362, 364
Tetramitidae, 182
Tetramitus, 182

bufonis, ij

lostiatus, 181

saJi'n us, 181
Tetramyxa, 230
Tetratoxum, 396

unifascicuJatum, 395
Tetratrichomastix, 187
Teutophrys, 353

tiisulca, 351
Texas cattle fever, 307
ThaJassicoJia, 218

nuc/eata, 216
Tha/assoJampe, 218
ThaJassophysa, 218
ThaJassothamnus, 218
Thaumatomastix, 170
Thaumatomonas, 170
Thaumatophrya, 423
Theca

fission, 58

structure, 9
Thecacineta, 420

baikaJica, 417
Thecamoeba, 240

orbis, 239
Thef/eria, 308

parva, 306, 307
Theileriidae, 309
The/ohanelJus, 317

notatus, 315
Thelohania, 322

c/adocera, 320
Theodor, O., 649
Theopera, 218
Theophormfs, 218
Thigmophrya, 374
Thigmophryidae, 374
Thigmotrichina, 370
Thoracomonas, 155
Thorocapsfs, 218
ThuricoJa, 411

obconica, ^08
Thurfcolopsis, 411
Thy/acidium, 383
Tiarina, 345

fusus, 343
TiUina, 355

canaJifera, 354
Tintinnidae, 393
Tintinnina, 391
Tintinnopsfs, 391

nucuJa, 9, 392

680 Index

Tintinnus, 393
ToJcophrya, 420

internal budding, 61
lemnaium, 414, 417
ToJcophryopsfs, 420

gigantea, ^ly
Tontonia, 390

gTscilUma, 390
Torodinium, 143

teredo, 142
Torquenympha, 190

octop/us, 191
Toxi cysts, 27

Toxins, effects on ciliates, 484
ToxogJugea, 322

vibrio, 320
Toxoplasma, 309, 310

canis, 509

human strain, 309
Toxoplasmosis, 310

complement-fixation tests, 648
Tracheliidae, 353
Trache/ius, 353

ovum, 352
Trache/ocerca, 348

entzi, 546
Trache/omonas, 167

hystrix, 163

voh'ocina, 161
TracheJophy/Jum, 348
Trache?ost}'Ja, 399

pediculifoTmis, ^00
Traumatiophora, 376
TrematophJyctfs, 230
Trentonia, 170
Trepomonas, 185

agiZis, 18^
Triactinomyxidae, 319
Triactinomyxon, 319

kgeri, 317, 518, 319
Triadfnium, 396

caudatum, 396
TrianguJomonas, 168

rigida, i6y
Tricarboxylic acid cycle, 458
Tricercomftus, 187

termopsidis, 186
Tricercomonas, 182

intestina/is, 547, 548
Trichamoeba, 240

pallida, 238
Tiichia, 233

decipiens, 233

inconsp/cua, 233
Trichites, circumpharyngeal, 31
Tiichloris, 152

paradoxa, J53
Trichochona, 412

Jecythoides, 413
Trichocysts, 26

flagellates, 27

functional significance, 27

types, 27
Trichodina, 410

spbeioidesi, 408

Trichoduboscqia, 322
Tricho/imax (see: Mastigina)
Tiichomastix (see: Monocerconionas)
Tnchonionadida, 185

food requirements, 448
Trichomonadidae, 188
Trichomonas, 190

buccals (see: tenax)

gaibnae, J89

gallinaium, 190

limacis, 189

tenax, 545

vagina/is, 564, 567

food requirements, 448
Trichonympha, 195

corbuia, 194
Trichonymphidae, 194
Trichonymphina, 191
Trichopeiiua, 362

spbagnetorum, 361
Trichopelmidae, 360
Tiichophiya, 423

epistyb'des, ^21
Trichorhyncbus, 285
Tricbospira, 362

inversa, 358
Trichospiridae, 362
Trichostomina, 353
Trichotaxis, 399
Trifasciculaiia, 396

parvum, 396
Trigonomonas, 185

compressa, 185
Trimastfgamoeba, 234
Trimastigidae, 180
Tiimastix, 1 80
Trimyema, 362

compressa, 361
Trimyemidae, 362
Trinema, 250

encbeJvs, 249
Tripabnaria, 396

dogfe/i, 396
Tripathi, Y. R., 316
Trip/agia, 218
Trip/umarfa, 396
Tripylina, 218
Tn'tricbomonas, 190

augusta, 1^, 189
budding, 72

foetus, 189

food requirements, 448

murfs, 13
Trocbe^a, 385
Tiocbilia, 345

marina, 344
Trochi/ioides, 345
Trog/od}'te/la, 396
Tropidoatractus, 385
Tropidoscyphus, 168

octocostatus, 167
Trypanodinium, 150
Tr}'panopJasma, 178
Tr)'panosoma, 178
biucei, ij6

Index 681

Trypanosoma (Cont.) :

CTuzi, 589, 591

gambiense, 584

glycolysis, 468

lewisi, ij6

pathogenicity, theories, 583

rangeJi, 591

rhodesiense, 585
Tr}panosoiniasis, 582

acute lethal infections, 638

causative organisms, 582

non-lethal infections, 639

relapsing lethal infections, 638
Trypanosoniidae, 175
Tubu/ina, 233
Turania, 364

TmiispiiilUna conoidea, 255
TurruJina andreaei, 255
TuscariJJa, 219
Tuscarora, 2x9
Tussetia, 153

po/ytomoides, 15^

UJivfna, 285
Umbih'cosphaera, 130
Uncinata, 399
Undelh, 393
Undellidae, 393
l/ndeJ/opsis, 393
Undulating membrane

ciliates, 19

flagellates, 13
UnicapsuJa, 316

muscu Jan's, 315
Unicapsulidae, 316
Unicauda, 317
L/radiophora, 285
Urceo/aria, 410

pateJ/ae, 408
Urceolariidae, 410
Urceohis, 168

cycJostomus, 166
Urfnympha, 193

taica, 192
UrnuJa, 423
Urocentrum, 364

tinho, 363
Uioglena, 127

UrogJenopsfs (see: UrogJena)
(JroJeptopsis, 399
Uwleptus, 399

mobiJis, 400
Uronema, 364

p/uricaudatum, ^6^
Uwnemopsis. 364
Uronychia, 398

heinrothf, 397
UropedaJium, 364
Urophagus, 185

rostratus, 18^
Urosoma, 399
Urospora, 290

rhyacodrfJi, 289
Urosporidae, 290

Urosporidium, 328
fu/iginosum, 328

UrostyJa, 399

h'mboonkengi, 399

Urotricha, 348
armata, 347

Urozona, 364

VacuoJaria, 170

vfrescens, 169
Vacuoles (see also: Contractile and sensory
vacuoles)

flotation, 32
Vacuome, 42

digestive granules, 43

volutin content, 43
VaginicoJa, 411

amphora, 408

annuJata, 408

Jongi'colh's, 9
Vaginicolidae, 410
VablkampBa, 234, 240

punctata, 238
Valkanov, A., 212
VaJ/acerta, 130
Vamp}reJ/a, 224

closterii, 223

Ja ten" tia, 223
Vampyrellidae, 223
VampyreJh'dium, 226

vagans, 225
Vampyrophr)'a, 376
Vasicola, 348

parvuJa, 349
Venoms, effects on ciliates, 485
Verneuih'na schizea, 255
Vitamin requirements, 438 (tabJe, 440)

analogues, 441, 442, 486

ascorbic acid, 442

biosynthesis, 443

biotin, 441

Bxo, 443

hematin, 443

nicotinic acid, 441

nucleic acid derivatives, 442

p-aminobenzoic acid, 442

pantothenic acid, 441

phytoflagellates, 438

protogen, 443

pteroylglutamic acid, 442

pyridoxine complex, 441

riboflavin, 439

sterols, 442

thiamine, 439
Volutin

effects of ribonuclease, 39

properties, 39
Volvocidae, 156

life-histories, 157

sexual reproduction, 159
Volvox, 157

development of colony, 158, 159

gametes, 8^
VoJvuJina, 157

682 Index

VoTticella, 411
consoma, ^11
mayeii, 409
microstoma, 409

conjugation, 89
picta, 409

VorffceZ/idae, 411

WagneieUa, 210
WaiJcsia, 392
Wangie?/a, 392
Waidia, 316

ovinocua, 315
Wardiidac, 316
Warner, K. L., 555
WebbineUa ciassa, 253
Wenrich, D. H., 546, 548, 553, 567
Wenyon, C. M., 109, 548, 557
WenyoneHa, 301
W islouchielh, 155
Woodruff, L. L., 91, 94, 96

Woodruffia, 355
metabolica, 354

XystoneUa, 393
Xystonellidae, 393
Xystonellopsis, 393

Zellenella, 336

fruncata, 335
Zonomyxa (see: AmphizoneJJa)
Zoomastigophorea, lyo
Zoofhamnium, 411

adamsi, 6, ah

aibuscuh, cyst, 411
Zooxanthellae (in Radiolarida), 214
ZschoJcJceJJa, 317

hiJdae, 315
Zygocystidae, 290
Zygocystis, 290

uenrfchi, 289
Zygosoma, 285

g/obosum, 284
Zygostephanus, 218

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