T OF

A COMPARISON OF THE LIFE CYCLE
OF CEITHIDIA WITH THAT OF
TRYPANOSOMA IN THE IN-
VERTEBRATE HOST

A THESIS ACCEPTED IN PAETIAL SATISFACTION OF
THE REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY
AT THE UNIVERSITY OF CALIFORNIA

BY

IRENE AGNES McCULLOCH

1919-

A Comparison of the Morphology and. the Life
Cycle of the Genus Gri thidia with that of
the crithidial stages of Tryanosoma.

Irene Agnes Lie Cul loch, A. B. , L. A.
Un i v e r s i ty of Y. an s as , 1 9 1 o .

A thesis submitted in partial fulfillment, c

.s for the degree of Doctor of Philosophy

-Committee :

UNIVERSITY OF CALIFORNIA PUBLICATIONS

IN

ZOOLOGY

Vol. 19, No. 4, pp. 135-190, plates 2-6, 3 figures in text October 4, 1919

A COMPARISON OF THE LIFE CYCLE OF

CRITHIDIA WITH THAT OF TRYPANOSOMA

IN THE INVERTEBRATE HOST

BY

IRENE McCULLOCH

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UNIVERSITY OF CALIFORNIA PUBLICATIONS

IN

ZOOLOGY

Vol. 19, No. 4, pp. 135-190, plates 2-6, 3 figures in text October 4, 1919

A COMPARISON OF THE LIFE CYCLE OF

CRITHIDIA WITH THAT OF TRYPANO-

SOMA IN THE INVERTEBRATE

HOST

BY

IRENE McCULLOCH

CONTENTS

PAGE

Introduction 136

Historical summary 139

The comparative morphology of Crithidia and the crithidial stages of

Trypanosoma 144

Nucleus 146

Blepharoplast 147

Parabasal body and rhizoplasts 149

Flagellum and undulating membrane 151

The life cycle of Crithidia euryophthalmi 152

The developmental series 153

Stomach phase 153

Extracellular crithidias 154

Oval spores 154

Developing crithidias 154

Multiple fission 155

Endogenous budding 155

Somatella 162

Binary fission 165

Intracellular crithidias 168

Rectal phase 170

Nectomonads 171

Haptomonads 174

Final spore forms 176

The degenerative series 177

Conclusions 178

Literature cited 180

Explanation of plates 182

136 University of California Publications in Zoology [VOL. 19

INTRODUCTION

In my work on the flagellate parasites of hemipteran insects during
the past five years the evidence indicating a close relationship between
Crithidia and the crithidial stages of Trypanosoma has been continu-
ously accumulating and has become more and more convincing. The
relationship is shown both in their morphology and in the stages of
their life cycles. This paper presents a comparison of the morphology
and the life cycle of the genus Crithidia with that of Trypanosoma in
its crithidial stages.

Before taking up this comparison a brief discussion of the phylo-
genetic relations of Leptomonas, Herpetomonas, Crithidia, and Try-
panosoma will be of value in clarifying the subject. In addition a
short discussion concerning the hosts, their food, and their methods of
infection will give a fundamental conception of the problem in hand.

The phylogenetic relation of these intestinal flagellates, Lepto-
monas, Ilerpetomonas, and Crithidia, to the haemoflagellates, or Try-
panosoma, has received the attention of many investigators and
consequently has been the subject of much controversy. Only a brief
statement of the historical side of this controversy need be given here.
Minchin (1908) discusses the possible sources from which the trypano-
somes may have been evolved, namely, from the herpetomonad-like
and the trypanoplasma-like ancestors. At that time as well as now
the evidence pointed to the crithidial (herpetomonad-like) forms as
the true recapitulative, developmental, primitive stage in the life
life cycle of Trypanosoma. Minchin (1912) stated:

The types denoted by the generic names, Leptomonas, Crithidia and Try-
panosoma form a perfect evolutionary series with monogenetic parasites of inver-
tebrates culminating in digenetic blood parasites. It must be emphasized, however,
that any such conclusions are of a tentative nature and can have no finality but
are liable to modification with every increase of knowledge concerning these
organisms.

Wenyon (1913) also discusses the whole question of the phylo-
genetic relationship of Leptomonas, Herpetomonas, Crithidia and
Trypanosoma. If the trypanosome be regarded as the highest stage
of development then his conclusion is that the phylogenetic order of
these flagellates would be : Leptomonas, Crithidia, Herpetomonas, and
Trypanosoma. He makes a distinction between the genera Lepto-

1919] McCulloch: Life Cycle of Crithidia and Trypanosoma 137

monas and Herpetomonas. Leptomonas is a flagellate having the non-
flagellated and herpetomonad form in its life cycle ; Herpetomonas
has these two stages, together with a crithidial and a trypaniform
stage. But such a distinction has not been generally accepted. His
chief reason for placing Herpetomonas next to Trypanosoma is the
presence of a trypaniform phase in the life cycle of a few herpeto-
monad flagellates, as, for example, those found in Musca domestica
and Drosophila confusa (Chatton and Leger, 1911).

From the viewpoint of the present study of these flagellates the
hemipteran insect hosts have been frequently divided, for convenience,
into the plant-feeding and the blood-sucking types. According to
Patton and Cragg (1913) some species of the three families of plant-
feeding Hemiptera, the Pentatomidae, the Lygaeidae, and the Coreidae
have been found to serve as hosts for either crithidial or herpetomonad
flagellates. One more family should now be added to this list, namely,
the Pyrrhocoridae, to which the " lupine bug" (Euryophthalmus con-
vivus) belongs. Among the blood-sucking Hemiptera some species of
the family Reduviidae and the family Cimicidae are parasitized by
haemoflagellates having a cycle in a vertebrate host and in some cases
by other flagellates having only the invertebrate or insect host.
Among the hosts discovered in the family Reduviidae we find Tria-
toma (== Conorhinus) megista Burm., the invertebrate host of Schizo-
trypanum cruzi (Chagas, 1909) and Triatoma protracta Uhler, the
invertebrate host for Trypanosoma triatomae (Kofoid and McCulloch,
1916).

In addition to the hemipteran insects the Diptera (flies, sheep
ticks, Siphonaptera (fleas), and Anapleura (lice) also serve as hosts
for some of the herpetomonad, crithidial, and trypaniform flagellates,
the rat-flea (Ceratophyllus fasciatus) is one of the invertebrate hosts
of Trypanosoma lewisi. Frequent reference will be made to this
flagellate in the comparison of Crithidia with Trypanosoma.

The methods of infection of the above hosts have been described
as casual, "cross," and hereditary. Of these three methods Patton
(1908) proved that nymphs of Lygaeus militaris became infected by
ingesting with their food the feces of infected adults. The feces con-
tained encysted or spore forms. Porter (1910) described two addi-
tional methods for the infection of sheep ticks, the "cross," and the
hereditary. The former occurs among insects with cannibalistic
habits, the contents and parasites of the digestive tract of one host
individual being eaten by another. The hereditary infection has been

140 University of California Publications in Zoology [VOL. 19

pathogenic to its vertebrate host, the rat. The invertebrate host of
this haemoflagellate is normally the rat-flea, which transmits the
flagellate from rat to rat. Both the vertebrate and the invertebrate
host of T. lewisi are widespread, abundant, easily procurable, and
adaptable to laboratory conditions. To make certain that none of the
stages of the life history of the so-called natural flagellates should
be confused with stages of the life history of T. lewisi a stock of un-
infected fleas was procured for the breeding cages. The rat-fleas have
frequently been found to be infected with Leptomonas pattoni.

At the time of the publication of the Minchin and Thomson paper
I was working on the morphology and life history of Crithidia eury-
ophthalmi (McCulloch, 1917), a form which occurs in the alimentary
tract of Euryophthalmus convivus. This material was of great in-
terest; the intracellular process of multiple fission was found, and
the initial infective spores were relatively abundant. Previously
some time had been spent in studying the morphology and life history
of Crithidia leptocoridis (McCulloch, 1915), which infects the digestive
tract of the box elder bug, Leptocoris trivittatus. It had also been
pointed out in a superficial way that the individuals of various forms,
shapes, and structures found in a typical infection of this crithidia,
C. leptocoridis, were apparently analogous to many of the correspond-
ing figures of Schizotrypanum cruzi (Chagas, 1909) in the invertebrate
host. As the investigation of C. euryophthalmi and C. leptocoridis
proceeded it became increasingly easy to link the life history of
Critkidia with the life history of Trypanosoma in the invertebrate
host. This was especially true of the life history of T. lewisi. In
order to demonstrate clearly the essential facts of the life cycle of one
of these important flagellates a brief outline of the life history of
T. lewisi will be given, based upon the work of Minchin and Thomson.

The developmental cycle of T. lewisi in the flea is divided into two
phases, characteristic of the parts of the digestive tract in which the
trypanosomes are found, viz., the stomach and the rectal phase. The
cycle in the rat-flea requires a minimum of five days for its complete
course. The trypanosomes enter the stomach of the flea with the
blood of an infected rat. They show the characteristic structure of a
trypanosome, the " kinetonucleus, " or parabasal body, being posterior
to the nucleus and the undulating membrane well developed. The
change in the medium is probably responsible for the physiological
changes which result in the bodies becoming more cylindrical. The
trypanosomes then penetrate the epithelial cells of the stomach and

1919] McCulloch: Life Cycle of Crithidia and Trypanosoma 141

undergo the process of multiple fission, a process which characterizes
the stomach phase. The initial steps in this process after the trypano-
some has entered an epithelial cell of the stomach, is the formation
of a "tailed" sphere. This is a spherical structure with the flagellum
and some cytoplasm protruding at one point. A little later the
the ' ' tailed ' ' sphere becomes ' ' tailless, ' ' and shows internally the form-
ation of a variable number of daughter individuals. In the forma-
tion of these daughter individuals, the nucleus, parabasal body, and
blepharoplast each divide an equal number of times, forming mero-
zoites of a crithidiomorphic type, i.e., a long, free type of trypano-
some whose external movements are crithidial but whose structure still
shows the parabasal body posterior to the nucleus. The crithidio-
morphic flagellates may do one of two things: enter other epithelial
cells and undergo the process of multiple fission, or collect at the
pyloric opening of the stomach, to be carried down through the intes-
tine into the rectum with the food. The intestine ordinarily serves as
a passageway for the parasites from the stomach to the rectum, but
under certain conditions rectal forms may migrate forward and attach
themselves to the wall of the intestine in the postpyloric region.

The rectal phase is established by the entrance of the crithidio-
morphic forms into this region of the digestive tract, and this region
becomes the permanent source of infection throughout the life of the
flea. During the migration from the stomach to the rectum structural
changes take place in the crithidiomorphic trypanosomes. The poste-
rior end of the body becomes club-shaped, and this shifting of the
cytoplasm assists in the forward movement of the parabasal body to
a position anterior to the nucleus. Following this condition, binary
fission brings about the production of the smaller, minute crithidias
which characterize this phase of the life history. The established rectal
phase is described as consisting of: (1) the attached or haptomonad
form, which is the multiplicative stage of the rectal development;
(2) the free or nectomonad form; and (3) the final trypanosome form
which is instrumental in infecting another host.

The above outline of the life history deals only with the develop-
mental series; but in both the stomach and rectum there are present
individuals which show degeneration and belong consequently to the
degenerative series. I have found that the life history of Crithidia
euryophthalmi can be correlated advantageously with the life his-
tory of T. lewisi in the invertebrate host, the flea. The life history
of C. euryophthalmi is not the life history of a haemoflagellate but

142 University of California Publications in Zoology [VOL. 19

apparently that of a more primitive flagellate (pis. 2-6) found living
in the medium of the fluid contents of the alimentary . tract of a
plant-feeding insect. As such, C. euryophthalmi (fig. A, 2) lacks in
the stomach phase the trypaniform characteristics, namely, the ble-
pharoplast (bl.), the parabasal (pi).) (or kinetonucleus) located poste-
rior to the nucleus (n.), and the well developed undulating membrane.
In this connection it is interesting to note that, regardless of the con-
trast in the initial stages of the stomach phase in each life cycle, the
process of multiple fission characterizes the stomach phase for both
the haemoflagellate and the more primitive crithidial flagellate. In
the stomach phase of the crithidial parasite there are also found the
"rounding up" forms (pi. 3, figs. 24, 25), the spheres (pi. 3, figs.
26-32), and the final stages of multiple fission wherein the resulting
merozoites are about to escape from the epithelial cell of the host (pi. 3,
fig. 39) . In addition to this I found numerous stages of an endogenous
process of multiple fission (pi. 2, figs. 11-23).

Correlation of the life histories of these forms (C. euryophthalmi
and T. lewisi) became more difficult in the stage following the process
of multiple fission, on account of the structure of the digestive tract
of the host, Euryophthalmus convivus. The digestive tract of the flea
(cf. Minchin and Thomson, 1915, text-fig. 1) shows the stomach as a
prominent enlargement of the tube, followed by a comparatively long,
slender intestine, at the posterior end of which is the rectal enlarge-
ment. The digestive tract of Euryophthalmus convivus, or lupine bug,
on the other hand, is quite unlike that of the flea (cf. McCulloch, 1917,
text-fig. 1 ) . The first enlargement of the mid-gut proper is followed by
a second and a third enlargement, a narrow constriction of the diges-
tive tract separating the three expansions. Posterior to these there is a
relatively long intestine which passes through the center of a ruffled,
ribbon-like gland. The intestine opens into the slight enlargement
near the entrance of the malpighian tubules, which in turn opens into
the rectum. Since the several parts of the digestive tract of the hem-
ipteran and other insects have not been satisfactorily homologized as
yet and the nomenclature used in describing the divisions is confused,
it is exceedingly difficult to ascertain the homology of these several
parts of the digestive tract. However, as indicated in my preliminary
paper (McCulloch, 1917), all three enlargements anterior to the intes-
tine were considered as parts of the stomach proper and were accord-
ingly designated as the "crop," mid-stomach, and pyloric expansion.
With this disposal of the three enlargements of the digestive tract, the

1919J McCulloch: Life Cycle of Crithidia and Trypanosoma 143

question immediately arose as to whether or not all the crithidial infec-
tions of these regions are a part of the stomach phase of the life
history of C. euryophthalmi. A careful study of the crithidial infec-
tion of each of these portions of the digestive tract together with many
examinations of the contents of the intestine, the gland, and the
rectum, convinced me that food conditions do not permit the parasites
to establish a permanent rectal phase in the rectum but that they
are forced to remain anterior to the intestine, in the pyloric expansion.
Preparations of the gland and intestine have as yet shown no infection
posteriorly, and the rectum has contained an occasional infection of
spore forms only.

To add to the difficulty in determining the extent of the stomach
phase and the beginning of the rectal phase no structural changes of a
striking nature occur in the stomach phase of C. euryophthalmi as is
the case in T. lewisi; nevertheless, the behavior of the crithidias is of
some assistance and the study of the serial sections and stained smears
of the several parts have added materially to our knowledge of the
phases. Taking into consideration the evidence from these sources
the crithidial infection of the "crop" is interpreted as the stomach
phase. The mid-stomach serves as a temporary location for the slowly
migrating forms of the stomach phase, and the pyloric expansion be-
comes the region of the permanent location of the "rectal" phase
during the life of the lupine bug. The established "rectal" phase
of the life history, of C. euryophthalmi in the pyloric expansion has
three general types of individuals, the attached, or haptomonad, the
free, or nectomonad, and the final spore forms, which probably serve
to infect another insect. Only the last type of parasite has been found
in the normal contents of the rectum.

With these brief explanatory outlines of the life histories of T.
lewisi and of C. euryophthalmi a detailed discussion of the more im-
portant points concerning the comparative morphology of the crith-
idial stages of Trypanosoma and of Crithidia will now be given. The
comparison of the life cycles which follows is based upon the work
of Minchin and Thomson on the life cycle of T. lewisi, and upon
the life history of C. euryophthalmi with special reference to the
accompanying plates (pis. 2-6). For further details of the life
history of T. lewisi the reader is referred to Minchin and Thomson's
paper (1915).

144 University of California Publications in Zoology [VOL. 19

THE COMPARATIVE MORPHOLOGY OP CRITHIDIA AND
THE CRITHIDIAL STAGES OF TRYPANOSOMA

The morphology of Crithidia and Trypanosoma has been the sub-
ject of careful investigation for a number of years, and our concep-
tion of the structure of these simple organisms has been modified from
time to time by additional discoveries. This is especially true of
the extranuclear organelles of these flagellates, owing to the recent
investigations carried on in the Zoological Laboratory of the Uni-
versity of California by Dr. C. A. Kofoid (1916) and Dr. Olive
Swezy (1916), the latter studying particularly the binuclear theory
of Hartmann (1911). The work of these investigators has centered
attention upon the extranuclear organelles of these flagellates, con-
sisting of the blepharoplast (fig. A, U.) at the base of the flagellum,
the parabasal body (pb.), or kinetonucleus, the rhizoplast (r/«.), the
parabasal rhizoplast (pb. rh.), the flagellum (fl.), and the undulating
membrane (und. m.). They have homologized the kinetonucleus of
the Protomonadina (Herpetomonas, Crithidia, and Trypanosoma}
with the parabasal body of the Polymastigina and the Hypermasti-
gina. Bearing this in mind, it at once becomes clear that this extra-
nuclear complex of organelles is the neuromotor apparatus of Crith-
idia and Trypanosoma (Kofoid, 1916). In a previous paper (Kofoid
and McCulloch, 1916) the term parabasal body was used in place of
kinetonucleus throughout and I shall employ this nomenclature in
the present paper.

The position of this extranuclear complex of organelles determines
largely whether the flagellate is a trypanosome or a crithidia. The
trypanosome is characterized by the presence of the parabasal body
and the blepharoplast posterior to the nucleus, and by a well developed,
undulating membrane which passes forward laterally along the edge
of the ribbon-like body. These characteristics are common to the
flagellates found in the blood, and modifications of this structure take
place as soon as the medium is changed, as in the transfer to the
stomach of the flea. The transition stages between a trypanosome and
a crithidia have been designated as crithidiomorphic trypanosomes by
Minchin and Thomson, as previously noted. In the transition forms
the parabasal body and the blepharoplast are still posterior to the
nucleus at a greater or less distance, but the movement and shape of
the body of the flagellate are distinctly like those of a crithidia. In

1919] McCulloch: Life Cycle of Crithidia and Trypanosoma 145

the crithidial forms the extranuclear complex of organelles is anterior
to the nucleus (fig. A, 1, 2) and the body has a tendency to be
cylindrical in cross-section and to show only a slight undulating mem-
brane (und. m., fig. A). These crithidial forms are common to the
life cycle of both Crithidia and Trypanosoma. The next step in our

Fig. A. Typical crithidial forms of: (1) Crithidia leptocoridis ; (2) C.
euryophthalmi; (3) Trypanosoma lewisi (after Minchin and Thomson, 1915, pi.
37, fig. 66); (4) T. triatomae; (5) Schizotrypanum crusi (after Chagas, 1909,
pi. 13, fig. 16) ; showing the similarity of the several flagellates in size, form
and structure. X 3500. 1)1., blepharoplast; ft., flagellum; n., nucleus; pb., para-
basal body; ph. rh., parabasal rhizoplast; n. rh., nuclear rhizoplast; und. m.,
undulating membrane; vac., vacuole.

discussion will be directed to the demonstration of the close resem-
blance of the crithidial forms of the genus Crithidia to those of
Trypanosoma in the invertebrate host.

A series of typical crithidial flagellates (fig. A) has been selected
from the stomach phase of the life histories of both Crithidia and

146 University of California Publications in Zoology [VOL. 19

Trypanosoma to show in detail the comparative morphology in this
particular stage. C. leptocoridis (fig. A, 1) and C. euryophthalmi
(fig. A, 2) have been selected to represent the structure of Crithidia
and Trypanosoma leivisi (fig. A, 3, after Minchin and Thomson, 1915,
pi. 37, fig. 66), T. triatomae (fig. A, 4), and Schizotrypanum cruzi
(fig. A, 5, after Chagas, 1909, pi. 13, fig. 16) that of Trypanosoma.

Each crithidial flagellate of this series has an elongate body,
cylindrical in outline but slightly flattened at the anterior portion,
which forms the undulating membrane. At the edge of this membrane
there is a sharply defined flagellum of variable length (fig. A, 1, 3).
The length of the flagellum has no particular significance since con-
siderable variation exists within each species. Posteriorly, however,
there is in the stomach phase a consistent difference between the
crithidial form of Crithidia and the crithidial form of Trypanosoma.
The posterior ends of the bodies of the true crithidias are more atten-
uate (fig. A, 1, 2) in the stomach phase, and do not show the slight
tendency to become club-shaped until the rectal phase is reached. In
figure A, 4, T. triatomae is quite blunt at the posterior end, and the
shifting of the cytoplasm into this region is increasing the width of
the body at the expense of the length. Another difference almost as
consistent as the one just noted is the variation in the nucleus (n.).
In Crithidia (fig. A, 1, 2) it is usually anterior to the center, and. in
the crithidial forms of Trypanosoma it is posterior to the center (fig.
A, 3, 4, 5).

Internally the similarity of the structure of the organelles and their
relationship in general is quite marked. The nucleus (n.), the ble-
pharoplast (bl.), and the parabasal body (ph.) are common to each
of these flagellates. The nuclear rhizoplast (rh.) and the parabasal
rhizoplast (pb. rh.) are found in all the above flagellates with the
exception of T. lewisi (fig. A, 3). The absence of these organelles in
the crithidial form of T. leivisi is questionable since they are present
in all the others. They may have been overlooked because of the
delicacy of their structure and the faintness with which they stain.

The nucleus and the extranuclear organelles will now be taken up
in detail.

Nucleus. — This organelle in each of the above flagellates may be
described as round or slightly oval in shape and of the vesicular type.
It varies somewhat in size from I/A in figure A, 3 to 1.7/* in figure
A, 5, but it is usually about two-thirds of the width of the body in
diameter. In Crithidia leptocoridis the nucleus shows clearly the ves-

1919] McCulloch: Life Cycle of Crithidia and Trypanosoma 147

icular type of structure, having a relatively large karyosome, a light
area outside of this, and a clearly defined nuclear membrane. In
figure A, 3 a slightly different condition exists ; the nucleus of C. euryo-
phthalmi is of the vesicular type, but the nuclear membrane is en-
crusted with considerable chromatin, and the central karyosome is
somewhat reduced as compared with that of C. leptocoridis in figure
A, 1. An examination of plate 5 indicates that the nucleus of this
flagellate (C. euryophthalmi) is characterized by the heavily encrusted
nuclear membrane, the small central karyosome, and the broad, clear
area between the membrane and karyosome.

In the trypanosomes (fig. A, 3, 4, 5) the nucleus is slightly poste-
rior to the center of the body, which has a tendency to be shorter and
more club-shaped at the posterior end than is that of the crithidias
(fig. A, 1, 2). The nucleus of Trypanosoma lewisi (fig. A, 3) shows
a peculiar structure. In this particular form it is difficult to demon-
strate the nuclear membrane, since the light area encircling the more
deeply stained portion could be interpreted as either intranuclear
or extranuclear. However, taking into consideration other figures of
T. lewisi (cf. Minchin and Thomson, 1915, pis. 37, 42) this light area
has been regarded as being extranuclear and the diameter is I/A
instead of 1.4/*. Within this relatively deep-staining, nucleus the
chromatin is divided unequally into a large, irregularly shaped granule
and a smaller one. The nucleus of T. triatomae (fig. A, 4) resembles
that of C. euryophthalmi. It is also characterized by the chromatin-
encrusted nuclear membrane, the wide clear area, and the small cen-
tral karyosome, as in C. euryophthalmi. A nucleus of a somewhat
more complex type is found in Schizotrypanum cruzi (fig. A, 5). A
faint nuclear membrane limits the clear nuclear space which sur-
rounds the central karyosome containing a centriole at the base of
the rhizoplast. This nucleus is 1.7/x in diameter and somewhat larger
than that of any other flagellate in the series.

Blepharoplast. — This structure is of great interest since it is the
center of the extranuclear organelles, or neuromotor apparatus. In
the crithidias this structure is not a sharply defined basal granule at
the base of the flagellum. For instance, in Crithidia leptocoridis a
slight enlargement at the base of the flagellum is noted (fig. A, 1).
Since this slight enlargement stains with almost the same intensity as
does the flagellum it is exceedingly difficult to get a satisfactory concep-
tion of its structure. By careful focusing of the binocular microscope
with Watson's no. 20 holoscopic oculars, however, the small enlarge-

148 University of California Publications in Zoology [VOL. 19

ment of slightly greater staining intensity can be observed at the
junction of the parabasal rhizoplast with the nuclear rhizoplast. In
C. euryophthalmi (fig. A, 2) even greater difficulty is experienced in
endeavoring to find the blepharoplast. In this form there is no en-
largement of the base of the fllagellum, but the area at the junction of
the rhizoplast is darker in appearance, and frequently a small granule
at one side can be seen by careful focusing.

Among the trypanosomes the blepharoplast is apparently a more
clearly defined structure. Our investigations of T. triatomae (fig.
A, 4) have yielded more tangible results. We found in this trypano-
some a slight enlargement of the base of the flagellum but no differ-
entiation in the staining capacity of the flagellum and basal granule.
In T. lewisi (fig. A, 3) and in Schizotrypanum cruzi (fig. A, 5) the
figures of these trypanosomes show what is evidently a well defined
basal granule, or blepharoplast. This is particularly true of T. lewisi
(fig. A, 3). In both the crithidias and the trypanosomes the blephar-
oplast frequently divides independently of the nucleus. In the binary
fission of C. euryophthalmi the blepharoplast and parabasal body
may divide before the nucleus divides (pi. 3, fig. 4), or the nucleus may
divide first (pi. 3, fig. 33). In figure 25, plate 3, the nucleus is divid-
ing but there are no indications of the division of blepharoplast and of
the parabasal body. A similar condition exists among the trypano-
somes, indicating that the blepharoplast is the kinetic center of the
extranuclear organelles, or neuromotor apparatus.. But this is not
the only kinetic center of these simple organisms. The nucleus also
contains a kinetic center which initiates division in the form of a
centrosome, at the base of the nuclear rhizoplast in those flagellates
containing this organelle. In the nucleus of S. cruzi (fig. A, 5) this
centriole, or centrosome, at the base of the nuclear rhizoplast is clearly
defined within the central karyosome of the nucleus. The origin of
the blepharoplast is still the subject of investigation and as yet 110
conclusive evidence is at hand to establish beyond doub't the way in
which this structure originates. In the endogenous buds, which are
exceedingly small and difficult to interpret, the blepharoplast seem-
ingly originates from the single nuclear structure as an outgrowth
rather than by a mitotic process. The outgrowth forms a second
deep-staining mass anterior to the nucleus, and at this stage the
nonflagellated forms have the appearance of "binucleated" spores.
As development proceeds the kinetic center of the blepharoplast is
probably established as the center of the neuromotor apparatus, and

1919] McCulloch: Life Cycle of Crithidia and Trypanosoma 149

from this blepharoplast the flagellum grows anteriorly and the para-
basal body to one side. Connecting the parabasal body with the
centrosome, or blepharoplast, is the parabasal rhizoplast (p~b. rh.).
Parallel cases indicating such an origin of the blepharoplast can be
found among other flagellates.

Parabasal body and rhizoplasts. — In all of these flagellates the
parabasal body (pb.) presents the same general appearance with
respect to its location, size and shape. In C. leptocoridis (fig. A, 1)
the parabasal body is a bar-shaped structure made up of two deeply
stained granules lying in close proximity. Immediately surrounding
this deep-staining bar is a clear area, limited by a sacklike covering
of dense cytoplasm, evidently continuous with the contractile cyto-
plasmic sheath of the flagellum. Between the parabasal body and the
blepharoplast region the cytoplasmic sheath takes on the appearance
of a cone-shaped mass of contractile fibers (p~b. rh.), the apex of
which is found at the base of the flagellum and continuous with the
outer contractile cytoplasm of the flagellum. The axial or central
part of this parabasal rhizoplast is presumably of blepharoplastic
origin and connects the blepharoplast and parabasal body.

The parabasal body of C. euryopkthalmi is similar to that of
C. leptocoridis. It is not so large comparatively (fig. A, 2) and it
seldom has the bilobed appearance. In C. euryophthalmi the most
interesting and valuable evidence has been found concerning the
relation of the parabasal body to the blepharoplast. In text figure
B, 1-5 a series of flagellates has been drawn from good iron-haema-
toxylin preparations to show clearly that the position of the organelle
is lateral and not axial. In figure B, 1 the end of the bar-shaped
parabasal body, which lies lateral to the nuclear rhizoplast, extends
outward to the periplast. From another angle the entire length of
this organelle is visible (fig. B, 2) peripheral to the nuclear rhizoplast,
which passes behind and underneath the parabasal rhizoplast. Still
another aspect of the possible position of this structure is indicated
in figures B, 3, 4. In each of these figures it is medial to the nuclear
rhizoplast and behind or beneath it as it passes from the centro-
some of the nucleus to the centrosome, or blepharoplast, at the base of
the flagellum. In such a figure as B, 5 the relationship of this organ-
elle to the others of the neuromotor apparatus can be seen most clearly.
The cytoplasm has become exceedingly clear and does not conceal the
other structure. The parabasal body in the flagellate stains very
deeply and has the appearance of a chromotoidal mass suspended in

150 University of California Publications in Zoology [VOL. 1!

a sacklike structure from the region of the blepharoplast. Unlike th<
suspensory apparatus of C. leptocoridis , C. euryophthalmi does no
show a fan-shaped mass of contractile cytoplasmic fibrils covering this
axial portion, which is of blepharoplastic origin (figs. A, 1, 2 ; figs. B
1-5). The outline of this cytoplasmic envelope in C. euryophthalmi i:
definite, slightly opaque, and continuous with the cytoplasmic sheatl
of the flagellum.

The parabasal body of the trypanosomes (figs. A, 3, 4, 5) is simi
larly a bar-shaped structure, which stains deeply, located to on<
side of the nuclear rhizoplast and blepharoplast. In T. leivisi (fig
A, 3) the parabasal body is relatively small while the blepharoplast ii
correspondingly large. As previously noted, no parabasal rhizoplas
has been figured by Minchin and Thomson in T. Lewisi. Here am
there suggestions of a connection might be pointed out in their figures
While it is possible that T. lewisi is exceptional in this respect, yet
since the structure is found in other flagellates of this group, we ma]
infer that a critical study of the preparations with a binocular micro
scope will reveal the presence of such a connection in the crithidia
stages of this flagellate.

Trypanosoma lewisi (fig. A, 3) is likewise the only flagellate her<
figured in the crithidial stages without a nuclear rhizoplast. Her<
again T. leivisi is either exceptional or the structure has perhaps beer
overlooked, since it is usually discerned with difficulty. Chagas (1909]
has figured a nuclear rhizoplast in Schizotrypanum cruzi and we hav<
found it also in T. triatomae. Therefore we may expect that it wil
be found in T. lewisi.

The parabasal body in Trypanosoma triatomae (fig. A) is a rela
tively large structure, its width approximately equal to one-half of it!
length. It is located a short distance anterior to the nucleus, and is sus
pended from the blepharoplast by a fan-shaped parabasal rhizoplas
like that of Crithidia leptocoridis, i.e., a suspensory apparatus with j
fibrous appearance. In the crithidial stage of T. triatomae a nucleai
rhizoplast was observed connecting the blepharoplast with the centriol<
of the karyosome, but such a connection was not found in the trypani
form individuals. If a nuclear rhizoplast be present in the trypaniforn
stage, in which we did not find it, it is possible that we may have beer
prevented from observing it because of the density of the cytoplasn
in this stage and a tendency in this delicate thread in the trypano
somes to stain lightly. In the crithidial stages of this trypanosome, or
the other hand, the cytoplasm is more or less vacuolated in appear
ance thus making the nuclear rhizoplast more evident. When a try

1919] McCulloch: Life Cycle of Crithidia and Trypanosoma 151

panosome rounds up for the process of multiple fission no nuclear
rhizoplast is in evidence, but after the complete transition into the
crithidial form the nuclear rhizoplast can be readily demonstrated
with the high power binocular microscope and Watson's holoscopic
eyepiece. It is also possible that further work will show that the
centrosomic structure of the trypaniform flagellate differs from that
of the crithidial form, and consequently that the rhizoplasts are absent
in the former.

In Schizotrypanum cruzi (fig. A, 5) the parabasal body is slightly
bilobed in appearance, and is suspended from the blepharoplast by a
clearly defined, fibrous rhizoplast. The suspensory apparatus of the
parabasal body is here relatively larger and apparently more highly
developed than in any of the other flagellates. A few of the trypani-
form stages also show a similar parabasal rhizoplast, according to the
figures of Chagas (1909). In the crithidial stages of this trypano-
some, as shown in figure A, 5, there is a sharply defined nuclear rhizo-
plast passing from the centriole of the nucleus to the blepharoplast.
The position of the parabasal body in 8. cruzi is similar to that of
Crithidia euryophthalmi and is situated apparently to one side of the
nuclear rhizoplast.

One of the most important discoveries in connection with the study
of these organelles is the fact that the parabasal body, or the so-called
kinetonucleus, is not axial in position in Crithidia euryophthalmi
(figs. B, 1-4) and apparently not in the other flagellates (figs. A,
1, 3-5). With the evidence at hand to show definitely that this body
is not axial but a lateral appendage of the blepharoplast in C. euryo-
phthalmi it is necessary to look upon this organelle as something other
than a second nucleus or a kinetonucleus. Reference has already
been made to the work of Dr: Kofoid (1916) concerning the homology
of this organelle with the parabasal body of other flagellates. The
observations concerning the origin of this structure, the location
and relation of the parabasal body to the other organelles as found
in the study of C. euryophthalmi offer some of the best evidence against
the binuclear conception of these flagellates (Hartmann, 1911).

Flagellum and undulating membrane. — Owing to the relationship
which exists between the flagellum (/?.) and undulating membrane
(und. m.) it is convenient to describe these two organelles together.
The flagellum consists of an outgrowth from the blepharoplast and is
surrounded by a cytoplasmic sheath which is continuous with the sack-
like sheath covering the parabasal body. In the ordinary preparations
no distinction can be made between this central portion originating

152 University of California Publications in Zoology [VOL. 19

from the blepharoplast and the cytoplasmic sheath surrounding it.
The entire flagellum stains as a single heavy line, almost chromatoidal
in appearance. It does not stain so deeply as does the nucleus, but
much deeper than the cytoplasm of the body and of the undulating
membrane. As the flagellum forms and lengthens, there is an accom-
panying elongation of the protoplasm which forms the undulating
membrane (und. m.). Both endoplasm and ectoplasm enter into the
formation of this organelle. Usually there is a thin, narrow band of
clear ectoplasm lying parallel to the flagellum (fig. A, 5). The length
of the membrane, and consequently of the intracellular part of the
flagellum, is greater among the crithidias of the stomach phase than of
the rectal phase owing to the shifting of the cytoplasm of the body
in the transition. This is usually true also of the crithidias as com^
pared with the crithidial stages of the trypanosomes (fig. A, 1, 2, 3, 4).
As previously noted, the length of the free flagellum (fig. A, 3) has
no significance from the viewpoint of comparative morphology, since
there is a wide variation in the length of this organelle for each species
of Crithidia and of Trypanosoma.

THE LIFE CYCLE OF CRITHIDIA EURYOPHTHALMI

The life cycle of C. euryophthalmi in Euryophthalmus convivus
begins, so far as known, with the casual ingestion with food of
spores from the fecal matter of infected insects. E. convivus com-
monly feeds upon the sap from the growing tips of the lupine, which
show many indications of excreta. In these same regions of the lupine
galls and other abnormal growths occur in great abundance. The pos-
sibility that these insects were getting their infection of C. euryo-
phthalmi from the sap of the lupine was suggested by the work of
Franga (1914). Examinations of the sap of the lupine were accord-
ingly made. Nematodes, numerous yeast-like spores, and bacteria
were found. No organisms were discovered, however, of any descrip-
tion which could be linked to the known stages of C. euryophthalmi
in the digestive tract of the host.

The large number of parasites in the life cycle of C. euryophthalmi
can be grouped readily into two series : the developmental, or infective,
and the degenerative, which are comparable to the developmental and
degenerative series of T. lewisi in the flea, as described by Minchin
and Thomson. That the correlation between these two life cycles is
marked will become clear in following the discussion of the life cycle

1919] McCulloch: Life Cycle of Crithidia and Trypanosoma 153

of C. euryophthalmi, notwithstanding the fact that the initial stages
of the two life cycles are entirely different in the insects. The initial
stages of the crithidial life cycle are small, oval spores, which develop
into elongate Crithidial flagellates, whereas the initial stages of the
trypanosome are trypanosomes from the blood of a rat, which indirectly
by a process of multiple fission, produce very similar elongate crithidial
flagellates. Subsequently all of the rectal stages in each life cycle are
very similar.

Fig. B. Five figures of Crithidia euryophthalmi to show the relation of the
parabasal body to the flagellum, blepharoplast, rhizoplast, and nucleus. X 3500.
Abbreviations same as in figure A.

The developmental or infective series of Crithidia euryophthalmi
consists of the stomach phase or the crithidias of the ''crop," mid-
stomach and upper part of the pyloric expansion. The degenerative
series includes the late rectal phase or the attached crithidias of the
pyloric expansion.

THE DEVELOPMENTAL SERIES
STOMACH PHASE

The stomach phase of Crithidia euryophthalmi is apparently init-
iated when the small, oval spores (pi. 2, figs. 1, 2) casually ingested-
with food begin to develop in the "crop." Such initial infective
spores develop rapidly into a swarm of multiplying crithidias (pi. 2,
figs. 2-7). Later as mature flagellates (pi. 2, figs. 9, 10) the crithidias

154 University of California Publications in Zoology [VOL. 19

may be carried immediately to the pyloric expansion through the mid-
stomach by the current of food, or they may undergo a process of
multiple fission extracellularly or intracellularly in the "crop." The
process of multiple fission may be similar to that of the spheres of
Trypanosoma leivisi (Minchin and Thomson, 1915), which have certain
characteristics in common with the somatella in the multiple fission of
some of the Polymastigina (trichomonad flagellates) described by
Kofoid and Swezy (1915). In addition to this type of multiple fission
there is a second and entirely different process of multiple fission, the
internal or endogenous budding (pi. 2, figs. 11-23). These two pro-
cesses of multiple fission have been described briefly in a preliminary
communication (McCulloch, 1917). Endogenous budding has been
emphasized in this paper and described in detail because of its interest
and importance.

EXTRACELULAR CRITHIDIAS

Oval spores. — The initial infective spores (pi. 2, fig. 1) are ovoid
and stain deeply. They are found in small numbers in the "crop,"
and present several distinguishing marks which serve as a means of
identification. They average 1.7/>t in width and about 3.4/A in length.
The nucleus stains diffusely and forms a solid mass of chromatin at
the extreme posterior end of the body. The parabasal body lies within
the anterior half of the spore, about equally distant from the nucleus
and forward end. One end of this bar-shaped structure, or parabasal
body, lies close to the thick periplast. On all the other sides of this
organelle there is the characteristic light area, which quickly destains
in iron-haematoxylin preparations. Careful focusing has revealed a
faint nuclear rhizoplast passing from the nucleus toward the region
of the parabasal body.

Developing crithidias. — When the initial infective spores begin
to develop in the "crop" a change occurs in their shape and staining
capacity (pi. 2, fig. 9). The forward outgrowth of the flagellum
assists in the elongation of the anterior end and the formation of the
undulating membrane (pi. 2, figs. 6-9). The posterior region elon-
gates less rapidly, but in time it frequently attains an even greater
length than the anterior end (pi. 2, fig. 9). The length of the free
flagellated crithidias which result from the developing forms varies
greatly at all times regardless of their location in the digestive tract.
In figure 9, plate 2, the crithidial flagellate has reached the extreme

1919] McCulloch: Life Cycle of Crithidia and Trypanosoma 155

length, comparatively, of 32/z. At the other extreme, a short crithidia
of 7ju (pi. 2, fig. 12) is evidently mature, since it is undergoing a
process of multiple fission of the endogenous-budding type. Between
these extremes is a series of intergrading forms. The sizes and
shapes of a swarm of free crithidial flagellates from the life cycle
of anyone of these species of flagellates under discussion show great
diversity, and Crithidia euryophthalmi is not an exception, as indi-
cated in plates 2 and 4. The change in the appearance of the nucleus
as development proceeds is noticeable. The deeply staining mass of
chromatin (pi. 2, figs. 1, 2) becomes a nucleus containing a prominent
central karyosome and a chromatin-encrusted nuclear membrane (pi.
2, figs. 3-10). Between the karyosome and membrane is a clear area,
which destains very readily after iron-haematoxylin.

MULTIPLE FISSION

Endogenous budding. — In looking over a large number of prepara-
tions of the digestive tract of Euryopkthalmus convivus, in the early
part of 1916, I chanced upon a splendid preparation of a "crop"
from a nymph which contained an exceedingly heavy infection of
crithidias of all shapes and sizes. The smear was well fixed and well
stained.

Among other things the nuclear structure was studied in detail
to determine whether the nucleus of these crithidias divided by a
mitotic process or by a more primitive method of mitosis. This search
led to the discovery of a flagellate which apparently contained two
nuclei (pi. 2, fig. 12), in a linear arrangement with respect to the long
axis of the body. Shortly after another crithidia (pi. 2, fig. 13) was
found in the same preparation, containing apparently three similar
nuclei, which were arranged in a like linear series. In the latter
(pi. 2, fig. 13) careful focusing revealed the outline of a fourth
partially concealed beneath the most anterior nuclear structure. In
each of these flagellates (pi. 2, figs. 12, 13) no indications of any
ordinary process of binary fission were detected. The blepharoplast,
parabasal body, and the rhizoplasts in each were still intact in so far
as could be determined. The alternative hypothesis that these nucleus-
like structures, which are relatively small, are internal parasites of a
bacterial or protozoan nature naturally was given full consideration.
Drawings of these flagellates were made with the camera lucida and
the readings were taken for future reference. Owing to the abun-

156 University of California Publications in Zoology [VOL. 19

dance of the parasites in this preparation of the "crop" ample
material was at hand for an extensive study of the morphology of this
phase of the life cycle of Crithidia euryophthalmi. From time to
time more flagellates were observed, apparently multinucleated, but
little additional light was thrown upon their peculiar nuclear struc-
tures until a large flagellate, such as shown in figure 22, plate 1, was
observed. This crithidia was relatively large and contained approxi-
mately twelve ' * binucleated " spore-like forms. The structure of the
sporelike forms within the periplast of the large flagellate was similar
to that of the numerous small, oval spores in the immediately sur-
rounding field, which were, beyond doubt, stages of the life cycle of
C. euryophthalmi. Another large flagellate containing six nuclear
structures within the periplast (pi. 2, fig. 21) was also drawn. The
flagellum and parabasal body were clearly outlined, as in the former,
multinucleated flagellates. Some of the enclosed nuclear-like struc-
tures were deeply stained, owing to the thickness of the preparation,
but the majority presented the same appearance as did the nuclear-
like structures in figures 12 and 13.

Investigation of the smear revealed more and more evidence of a
possible endogenous, or internal budding, in the life cycle of Crithidia
euryophthalmi. Past experience indicated that the preparations of
the ' ' crops ' ' of young nymphs furnished the best smears for the study
of the initial infections, of which the endogenous budding forms
were evidently a part. An effort was accordingly made during the
next breeding season of Euryophthalmus convivus to collect as many
young nymphs as possible in order to obtain additional prepara-
tions of the digestive tract, with reference to the * t crop ' ' in particular.

Out of a large number of preparations of the "crops" prepared
during the following season, only two contained additional stages of
the process of endogenous budding. The percentage of infection of
the "crops" of young nymphs was found to be approximately twenty
per cent as compared with two per cent among adult insects.

In the two preparations there were numerous small, "binucleated,"
spore-like forms, grouped near discarded flagella, with or without ble-
pharoplasts, and with parabasal bodies still attached (pi. 2, fig. 23).
It was easy to conceive of the degeneration of the cytoplasm surround-
ing the spore-like forms, leaving a field covered with the internal
spores, or zooids, and the extranuclear organelles of the parent cell.
In the earlier stages of degeneration presumably the parabasal bodies
were still attached by the parabasal rhizoplasts to the blepharoplasts

1919] McCulloch: Life Cycle of Crithidia and Trypanosoma 157

at the bases of the flagella. Later stages showed only flagella among
the zooids. This additional evidence from the two smears at once
suggested the work of Moore and Breinl (1907), in which " latent
bodies ' ' were described in the life cycle of a haemoflagellate, Trypano-
soma gambiense.

According to these investigators T. gambiense in the blood of a
vertebrate host formed latent bodies in the nuclear region, under
certain conditions. These latent bodies are pictured and described
as being small nucleated structures having a nuclear membrane and a
single mass of chromatin in the center. The exact method of the
formation of the bodies in the nucleus or from the nucleus is not
clear, since none of the early stages in the formation of the latent
bodies are figured or described in detail. Apparently only the results
of an endogenous process in the life cycle of T. gambiense were ob-
served by these investigators. The final stages of the process in the
life cycle of T. gambiense are the presence of large, degenerating try-
panosomes with minute, nucleated latent bodies in the nuclear area.
The flagellum, kinetonucleus, or parabasal body and cytoplasm are in
various stages of degeneration. Their figures of the latent bodies
(Moore and Breinl, 1907) should be compared with figure 23 of this
paper.

The latent bodies in the life cycle of Trypanosoma gambiense and
the endogenous zooids of the life cycle of Crithidia euryophthalmi are
not the only instances of an endogenous budding in the life cycle of
the Protomonadina (Trypanosoma, Crithidia, Herpetomonas, Lepto-
monas). Minchin and Thomson (1915) searched for evidence of an
endogenous budding in the life cycle of T. lewisi but failed to find any
indications of the process. However, in the life cycle of Leptomonas
pattoni, one of the so-called natural flagellates frequently found in
the digestive tract of the flea, they found several crithidial-like flagel-
lates containing a nucleus and an endogenous bud. These authors
suggested that the endogenous buds found therein were doubtless com-
parable to the latent bodies of Moore and Breinl.

The early stages of the endogenous, or internal budding of Crith-
idia euryophthalmi are shown in figures 11 and 12, plate 2. In figure
11 there is a relatively short flagellate undergoing multiple fission in
this way. The nucleus has budded off two circular, nucleus-like buds,
each showing a chromatin-encrusted nuclear membrane. The para-
basal body and the flagellum of this flagellate are still intact and have
no indications of binary fission. Somewhat similar to this flagellate

158 University of California Publications in Zoology [VOL. 19

is the one shown in figure 12, plate 2. The anterior end of the body
is slightly shortened but the nuclear structure has the same general
appearance. Only one bud has been given off, and this lies directly
posterior to the nucleus proper. As in the former case, the chromatin
is collected on the inner surface of the nuclear membrane of both the
nucleus and endogenous bud. In figure 13, plate 2, a more complex
organization was observed. The nucleus and two clearly defined
nuclear buds are arranged in a linear series, the two buds being pos-
terior to the nucleus proper. Partially concealed by the nucleus is a
third bud, which is being constricted off from the nucleus. This par-
ticular view of the process is in all probability an end view and only
a portion of the bud is visible. If the observations and interpretation
of this structure be correct, the nucleus, with all of its chromatin col-
lected on the inner surface of the membrane, repeatedly constricts or
buds off a portion, forming a series of nuclear buds (pi. 2, figs. 13,
20, 21). There is at hand at present no evidence of a central karyo-
some being present in the nucleus when endogenous buds are formed.
The nuclear buds, therefore, in so far as our observations have gone,
are due neither to a clearly defined amitosis nor to a primitive form
of mitosis of the chromatin material which normally occurs in a
central karyosome.

Although evidence of a mitotic process in this nuclear division is
wanting, nevertheless some evidence of a promitosis has been found in
the division of the nucleus in an early stage of the formation of a soma-
tella (pi. 3, fig. 25). The formation of the somatella involving the
" rounding up " of an elongate flagellate into a sphere will be described
shortly; it will suffice for our purpose here to point out salient fea-
tures of the internal structure of the ''rounding up" flagellate (pi. 3,
fig. 25). The blepharoplast, parabasal body, and the rhizoplast of this
flagellate have not yet begun to divide but the nucleus is beginning to
form two daughter-nuclei. The central karyosome which is normally
present in C. euryophthalmi has formed two unequal masses of chro-
matin connected by a centrodesmose. No critical evidence was found
showing that there is present at each end of the controdesmose a cent-
riole or centrosome differentiated from the chromatin material in this
minute form. Peripherally there is the nuclear membrane still present
and intact, but it is constricting in the center to form two nuclei. If a
similar division of the chromatin material occurs in the nuclear bud-
ding it has thus far escaped observation. The number of early stages
of nuclear budding studied has been small, owing to the rarity of

1919] McCulloch: Life Cycle of Crithidia and Trypanosoma 159

preparations showing the process. In every instance of nuclear bud-
ding so far observed the chromatin has been peripheral, with a more
or less unequal distribution on the nuclear membrane. Frequently
there is one heavy mass with a uniform amount elsewhere on the
membrane (pi. 2, fig. 15).

A somewhat larger, elongate flagellate, with sharply defined endo-
genous buds, is shown in figure 14, plate 2. Unlike the endogenous
forms just described the buds here are not posterior to the nucleus.
In figure 14, opposite the parabasal body, there is a bud decidedly
anterior and lateral in position. A second endogenous bud is also ante-
rior between the nucleus and parabasal body. Within all of these
nuclear structures the chromatin is massed irregularly upon the nuclear
membrane.

In addition to these elongate flagellates undergoing multiple fission
of this type there are also pear-shaped crithidias (pi. 2, figs. 15-18)
containing several or numerous endogenous buds. Possibly it is the
greater thickness of these flagellates, with consequent decrease in
destaining capacity, which makes the buds within stand out so dis-
tinctly. Another marked characteristic of the pear-shaped flagellates
is the lack of differentiation between the nucleus and the buds. There
is no evidence of a parent nucleus which has given rise to the buds.
The distribution of the chromatin within these buds presents some
interesting variations. In figure 15 the buds contain a clearly defined
chromatin encrusted nuclear membrane together with one distinct mass
or chromatin granule. In figure 17 the massing of the chromatin may
mean a more advanced stage since it is no longer on the entire nuclear
membrane but has been segregated into two masses, which give the
buds a bipartite appearance. Proceeding to figure 18 in this pear-
shaped individual more steps in advance are portrayed, namely the
unused portions of the flagellate are beginning to degenerate around
the endogenous buds. The parabasal body has already disappeared,
and only a portion of the discarded flagellum remains near by. The
structure of the endogenous buds of this spherical crithidia is remark-
ably uniform. In each bud the chromatin granule is adherent to
the nuclear membrane.

Other elongate flagellates undergoing multiple fission are observed
in figures 19, 20, and 21. Some interesting variations can be pointed
out in these crithidias. In figure 19 there is a nuclear rhizoplast,
which can be traced from the edge of the nuclear membrane to the
blepharoplast. Of greater interest are the variations shown in figures

160 University of California Publications in Zoology [VOL. 19

20 and 21. In figure 20 the binucleated appearance of the nucleus and
buds is very prominent. The chromatin granules have a paired effect
which is difficult to interpret. In figure 21 a more complex structure
is represented. Anterior to the nucleus there is a group of five
endogenous buds. Two of these, owing to their position, are not de-
stained sufficiently to make clear their nuclear structure. This large
flagellate has a sharply defined parabasal body and flagellum, similar
to those in figures 19 and 20. The large size of such forms has aided
materially in the interpretation of the endogenous buds, which, upon
developing, form the so-called zooids.

The most mature stage of endogenous budding yet found, wherein
the resulting zooids are still within the periplast, is pictured in
figure 22. There are approximately twelve clearly defined zooids
massed near the central part of this large flagellate. The structure
of the zooids could be studied readily. The nucleus and parabasal
body in each, because of their deep stain, helped to distinguish the
several zooids. The other organelles were not visible. Another inter-
esting feature concerning these zooids is the difference in the stages
of their development. Some are larger and more mature and were
doubtless budden off from the nucleus first. Probably the zooids of
the flagellate have been retained within the periplast of the parent
for a longer period than usual. If the size of these zooids be compared
with that of the free zooids shown in figure 23 the former, on the
whole, are larger and more fully developed. There are, moreover,
no marked signs of degeneration of the parabasal body, of the flagel-
lum, or of the cytoplasm. The parabasal body is almost hidden by
the zooids in that region.

The final step in the endogenous-budding process comes with the
degeneration of the body-plasm of the parent flagellate, leaving a mass
of zooids, together with the flagellum and parabasal body of the parent.
Portions of smears have been observed to be literally covered with
zooids and discarded flagella. As degeneration proceeds the parabasal
bodies next disappear, and finally the flagella, leaving only numerous
zooids of various sizes and structure. The last phase is the one most
frequently observed in preparations of Crithidia euryophthalmi. I
had been working with C. euryophthalmi more or less for a period
of almost two years before the clue as to the origin of these small
zooids was found. It had been most puzzling to find so many of these
small, non-flagellated, binucleated forms (pi. 4, fig. 40), which were
obviously unlike the initial infective spores (pi. 2, figs. 1, 2). They

1919] McCulloch: Life Cycle of Crithidia and Trypanosoma 161

were smaller in size, their periplast was thinner, and they destained
much more rapidly after iron-haemotoxylin. Serial sections of the
mid-stomach showed them grouped in pockets between the epithelial
cells. Possibly the endogenous process occurred there or they were
lodged there as the current of food carried the others down into the
pyloric expansion. They could be arranged in a series, beginning
with those averaging about 1.7/x, in length (pi. 4, fig. 40) and gradating
to the size of the spores which were regarded as the initial infective
spores (pi. 2, fig. 1; pi. 4, figs. 40-54).

The development of the endogenous buds into binucleated zooids
is not easy to interpret. In figure 23, plate 2, the numerous zooids
present variations in both size and structure. Unlike the latent bodies
of Trypanosoma gambiense of Moore and Breinl (1907) and the endo-
genous buds of Leptomanas pattoni of Minchin and Thomson (1915),
these endogenous buds show no central chromatin granule within the
nuclear membrane. Their chromatin is distributed at the periphery
of the nucleus usually in one of three ways. In the elongate flagellates
(pi. 2, figs. 11, 14, 19) the buds have their chromatin material massed
irregularly on the nuclear membrane. A second form of peripheral
chromatin in the nucleus is a noticeable mass or granule at one point.
This is characteristic of the buds within the pear-shaped flagellates
(pi. 2, figs. 15-18). The third type of nuclear structure in the buds
is possibly only a slight modification of the others. In figure 20,
plate 2, the internal or endogenous buds appear to be binucleated
because of the peculiar segregation of the chromatin material on the
nuclear membrane. At present these several modifications of the
nuclear structure are not regarded as having any special sequence or
significance. In some of the smallest zooids of figure 23 there are
still other modifications wherein a single, deeply staining mass is fre-
quently observed. Owing to the size of these zooids it is extremely
difficult, even with a binocular microscope, to form any adequate
conception of their structure.

A diligent search has been made among these small forms to find
a series of developing zooids which would clearly show the whole
process of the formation of the several organelles. While Crithidia
euryophthalmi undoubtedly furnishes the necessary material for such
a study, the size of the endogenous buds makes the interpretation
exceedingly difficult. It is therefore disappointing that a complete
series has not yet been accumulated showing the steps which are
thought to take place in the development of a bud into a typical

162 University of California Publications in Zoology [VOL. 19

crithidia. The results of the study now indicate that the extranuclear
organelles are apparently formed as outgrowths from the nucleus.
It is conceived that the centrosome of the nucleus divides, giving rise
to the extranuclear centrosome, or blepharoplast. From the blepharo-
plast, which is the dynamic center of the nuclear outgrowth, two
other organelles are formed. The flagellum grows forward anteriorly
and the parabasal body to the side. The connection which persists
between the blepharoplast and the nucleus is the nuclear rhizoplast.
Between the blepharoplast and the parabasal body is the parabasal
rhizoplast. Careful observation of some of the larger forms reveals
a cytoplasmic sheath around the flagellum, which is continuous with
a similar sheath around the parabasal body. The latter sheath is
like a sack, in which the deeply staining, bar-shaped parabasal body
is suspended. The variation in size of the parabasal body may
explain the light area which is frequently observed to surround this
organelle. The fan-shaped appearance of the parabasal rhizoplast
is due in all probability to this sacklike sheath. Any general con-
clusions concerning the origin of these organelles would, at this time
however, be premature, although the observations of the zooids under
the binocular microscope tend to give this conception of their origin.
It is necessary to keep in mind constantly the fact that the material
with which we are dealing is the complex life cycle of a flagellate and
that several stages of the life cycle have not yet been followed, step
by step, in the living material. The possibility of confusing two life
cycles is always present, and the fact that all of the work is done
near the limits of microscopical magnification adds further possibility
of misconception.

Aside from these difficulties and doubtful points, however, the
discovery of all of these stages of what has been interpretated as a
process of endogenous budding, opens up further problems for investi-
gation in the life cycles of these flagellates. The origin of the para-
basal body in the endogenous bud is a big problem in itself. In addi-
tion, the light thrown upon the probable origin of the numerous
binucleated spores or Leishmanw-like bodies, which occur so abun-
dantly in the life cycles of such flagellates as Schizotrypanum cruzi
(Chagas, 1910), Crithidia melophagia (Porter, 1910), and C. lepto-
coridis (McCulloch, 1915), is very suggestive.

Somatella. — Previous to the discovery of the multinucleated flag-
ellates which were undergoing a process of internal or endogenous
budding, another type of multiple fission had been studied in prepara-

1919] McCulloch: Life Cycle of Crithidia and Trypanosoma 163

tions of the "crop" of Euryopthalmus convivus, namely, that which
leads to the formation of the somatella (pi. 3, figs. 28-32). These
spherical crithidias have certain characteristics in common with the
tailed and tailless spheres described in the life cycle of Trypanosoma
lewisi by Minchin and Thomson (1915). In a general way they re-
semble also the somatellas in the life cycle of some of the Polymastigina
described by Kofoid and Swezy (1915). Beginning with the earliest
stages of this type of multiple fission a series of flagellates can be ar-
ranged which parallels the successive stages of multiple fission of T.
lewisi (cf. Minchin and Thomson, 1915, pi. 37). At the beginning
of this series such flagellates as shown in figures 24 and 25 of plate 3
are to be found. In figure 24 the elongate flagellate is beginning to
round up. The attenuate ends are being drawn up to the central part
of the body and the flagellum has become intracellular throughout its
length. A somewhat different formation of the sphere is shown in
figure 25, plate 3. The long, attenuate ends are being wrapped about
the body and the flagellum is likewise entirely intracellular in this
flagellate. Other important features to be observed in connection
with this flagellate are the first indications of nuclear division in the
spherical formation. The centriole or centrosome within the nucleus
of the rounding-up flagellate is initiating the division of the organ-
elles. It has divided into two daughter-centrosomes, which are still
connected by a centrodesmose, and with each daughter-centrosome there
is present a varying amount of chromatin material. No chromosomes
are present at this stage of the division, and their presence at any
period throughout the process in Crithidia euryophthalmi has not been
established. Indications of chromosomes in the ordinary binary fission
have been observed, but no definite number has been determined. The
nuclear membrane has begun to constrict on either side of the centro-
desmose. The entire division of the chromatin material takes place
within the nuclear membrane, and the process is apparently a primi-
tive type of promitosis. The centrosome within the karyosome is not
always the first to divide ; on the contrary, in many instances the first
indication of the division of the organelles is shown by the blepharo-
plast or extranuclear centrosome. When the blepharoplast divides
the division of the parabasal body follows immediately. A repetition
of the division of the several organelles occurs and the spheres finally
break up into a number of merozoites, or daughter individuals.

The spherical formation is completed in figure 26, plate 3. A
flagellum is protruding beyond the surface of the sphere. Internally

164 University of California Publications in Zoology [VOL. 19

division of the blepharoplast, the parabasal body, and the nucleus
has occurred. A second flagellum growing out from the daughter-
blepharoplast cannot be observed. Another more advanced stage of
multiple fission is shown in the somatella in figure 27. The number of
nuclei and of the parabasal bodies is the same as in figure 26, but the
outgrowths of the daughter-flagella are clearly shown in this sphere.
Spheres without protruding flagella are to be found in figures 28 and
29, plate 3. The formation of the merozoites within the spheres, as
presented in figure 29, has advanced to the point where they are
clearly defined. In figure 28 another important observation can be
made, namely, that the divisions of the blepharoplast and the nucleus
do not occur simultaneously. In this particular sphere there are three
parabasal bodies present but only two nuclei. In figure 29 there are
four merozoites visible, each of which shows the outgrowth of a flag-
ellum, and a similar spherical formation is shown in figure 30, wherein
the four merozoites are somewhat larger and more developed. In the
latter somatella, however, the thickness of the sphere prevented the
usual amount of destaining necessary to show the nuclear structure.
In each of these merozoites the body is elongating and the anterior
end is becoming attenuate.

Another thick sphere is found in figure 31, and all the nuclei
therein have the appearance of being a solid mass of chromatin. In
this somatella the number of merozoites which could be counted is
twelve. The irregular outline of the sphere indicates that the breal\-
ing up or plasmotomy of the sphere is about to occur. Possibly some
of the merozoites have already escaped. In the investigation thus
far the number of merozoites in a somatella has been exceedingly
variable. In some of the Polymastigina, Kofoid and Swezy (1915)
found the number of merozoites to be eight, which is apparently con-
stant for the somatellas of these flagellates. Minchin and Thomson
(1915) report a variable number of merozoites in the spheres of
Trypanosoma lewisi but they found the average number to be approxi-
mately ten. In the spherical mass of flagellates shown in figure 31,
plate 3, the number is twelve, but in a still larger sphere (pi. 3, fig.
32) the number is probably double that, or twenty-four. The dense-
ness of the latter may have obscured some of the parabasal bodies and
nuclei. Protruding from the surface of this sphere are numerous flag-
ella which are outgrowths from the daughter-blepharoplasts. In the
somatellas of the Polymastigina the nucleus and the extranuclear
organelles may divide simultaneously, but in the spheres of T. leivisi

1919] McCulloch: Life Cycle of Crithidia and Trypanosoma 165

and in the somatella of Crithidia euryophthalmi either the extra-
nuclear or the nuclear organelles may divide first. The ultimate
number of nuclei is equal to the number of parabasal bodies and of
the blepharoplasts.

The stage of development of the merozoites when plasmotomy
occurs, is variable. Usually the rupture of the somatella occurs when
the merozoites are just beginning to elongate, as in figure 31. All
the organeles of these merozoites are definitely outlined, and the
flagella are free for a certain distance of their length. In a few
instances extremely elongate flagellates have been observed wriggling
about within the spherical structures. There is no evidence of a
residual mass of cytoplasm.

A comparison of these two methods of multiple fission in the
life cycle of Crithidia euryophthalmi reveals the fact that they are
fundamentally different, In the first place, the former method, endo-
genous budding, involves only one organelle of the crithidia, namely,
the nucleus. In the latter method a somatella results from a repeated
division of the nucleus, blepharoplast, parabasal body, and, in all
probability, the nuclear and parabasal rhizoplasts. The flagella, how-
ever, in each case are new outgrowths from the newly formed blepharo-
plasts in endogenous buds or from the daughter-blepharoplasts in the
somatella. Secondly, the flagellates undergoing endogenous budding
retain their normal shape as elongate or pear-shaped crithidias. In
some instances their size increases as the buds develop. The flagellates
forming somatellas round up into spheres. The comparative efficiency
of the two processes from the standpoint of the multiplicative phase
in the host cannot be estimated. The length of time necessary 'to com-
plete each process and the conditions under which each occurs are at
present unknown. On the whole, the number of individuals resulting
from an equal number of endogenous flagellates or of somatellas is
approximately the same. Our conclusion, however, concerning the
two methods is that the endogenous budding is of greater importance
in the life cycle of C. euryophthalmi since in the preparations there
are relatively many more evidences of the endogenous budding than
of the somatella.

BINAEY FISSION

Another method of multiplication of Crithidia euryophthalmi in
the "crop" is binary fission. This process has also been observed in
the pyloric expansion. While the number of crithidias found dividing

166 University of California Publications in Zoology [VoL- 19

in this way is relatively small, on the whole, yet crithidias of almost
any stage of development apparently may thus increase their number.
Some conception of the prevalence of the process throughout the
life cycle may be gained by studying figures 33 to 37. In addition
to these smaller crithidias in various stages of development many
instances of binary fission have been observed among the elongate or
mature flagellates.

The blepharoplast or the centrosome of the nucleus may initiate
the division of the several organelles. In figure 33, plate 3, the
centrosome of the nucleus is in the process of binary fission and there
are present two daughter-nuclei. In each of the nuclei the chromatin
is peripheral on the nuclear membrane. The blepharoplast and the
parabasal body have not yet begun to divide. The general appear-
ance of this binary fission form is very similar to that of the smaller
somatella. In the unflagellated crithidia shown in figure 34, plate 3,
the blepharoplast and parabasal body have divided but the nucleus
is still intact. A more advanced stage of binary fission is found in
figure 35. In this small, unflagellated crithidia the blepharoplast,
parabasal body, and the nucleus have divided, and a light, thin area,
which is preliminary to the cleft in the cytoplasm, extends longi-
tudinally between the two sets of organelles. The flagella can also
be observed. The longer, more clearly defined flagellum is evidently
that of the parent since the second is shorter and less distinct. The
last stages of binary fission are shown in figures 36 and 37. In figure
36 the cleft in the cytoplasm can be traced from the anterior to the
posterior end, and the daughter flagellum has also attained a greater
length. ' The two flagella in figure 37 are the same length; the separa-
tion of the two daughter individuals is more marked and almost com-
plete in figure 38. The posterior ends are the last to remain attached
and the lashing about of the anterior ends assists efficiently in the
final tearing apart.

One of the most interesting problems in connection with this
method of reproduction has always been the origin of the daughter-
flagellum. Is it due to the division of the flagellum of the parent or
to a new outgrowth from the daughter-blepharoplast ? A review
of the work already done on this genus indicates that with the excep-
tion of Porter (1909, 1910) all authors regard the daughter-flagellum
as a new outgrowth and consider that the parent-flagellum does not
split to form two daughter flagella. My work on Crithidia leptocoridis
and C. euryophthalmi, unlike that of Porter, is an agreement with

1919] McCulloch: Life Cycle of Crithidia and Trypanosoma 167

the results of other investigators. For some time the evidence of the
new outgrowth was difficult to obtain (McCulloch, 1915), but very
clear evidence of such an outgrowth from the blepharoplast has finally
been discovered. Text-figure C gives a very clear picture of the origin
of the daughter-flagellum in C. leptocoridis. The larger size and the
greater abundance of crithidias undergoing this process make C. lepto-
coridis the most desirable material to illustrate this point.

The importance of binary fission in increasing the number of
parasites in the multiplicative phase in the "crop" is not great. At
the present time no evidence of the process has been found in the mid-
stomach, which serves chiefly as a passage way for the crithidias mi-

Fig. C. Flagellate stage of Crithidia leptocoridis to show the outgrowth of
the new flagellum in binary fission. X 3500. Abbreviations the same as in
figure A.

grating from the "crop" to the pyloric expansion. In the pyloric
portion of the digestive tract, however, binary fission is of great im-
portance in increasing the number of crithidias, which attach them-
selves to the epithelial lining of the pyloric expansion.

In connection with the process of binary fission in Crithidia eury-
ophthalmi another problem of interest has occurred, namely, the nature
of the difference, if there be any, between the division of the several
organelles in binary fission and the division of these same organelles in
the somatella. Minchin and Thomson (1915) have regarded the in-
crease in the number of nuclei and kinetonuclei in the spheres of Try-

168 University of California Publications in Zoology [VOL. 19

panosoma lewisi as being due to a repeated binary fission wherein the
resulting individuals failed to separate immediately. Kofoid and
Swezy (1915) have described in detail the process of binary fission and
of multiple fission for the trichomonad flagellates, and these authors
found the increase of organelles in multiple fission to be due to thrice
repeated mitosis. The larger size of those flagellates together with
the correspondingly increased size of the organelles presents better
material for the study of binary and multiple fission than do the
trypanosomes or the crithidias. In C. euryophthalmi the minute size
of the flagellates undergoing either process have made accurate inter-
pretation thus far impossible. For this reason the relation of binary
fission to multiple fission in a somatella must remain an open question
for the present.

INTRACELLULAR CRITHIDIAS

Another salient similarity between the life cycle of Crithidia
euryophthalmi and the life cycle of Try panosoma lewisi in the inverte-
brate host is the appearance in each life cycle of a stage of intra-
cellular multiple fission. In C. euryophthalmi there is figured for
the first time an epithelial cell in the life cycle of a crithidia taken
from a '"crop" containing numerous crithidial parasites. Careful
examination of this infected cell shows that there are three distinct
groups (pi. 3, fig. 39, a, b, c) of parasites and a number of scattered
crithidias. Altogether there are approximately seventy parasites in
this one epithelial cell. In figure 39a the crithidias are of two sizes:
long, slender flagellates, and short, non-flagellated forms. Since mul-
tiple fission of either the endogenous or somatella type produces zooids
or merozoites of approximately the same size, it appears that several
infections have occurred in this cell. It is conceivable that all the
elongate flagellates are due to one infection while the short, non-flag-
ellated crithidias are due to a second infection. In figure 39& there
are ten small oval forms. In structure they show a diffuse nucleus,
as do the other parasites within this host-cell, which is probably due
in part to the thickness of the cell. A nuclear rhizoplast can be
observed passing from the nucleus to the blepharoplast, and a short
intracellular flagellum extends forward to the anterior end of the
body. The parabasal body of each is a relatively small and deeply
staining structure. At c and e of figure 39 are more of the small oval
forms. In the former region the cytoplasm of the host-cell has been

1919] McCulloch: Life Cycle of Crithidia and Trypanosoma 169

completely destroyed within a certain radius of the parasites. In
the latter the small forms are scattered in the cytoplasm of the host-
cell. At d there is still another group of elongate flagellates, which
are probably the results of another process of multiple fission. They
are approximately the same size and shape, and are in the same stage of
development. Whether they are merozoites from a somatella or zooids
from endogenous buds is not clear, but they have broken apart some-
what and are making their way to the periphery of the host-cell. At /
a flagellate is protruding through the cell wall and the posterior end
is directed forward to penetrate the tissue. In the study of the living
material of both C. leptocoridis and C. euryophthalmi crithidias have
been observed to direct their posterior ends forward and to use the
flagellated ends as propellers in penetrating tissues. .

The early stages of the intracellular multiple fission have not yet
been found within the epithelial cells of the crop and the more ad-
vanced stages, such as shown in figure 39, do not indicate definitely the
method of multiple fission. These intracellular crithidias had been
described as the results of multiple fission within somatellas before
the discovery of the stages of the endogenous process of multiple
fission in this life cycle. The evidence is not absolutely convincing
either way, but there is at the present state of the investigation a
preponderance of evidence in favor of their probable origin by the
plasmotomy of a somatella. The circular outlines of the vacuoles in
which the groups of parasites are found in figure 39 suggest the
formation of the spheroidal somatella. Further there are no evi-
dences of discarded flagella within these cells which suggests the possi-
bility that endogenous budding might have occurred with the forma-
tion of circular cavities in the cytoplasm of the host-cell. On the
other hand, if the numerous small oval spores are due to a process of
multiple fission within a somatella the subsequent plasmotomy has
taken place very early. Usually the breaking apart of the merozoites
does not occur until they have become elongate flagellates. Moreover,
the exact method of the process of intracellular multiple fission is
not so important as the fact that under certain conditions crithidias
become intracellular and destroy the epithelial lining of the digestive
tract which they penetrate. Each destruction of a host-cell thus also
means a tremendous increase apparently in the number of the para-
sites. The intracellular crithidias are not found frequently in the
preparations. We have no proof that it is an obligatory phase, though
it might well be so. Nor have we evidence that it follows the forma-

170 University of California Publications in Zoology [VOL. 19

tion of a zygote, as does the somatella of Plasmodium in the wall of
the digestive tract of a mosquito. There is no evidence that the zygote
precedes the somatella in the Polymastigina. In the life cycle of
Trypanosoma lewisi the process of intracellular multiple fission is
apparently obligatory. The trypanosomes, or haemoflagellates, pene-
trate epithelial cells of the "crop," undergo multiple fission, and
crithidiomorphic merozoites are produced. This phase brings about the
early stages of transition from a trypanosome to a crithidia. A similar
need for such a phase is not present in the life cycle of Crithidia
euryophthalmi. At present our knowledge of intracellular multiple
fission in the life cycle of C. euryophthalmi is too meager to permit of
extended correlation between the life cycle of this more primitive
flagellate with that of the more highly developed haemoflagellate T.
leivisi. It is conceivable that in the evolution of trypanosomes from
the crithidial-like flagellates the intracellular multiple fission was
carried over and became more specialized and more important in the
life cycle of the haemoflagellate.

RECTAL PHASE

The stomach phase of Crithidia euryophthalmi, beginning with the
initial infective spores and ending with the great swarm of parasites
resulting from binary, extracellular, and intracellular multiple fission
in the "crop," is followed by the established rectal phase of the life
cycle in the pyloric expansion.

Owing to the structure of the digestive tract of Euryophthalmus
convivus, with its three divisions separated only by a narrow con-
striction, which allows a possible intermingling under normal condi-
tions of the crithidias in the "crop" with those of the mid-stomach
and pyloric expansion, it is extremely difficult to say where the stomach
phase ends and the rectal phase begins. In Trypanosoma lewisi the
transition between the stomach and rectal phase, as has already been
pointed out, is marked by definite structural changes. The trypano-
somes, brought into the stomach of the flea with blood from a rat, enter
epithelial cells and undergo multiple fission, producing merozoites of
a crithidiomorphic type. The merozoites thus produced may do one
of two things, either enter other epithelial cells of the stomach or
collect at the pyloric opening and be carried down the intestine to the
rectum with food. As they pass through the intestine the structural

1919] McCulloch: Life Cycle of Crithidia and Trypanosoma 171

changes which convert crithidiomorphic forms into crithidias are
taking place.

The frequency with which we find preparations of the "crop"
free from infection with C. euryophthalmi, the less frequency of infec-
tion of the mid-stomach, together with the almost invariably heavy
infection in the pyloric expansion, lead us to the conclusion that the
crithidias of Euryophthalmus convives have the same tendency to
migrate posteriorly as does T. lewisi in the flea. In C. euryophthalmi,
however, the movement is less marked than in T. lewisi. This tendency
of slow progression posteriorly doubtless depends upon the move-
ment of food contents from the "crop" into the mid-stomach and
pyloric expansion.

The migrating mass of crithidias from the ' * crop ' ' soon establishes
three distinct types of parasites in the pyloric expansion: the necto-
monads or free flagellates, the haptomonads or attached flagellates,
and the infective spores which serve for transmitting C. euryophthalmi
to another host. These types or classes of parasites are comparable in
almost every way to the nectomonads, or free flagellates, the hapto-
monads, or attached flagellates, and the final trypaniform stage of the
rectal phase of T. lewisi. The nectomonads and haptomonads of each
life-cvcle are almost identical in structure and behavior.

NECTOMONADS

Smear preparations of the mid-stomach and of the pyloric expan-
sion show little difference in the morphological structure of their
crithidial infection. The serial sections of these two parts, however,
show a sharp distinction in the structure of haptomonad forms and
the nature of the epithelial lining to which they are attached, and
a slight distinction in the structure of the nectomonads in the mid-
stomach and in the pyloric expansion. The nectomonads of the
former are usually of the elongate, slender type (pi. 5, figs. 73-80).
The zooids, the result of the processes of multiple fission (pi. 4,
figs. 40-54) are abundant in the smear preparations of both regions.
The series sections thus far have shown numerous zooids (pi. 4,
figs. 40-50) in the anterior portion of the mid-stomach. These
zooids (pi. 4, fig. 40) are frequently grouped together in the grooves
between epithelial cells. For this reason it is possible that the cur-
rent of food in passing down the digestive tract failed to carry
them on into the pyloric expansion. The zooids are small forms,

172 University of California Publications in Zoology [VOL. 19

(pi. 4, figs. 40-47) from I/* (pi. 4, fig. 40) to 1.5ft (pi. 4, fig. 47) in
length and from 0.7 to I/A in diameter. In comparing them with
the initial infective oval spores (pi. 2, figs. 1, 2) it is noticed that they
are smaller, stain less densely, and do not show a heavy periplast. The
nuclear structure also is unlike that of the initial spore forms, and the
location of the nucleus within the zooid adds another distinguishing
character. These zooids have always been found in the anterior por-
tion of the mid-stomach in the serial sections while the sections imme-
diately posterior contain developing crithidias (pi. 4, figs. 59-64).
Groups of elongating forms are frequently found (pi. 4, fig. 59).
With the elongation of the body of the zooids, especially of the ante-
rior end, the nucleus instead of being filled with chromatin is now
vesicular and contains a distinct karyosome. This group (pi. 4,
fig. 59) shows the parabasal bodies in close proximity to the nuclear
membranes. Judging from the conditions found within the great
majority of forms, figure 59 is probably an exceptional case in this
respect. The parabasal body normally moves anteriorly in the early
development of the zooid (pi. 6, fig. 40) before the flagellum and the
anterior end grow out (pi. 4, figs. 41-53). In the figures just noted
the flagellum' is not yet visible. The nuclear rhizoplast, extending
from the nucleus forward to the region of the blepharoplast and para-
basal body, is found by focusing carefully. In figure 54 the flagellum
is growing out from the blepharoplast but there is no noticeable
lengthening of the anterior end of the body. In figures 55 to 58 the
flagellum and anterior end of each are lengthening simultaneously.
In these same figures the nuclei are like the nuclei of the zooids, being
completely filled with chromatin. Farther on posteriorly are found
crithidias such as are shown in figures 63 to 72. Beginning with
figure 65 there is also an elongation of the posterior end, which is equal
to that of the anterior.

The majority of these developing crithidias have the vesicular
type of nucleus. Figures 65, 67, and 68 are exceptions, but no sig-
nificance can be attached to them since the position, relative thickness
of the body, or the technique, could explain these exceptions in this
region of the digestive tract. The study of the serial sections leads
us to think that the time necessary for the stomach crithidias to
reach the rectum is approximately the amount of time required for
the zooids to develop into mature flagellates. Not all of the develop-
ing zooids become mature in the mid-stomach. Under certain con-
ditions the food current probably carries many of the non-flagelated
stages or zooids back into the pyloric expansion before they have

1919] McCulloch: Life Cycle of Crithidia and Trypanosoma 173

scarcely begun to develop. As the developing forms and the fully
matured forms enter the pyloric expansion, they become part of the
permanent crithidial infection therein. The content of the crithidial
infection of this region varies from time to time.- Nectomonads
may predominate or all nectomonads may become haptomonads, or
attached forms. Usually the nectomonads and haptomonads are both
present in large numbers. Mature nectomonads in the mid-stomach
are usually the elongate, slender crithidias (pi. 4, figs. 73-79). In
figure 74 the body size is approximately 25/x in length and 1.7/* in
width. Figures 75 to 78 are yet narrower, averaging about I/A in
width. The nectomonads of the pyloric expansion are frequently
like figures 73 to 79, long, slender forms together with numerous
shorter and stouter erithidias (pi. 4, figs. 80-90). It is conceivable
that the elongate slender forms give rise to the shorter, stout forms.
The length of the body is decreased and the width is increased. Both
the anterior and the posterior ends of the body become less attenuate.
The structure of the nucleus of the elongate forms varies con-
siderably. In figures 73 and 80 there are distinct central karyosomes
with chromatin-encrusted membranes. In figure 74 the chromatin
is in five granules scattered within the nuclear membrane. The
chromatin is broken up into granules in figures 76 and 79. The break-
ing up of the chromatin material in these nectomonad forms doubtless
means the beginning of a degeneration, which will be discussed shortly.
The nuclear structures in figures 77 and 78 are unique. In figure
77 the nuclear membrane is rather densely encrusted with chromatin
and is elongate and irregular in outline. In figure 78 the nuclear
membrane is oval, chromatin-encrusted, with two masses of chromatin
at the anterior and posterior regions of the nucleus. In the series
of figures 81, 83 to 86 a karyosome of a variable size is found in
each and its location is not always central. In figure 82 a distinct
chromidial fragmentation of the chromatin has taken place, which is
another indication of degeneration. The short, stout forms (pi. 4,
figs. 87-90) are transition forms from the nectomonads to hapto-
monads. While these flagellates are still free forms they resemble the
haptomonads of the pyloric expansion, which are attached in the mid-
region of this division. In the anterior part of the pyloric expansion
the haptomonads are relatively long, slender flagellates while posterior
to the middle portion they are still short and more pear-shaped. The
nuclear structure of these transition forms indicates no degeneration
as yet. They all have a central karyosome and a more or less encrusted
nuclear membrane.

174 University of California Publications in Zoology [VOL. 19

HAPTOMONADS

One of the most characteristic features of the life cycle of a crithi-
dial flagellate is the great mass of attached forms which line definite
parts of the digestive tract of the host. Minchin and Thomson found
three regions of the digestive tract of the rat-flea where the crithidias
of Trypanosoma lewisi might attach themselves, namely, the prepyloric,
post-pyloric, and the rectal regions. In the lupine bug there are like-
wise three regions where the haptomonads may attach themselves : ( 1 )
in the posterior part of the "crop," where they are possibly com-
parable to the prepyloric crithidias in the posterior part of the
stomach of the flea; (2) in the posterior half of the mid-stomach,
where they are probably comparable to the post-pyloric crithidias
of the rat-flea, attached to the anterior part of the intestine; and (3)
in the pyloric expansion, where they are comparable to the hapto-
monads of the rectum of the rat-flea. In Trypanosoma lewisi in the
flea these investigators regard the prepyloric haptomonads as being
due to a forward migration from the rectum,. possibly as a result of
the food conditions. The prepyloric haptomonads are not found fre-
quently in the flea, and in only one preparation of the "crop" of the
lupine bug were haptomonads found. In the lupine bug haptomonads
were found in a number of preparations of the mid-stomach, but they
were commonly present in preparations of the pyloric expansion. The
haptomonads observed in the single preparation of the "crop" were
of the rectal type, small oval forms similar to those from the pyloric
expansion shown in plate 6, figures 107 and 117. The serial sections of
the digestive tract, however, showed no haptomonads in the "crop."

From the serial sections of the mid-stomach abundant material
was obtained for the study of the haptomonad of this region. The
attached forms here (pi. 6, figs. 93-96) are relatively small, slender
flagellates. They are uniform in size and shape, on the whole, and
form a definite fringe on the inner surface of the epithelial lining of
the sections of the mid-stomach. These flagellates attach themselves
to the epithelial cells by means of the flagella. They frequently almost
surround the elongate, columnar epithelial cells, which project into
the lumen of the digestive tract. The grooves between masses of epi-
thelial cells evidently afford a particularly good place for the hapto-
monads, since they are found in compact layers in such places. Hapto-
monad crithidias of this type are the only ones found in the mid-
stomach and they continue to line the digestive tract posteriorly

1919] McCulloch: Life Cycle of Crithidia and Trypanosoma 175

into the pyloric expansion. Considering the attached crithidias the
most anterior sections of the pyloric expansion are almost identical
with the sections from the posterior part of the mid-stomach. The
lumen of the narrow constriction separating the mid-stomach from
the pyloric expansion is frequently almost blocked with the bodies of
the flagellates extending out into the passageway between these two
enlargements of the digestive tract.

The structure of these haptomonads (pi. 6, figs. 93-96) is also
relatively uniform. The majority of the crithidias show the vesicular
type of nucleus, with a central karyosome and a nuclear membrane
containing an initer lining of chromatin material. In figure 94 the
nuclear membrane is slightly distorted in appearance and the karyo-
some is excentric, being posterior. In figure 96 the nuclear membrane
is not distinct and the enlarged mass of chromatin material is in the
form of two granules. These haptomonads are usually characterized
by their attenuate anterior and posterior ends. The posterior ends of
these crithidias more than of any others show relatively extreme
attenuation.

Beginning in a region just posterior to the mid-stomach end of
the pyloric expansion a series of transition haptomonads is found
(pi. 6, figs. 97-106), lining the middle part of this division of the
digestive tract, these transition haptomonads, which vary consider-
ably in size. Figure 97 shows a broad, stout form. Such forms undergo
binary fission, producing two smaller, more slender individuals. Pos-
sibly figures 100 and 101 are the products of such a division. The
binary fission of the broad, stout forms rapidly increases the number
of haptomonads which make up the dense and compact layer of attached
crithidias. The nuclear structure of these forms shows compara-
tively little variation. They have the vesicular nucleus with the small
central karyosome and chromatin-encrusted nuclear membrane.

At the posterior portion of the pyloric expansion are found the
normal rectal forms (pi. 6, figs. 107-124) which are common to the
life cycles of so many of these flagellates. One of the interesting
things observed in connection with these forms is that the wall of the
pyloric expansion becomes exceedingly thin. There are few indica-
tions left of the epithelial cells lining this part of the mid-gut. Sec-
tions of this portion of the tract previous to any infection by the
flagellate are not at hand, unfortunately, and consequently it is diffi-
cult to estimate the total amount of destruction incurred. The pyloric
expansion of an infected adult bug, however, is extremely weak and

176 University of California Publications in Zoology [VOL. 19

becomes torn very readily ; it has every appearance of having been
almost entirely destroyed by the crithidias. Among these small hapto-
monads numerous variations of size and shape are noted. The length
of the free flagellum (pi. 6, figs. 109, 110) is exceedingly variable.
In other crithidias whatever flagella are present are intracellular
throughout their length. The changes brought about in the flagella
are probably due to their absorption. In every case the flagellum
attaches the crithidia to the wall of the digestive tract. The nuclear
structure of the haptomonads in this region, with the exception of
the forms in figure 91, shows no indication of degeneration. The round
haptomonads finally become free forms. They can be seen to drop
off in the living preparations and to roll up into round or oval forms
(pi. 6, figs. 118, 119, 122). The cytoplasm of these forms is vacuolate
and stains lightly. These round crithidias then degenerate along with
the nectomonads, which are constantly degenerating. The degenera-
tion of the haptomonads and nectomonads will be described under
the degenerative series.

FINAL SPOEE FORMS

The structure of the digestive tract of EuryophtJialmus co-nvivus,
including three portions of the stomach proper and the intestine with
its gland, differentiates very clearly between the degenerative series
of crithidias and the final spore forms which can be transmitted to
another host. In the anterior part of the digestive tract is found the
developmental series which becomes the degenerative series together
with the final spore forms. Posterior to the gland only the final
spore forms have been observed. The preparations of the rectum
show only these final spore forms (pi. 6, figs. 125-131). These are
oval, non-flagellated forms containing a thick periplast, within which
are the nucleus and parabasal body. In figure 126 the characteris-
tics of these final spore forms may be noted. They are approximately
2.8/>t in length and 1.4ft in width. The nucleus lies in the extreme
posterior end of the body and stains deeply. The parabasal body is
sharply outlined within a vacuolate area. A faint nuclear rhizoplast
may be visible, extending from the nucleus toward the parabasal
body. As previously indicated in a preliminary paper (McCulloch,
1917) it was some time before the true rectum was discovered, and
consequently the significance of these final spore forms was not en-
tirely clear in the beginning of the investigation. However, with the
discovery of the true rectum and the fact that it contained only these

1919] McCulloch: Life Cycle of Crithidia and Trypanosoma 177

final spores the characteristics of these rectal forms became clear and
it was relatively easy to find them in both the mid-stomach and pyloric
expansion. In the "crop" of the lupine bug the multiple fission
gives rise to numerous small zooids. Some few of these zooids, on
reaching a certain stage of development, become the well protected
final spore forms. Their development into mature flagellates is ar-
rested for an indefinite time, perhaps in response to some unfavorable
internal or external chemical condition. The periplast becomes thick-
ened and these zooids stain deeply and retain the stain much better
than the unprotected zooids. Since only a few of the zooids be-
come thus encysted the change cannot be regarded as a general
change in response to some general stimulus. In the infections of
the mid-stomach and pyloric expansion some of the final spore forms
can be found at almost any period in the life history of the flagellates.
Considering that these are the only stage of the life cycle of the
flagellate which have been found in the rectum they have been re-
garded as the final spore forms, which upon being ingested with food
by another insect host become the initial infective spores. Our observa-
tions upon these cannot be regarded as conclusive as yet, owing to
the fact that experimentally we have not produced infection with
these spores.

THE DEGENERATIVE SERIES

The degenerative series includes all individuals of the life cycle
other than the final spore forms and their antecedents which have
just been discussed. It has already been pointed out that the rectal
phase of C. euryophthalmi in the pyloric expansion of the lupine bug
is comparable to the rectal phase of T. lewisi in the rectum of the
flea. In the lupine bug the degenerating forms abound in the poste-
rior portion of the pyloric expansion and their number is apparently
not decreased by constant elimination under normal conditions of
such parasites from the intestinal contents, which pass through a
relatively long intestine before reaching the colon and rectum. If
any flagellates succeed in passing out with the intestinal matter into
this long intestine they are evidently destroyed by the new chemical
medium before they reach the rectum. In the flea the degenerating
forms are in the rectum and it is possible that their number is
repeatedly being decreased with each discharge of the feces.

There is little danger of confusing the developmental and degenera-
tive series in the life-cycle of C. euryopkthalmi since the differentia-

178 University of California Publications in Zoology [VOL. 19

tion of the parasites into the final spore forms and the ordinary
propagative forms takes place apparently in the stomach phase in
the * * crop. ' ' The majority, in fact, nearly all of the parasites become
ordinary propagative forms which develop and increase their num-
bers in the digestive tract of the lupine bug. After passing through
the various processes of the life cycle they degenerate in the late
rectal phase in the pyloric expansion. The permanent rectal phase
persists through the life of the lupine bug after the first infection;
the degenerative series is soon formed, and likewise persists throughout
the life of the host.

The individuals of the degenerative series are distinguished among
the mass of living crithidias by a sticky periplast, to which bacteria
frequently adhere by virtue of the tendency of crithidias to adhere to
each other, by slow sluggish movements, and by odd sizes and shapes.
The degenerating forms are detected in stained preparations by nuclei
with diffused chromatin or by a vesicular nucleus breaking up into a
number of chromatin granules, and by the vacuolated cytoplasm. The
size, shape, and location of the crithidias also assists in distinguishing
between the developmental and degenerative crithidias.

CONCLUSIONS

1. The crithidial flagellates of the life cycle of Trypanosoma are
structurally like the crithidial flagellates of the life cycle of Crithidia.
The extranuclear organelles, the blepharoplast, parabasal body, para-
basal rhizoplast, nuclear rhizoplast, and the flagellum are all common
to the crithidial flagellates of both Trypanosoma and Crithidia.

2. From the viewpoint of comparative morphology the differences
existing between the crithidial forms of C. euryophthalmi and the
crithidial forms of T. lewisi are less marked than are the differences
between similar stages of T. lewisi and Schizotrypanum cruzi.

3. Using the life cycle of T. lewisi as a standard for comparison of
the life cycle of a haemoflagellate or a trypanosome and the life cycle
of C. euryophthalmi as the standard of the life cycle of a more primi-
tive crithidial flagellate, there are more parallel stages and phases in
these two life cycles than exist between the life cycle of any trypano-
some and the life cycle of any herpetomonad or of any leptomonad now
known. Furthermore the close correlation between these two life
cycles of T. lewisi and of C. euryophthalmi affords new evidence that

1919] McCulloch: Life Cycle of Crithidia and Trypanosoma 179

the evolution of a trypanosome has probably taken place from a
crithidial flagellate rather than from a herpetomonad or leptomonad
flagellate.

4. The process of multiple fission in the somatella of C. euryoph-
thalmi is fundamentally like the multiple fission (sphere formation)
of T. lewisi and also like the multiple fission (somatella) of the tricho-
monad flagellates. In each of these flagellates the nucleus, parabasal
body, blepharoplast, and flagellum, or flagella, are concerned in the
process. In each after multiple fission plasmotomy occurs.

5. The process of multiple fission by endogenous budding in the
life-cycle of C. euryophthalmi tends not only to establish another link
in common between the life cycles of Trypanosoma (e.g., T. gambiense)
and the life cycle of Crithidia but also to link the life cycle of
Crithidia more closely to the lower protozoan forms which contain
numerous Leishmania-like bodies in their life cycles.

6. The endogenous buds in the life of C. euryophhtalmi are com-
parable to the latent bodies in the life-cycle of T. gambiense.

180 University of California Publications in Zoology [VOL. 19

LITERATURE CITED

CHATTON, E., and LEGER, A.

1911. Eutrypanosomes, Leptomonas et d'un Trypanosoma et leptotrypano-
somes chez Drosophila confusa (Muscide). C. E. Soc. Biol. Paris,
70, 34-36.
CHAGAS, C.

1909. Ueber eine neue Trypanosomiasis des Menschen. Mem. Tnst. Osw.
Cruz, 1, 159-218, pis. 9-13, 10 figs, in text.

FANTHAM, H. B., and PORTER, A.

1915a. Further experimental researches on insect flagellates introduced into

vertebrates. Proc. Cambridge. Philos. Soc., 18, 137-148.
1915b. On the natural occurrence of herpetomonads (leptomonads) in mice.

Parasitology, 8, 128-132, 7 figs, in text.
1915c. Some experimental researches on induced herpetomoniasis in birds.

Ann. Trop. Med., 9, 543-558, pi. 39.
FRANQA, C.

1914. La flagellose des Euphorbes. Arch. f. Prot., 34, 108-132, pi. 5, 4

figs, in text.

HARTMANN, M.

1911. Die Konstitution der Protistenkernei Jena, Fischer, pp. 1-54, 13

figs, in text.
KOFOID, C. A.

1916. The biological and medical significance of the life-history of intes-
tinal flagellates. Proc. Sec. Pan-Amer. Sci. Cong., Washington,
1915-16, 10, 546-565.

KOFOID, C. A., and McCuLLOCH, I.

1916. On Trypanosoma triatomae, a new flagellate from a hemipteran bug

from the nests of the wood rat, Neotoma fuscipes. Univ. Calif.
Publ. Zool., 16, 113-126, pis. 14-15.

KOFOID, C. A., and SWEZY, O.

1915. Mitosis and multiple fission in trichomonad flagellates. Proc. Am.

Acad. Arts and Sci., 51, 287-379, pis. 1-8, 7 figs, in text.

MCCULLOCH, I.

1915. An outline of the morphology and life history of Crithidia leptocori-
dis sp. nov. Univ. Calif. Publ. Zool., 16, 1-22, pis. 1-4, 1 fig.
in text.

1917. Crithidia euryophthalmi, sp. nov., from the hemipteran bug, Euryo-

phthamus convivus, Stal. Ibid., 18, 75-88, 35 figs, in text.

MINCHIN, E. A.

1908. Investigations on the development of trypanosomes in tse-tse flies
and other Diptera. Quart. Jour. Micr. Sci., 52, 159-260, pis. 8-13-,
2 figg. in text.

1912. An introduction to the study of Protoza. London, Arnold, pp.

1-520, 194 figs, in text.

1919] McCulloch: Life Cycle of Crithidia and Trypanosoma 181

MINCHIN, E. A., and THOMSON, J. D.

1915. The rat-trypanosome, Trypanosoma lewisi, in its relation to the rat-

flea, Ceratophyllus fasciatus. Quart. Jour. Micr. Sci., 60, 463-692,
pis. 36-45, 24 figs, in text.

MOORE, J. E. S., and BREINL, A.

1907. Cytology of the trypanosomes, I. Ann. Trop. Med., 1, 441-480, pis.

38-42.

PATTON, W. S.

1908. Herpetomonas lygaei. Arch. f. Prot., 13, 1-18, pi. 1, 2 figs, in text.

1908. The life cycle of a species of Crithidia parasitic in the intestinal

tract of Gerris fossarum Fabr. Ibid., 12, 131-146, pi. 9.

1909. The life cycle of a species of Crithidia parasitic in the intestinal

tract of Tabanus hilarius and Tabanus spJ Ibid., 15, 333-362, pi.
30, 2 figs, in text.

PATTON, W. S., and CRAGG, F. W.

1913. A text book of medical entomology. London, Christian Literature

Society for India, pp. 1-768, pis. 1-89.
PORTER, A.

1909. The morphology and the life cycle of Crithidia gerridis as found in
the British water-bug, Gerris paludum. Parasitology, 2, 348-366,
pi. 4.

1909. The life cycle of Herpetomonas jaculum (Leger) parasitic in the

alimentary tract of Nepa cinera. Ibid., 2, 367-391, pi. 5, 1 fig.
in text.

1910. The structure and life history of Crithidia melophagia (Flu) an

endoparasite of the sheep-ked, Melopliagus ovinus. Quart. Jour.
Micr. Sci., 55, pis. 12-13, 15 figs, in text.

PROWAZEK, S.

1904. Die Entwicklung von Herpetomonas. Arb. kais. Gesundh., 20, 440-
452, 7 figs, in text.

S\VEZY, O.

1916. The kinetonucleus of flagellates and the binuclear theory of Hart-

mann. Univ. Calif. Publ. Zool., 16, 185-240, 58 figs, in text.

WENYON, C. M.

1913. Observations on Herpetomonas muscae domesticae and some allied
flagellates with special reference to the structure of their nuclei.
Arch. f. Prot., 31, 1-36, pis. 1-3, 6 figs, and 1 diagram in text. .

EXPLANATION OF PLATES

All figures were outlined with a camera lucida using a ^ objective on the
binocular microscope and a Watson no. 20 holoscopic eye-piece. The magnifica-
tion is in all cases approximately 3500. Unless otherwise stated all figures were
made from iron-haematoxylin preparations.

PLATE 2

Crithidia euryophthalmi from the ' ' crop ' ' of Euryophthalmus convivus.

Fig. 1. Small, oval infective spore which has been casually ingested with
food. This spore shows the structure common to these phases, thick periplast,
deeply staining nucleus in extreme posterior end, heavily stained parabasal
body. Faint nuclear rhizoplast.

Figs. 2-8. A series of developing crithidias showing successive stages of
development. Both anterior and posterior ends are becoming attenuate. The
nucleus changes from a solid mass of chromatin to a vesicular nucleus, chro-
matin-encrusted membrane and central karyosome. Flagellum grows forward
and carries anterior end of body along with it, which forms undulating membrane.

Figs. 9-10. Two mature flagellates illustrating extremes in length, width,
and shape, common to C. euryophthalmi. A whole series of intergrading forms
occur between these two extremes.

Figs. 11-23. Multiple fission; endogenous budding. (Fig. 11). Flagellate
with a nucleus, two endogenous buds posterior to nucleus. Chromatin of both
nucleus and buds massed on nuclear membrane. Blepharoplast and parabasal
body intact, no indications of division.

Fig. 12. A short flagellate; one endogenous bud; chromatin peripheral in
each nuclear structure.

Fig. 13. Flagellate with three clearly defined nuclear structures and the
beginning of a third bud from the most anterior, the nucleus proper. Chromatin
peripheral in each of these nuclear structures.

Fig. 14. Elongate flagellate nucleus; two endogenous buds anterior to the
nucleus; chromatin peripheral.

Figs. 15-17. Pear-shaped crithidias with endogenous buds. If nucleus be
present in each there are no differences in structure between nucleus and bud.
Buds in these flagellates are always sharply defined. Chromatin material on
membrane but most of it is in definite granules.

Fig. 18. Pear-shaped form with numerous endogenous buds but no definite
nucleus. First stages of degeneration present, parabasal body has disappeared
and only a fragment of discarded flagellum is near. Chromatin peripheral
but also in form of one granule.

Fig. 19. Elongate flagellate; two endogenous buds posterior to nucleus.
Nuclear rhizoplast still present. Chromatin distributed irregularly on the
nuclear membranes.

Fig. 20. Elongate flagellates with nucleus and two endogenous buds; chro-
matin massed in the anterior and posterior portion of each nuclear structure.
Parabasal body has taken no part in this multiple fission.

Fig. 21. Large endogenous flagellate with five buds, all anterior to nucleus
proper. Two of the buds are not destained sufficiently to see their structure.
The organelles other than nucleus have taken no part in the process of endo-
genous budding. Flagellum, blepharoplast, parabasal rhizoplast and parabasal
body are still intact and clearly shown.

Fig. 22. Late stage in endogenous budding. Buds have formed zooids,
each of which has a nucleus and a second, deeply-staining structure anterior
to nucleus. Flagellum, parabasal body, parabasal rhizoplast, and the blepharo-
plast are still intact. No signs of degeneration are to be observed in this
flagellate.

Fig. 23. Drawing made from a field literally covered by discarded flagella
and small zooids. The blepharoplast and parabasal body are still attached to
8ome of the flagella, Zooids show various stages of nuclear structure. Some
have a single mass of chromatin, others two granules within a chromatin-en-
crusted membrane. Some few of the zooids contain a nucleus, nuclear rhizo-
plast, and a second mass of chromatin anterior to these.

[182]

UNIV. CALIF, PUBL, ZOOL. VOL, 19

[McCULLOCH] PLATE 2

PLATE 3

Figs. 24-32. Multiple fission; somatellas. (Fig. 24). An early stage in the
formation of a somatella. The flagellate is rounding up, and the flagellum is
entirely intracellular. No indications of division of the nucleus, parabasal
body, or the blepharoplast are present.

Fig. 25. A somewhat different type of rounding up. The attenuate anterior
and posterior ends are being wrapped about the body. The nucleus has begun
to constrict or to divide by a process of primitive promitosis. The blepharo-
plast and parabasal have not yet begun to divide.

Fig. 26. A more advanced stage in the formation of the somatella. The
flagellum of the rounded up flagellate is protruding and both the nucleus and
blepharoplast, together with the parabasal body, have divided. The new
daughter-flagellum is not yet visible.

Fig. 27. A sphere, or somatella, still more advanced in its development.
Not only have the blepharoplast, parabasal body, and the nucleus divided but
the new outgrowth of the second flagellum from the daughter-blepharoplast is
clearly visible.

Fig. 28. A spherical crithidia showing a repeated process of division on
the part of the nuclei and parabasal bodies. Three parabasal bodies and two
nuclei are present. There are possibly indications in one of the nuclei wherein
the chromatin has been divided that another division was about to occur.

Fig. 29. A sphere, or somatella, without protruding flagella, containing at
least four definitely outlined merozoites. The nuclei, parabasal bodies, nuclear
rhizoplasts, and the outgrowths of the flagella are clearly visible.

Fig. 30. A densely stained, small somatella containing four relatively large
merozoites which are beginning to elongate.

Fig. 31. A sphere, or somatella, breaking up and the merozoites about to
escape. The destruction of the sphere has occurred later than usual and the
merozoites have become almost mature flagellates. All nuclear structures are
deeply stained, owing to the thickness of the sphere.

Fig. 32. An exceedingly large sphere, comparatively, showing many pro-
truding flagella. Here again the nuclear structures are deeply stained because
of the thickness of the sphere. The exact number of merozoites cannot be
determined, but approximately twenty-four nuclei and parabasal bodies can
be counted.

Figs. 33-38. Binary fission. (Fig. 33.) A small spherical crithidia under-
going binary fission. The nucleus has divided but the blepharoplast and para-
basal body show no indications of fission.

Fig. 34. Binary fission, in which the blepharoplast and parabasal body have
divided but the nucleus has not yet divided. A flagellum from the daughter-
blepharoplast has already grown forward.

Fig. 35. Binary fission taking place in a developing crithidia. Both the
nucleus and parabasal body have divided, and a new flagellum can be observed
growing from the daughter-blepharoplast.

Fig. 36. Simple binary fission; blepharoplast, parabasal body, and nucleus
have divided. The chromatin in the nuclei is peripheral, about the membrane.

Figs. 37, 38. A more advanced stage of binary fission, showing in addition
to the division of the blepharoplasts, parabasal bodies, and nuclei a cleavage
in the cytoplasm to form two crithidias in each case.

Fig. 39. Intracellular multiple fission: one of the many infected cells
from the "crop" of Euryophthalmus convivus. This cell is in a degenerating
condition. The nucleus stains a blue-gray color in iron-haemotoxylin. There
are at least three and possibly five intracellular infections by C. euryophthalmi
in this cell. There are approximately seventy parasites within this cell: (a)
A group of parasites of two sizes, small oval forms, non-flagellated and elon-
gated Crithidia. Nuclei of all crithidias are diffuse, possibly due to thickness
of smear. Nucleus and parabasal body readily observed but the other organ-
elles are not always clear, (b) Another group evidently the result of a process
of intracellular multiple fission, (c) Similar to b. Circular cavity about these
non-flagellated crithidias may indicate the outline of a former somatella
wherein plasmotomy has occurred early, (d) Elongate merozoites probably
resulting from another intracellular somatella. Plasmotomy has occurred and
the merozoites are about to make their way out of the host cell, (e) A scattered
group of oval merozoites. Considerable variation in size is noted. Parabasal
body, nucleus, and intracellular portions of flagellum clearly shown. (/) A
mature merozoite making it way out of the host cell. The non-flagellated, or
posterior, end directed first.

[184]

UNIV, CALIF, PUBL. ZOOL VOL. 19

^3^

V

[McCULLOCH] PLATE 3

I
(?

26

25

33

29

35

34

36

li

38

'

32

''i<!ov Is

- *

.V <•-•:'.. .V

*- *

*•

^ '
— *

39

PLATE 4
Crithidia euryophthalmi from mid-stomach of Euryophthalmus convivus.

Figs. 40-92. Nectomonads (Figs. 40, 41). Small oval forms at the begin-
ning of the developmental series of merozoites or zooids in the early rectal
phase, showing nucleus, parabasal body, nuclear rhizoplast, and light area around
the parabasal body. Nucleus at posterior end of the body. Anterior end
pointed or blunt.

Figs. 42-47. More advanced stages in the developmental series; width of
body and distance between nucleus and parabasal are increasing.

Figs. 48-54. Size of the oval forms increasing; nucleus still diffuse and
located at extreme posterior end of body. Nuclear rhizoplast present, but no
indication of the flagellum except in figure 54.

Figs. 55-58. Developing forms. Anterior end elongating and extending
out along the flagellum. Nucleus still diffuse. In figure 58 the posterior end
of the body is slightly elongated.

Fig. 59. A group in upper part of mid-stomach. Note vesicular type of
nucleus, with the centriole-like karyosome. Both ends of the body are more
or less pointed. The position of the parabasal body near the nucleus is unusual.

Fig. 60-62. Both anterior and posterior ends are elongating. Vesicular
nu'cleus, nuclear rhizoplast, parabasal body, parabasal rhizoplast, and flagellum
very distinct.

Fig. 63. Posterior end blunt; vesicular type of nucleus. Parabasal body
near the nucleus. Anterior end of the body well developed; a slight undulating
membrane present.

Fig. 64. Form a little more developed. Posterior end of the body is
blunt or round, and nucleus is diffuse.

Fig. 65. Almost mature flagellate; nucleus diffuse; posterior end elongated
and quite broad.

Fig. 66. Free form, showing vesicular nucleus and a distinct undulating
membrane.

Fig. 67. Nectomonad developing into elongate flagellate; nucleus diffuse.

Figs. 68-72. Nectomonads of mid-stomach, long, slender, flagellates show-
ing the common variations in the nuclear structures. The diffuse type in
figure 68; vesicular in figures 69 and 71; chromatin-encrusted nuclear membrane
in figures 70 and 72.

[186]

UNIV. CALIF. PUBL. ZOOL. VOL. 19

[McCULLOCH] PLATE 4

»

40

* .

43 44 45 46

47

48

51

52

53

54

55

56

58

59

63

61

62

66

64

\

65

69

70

PLATE 5

Figs. 73-92. Nectomonads from pyloric expansion (Figs. 73-80). A series
of elongate, slender flagellates from crithidial infection of the pyloric expan-
sion. Vesicular types of nuclear structure in figures 73, 75, 80. Chromatin
broken up into granules in figures 74, 76, and 79. Chromatin of nucleus on
the membrane in figures 77, 78.

Figs. 81-86. Another group of nectomonads from pyloric expansion, illus-
trating the changes which take place in shape of body, length decreases, width
increases. These are shorter, broad types of crithidias, and probably come
from the longer, more slender forms. Nuclear structure usually shows the
central karyosome. Figure 82 shows the chromatin broken up, a condition which
may indicate an early stage of degeneration of the nectomonads.

Figs. 87-90. Transition flagellates from nectomonads to haptomonads. By
a process of binary fission these transition forms become reduced in size. Cer-
tain nectomonads become attached to wall of the pyloric expansion or they
form haptomonads. Others degenerate before reaching this stage.

Figs. 91-92. Free flagellates. Their size is doubtless due to a process of'
binary fission and they resemble the attached haptomonads of the pyloric
expansion. They are either nectomonads which are about to become hapto-
monads or vice versa. If the latter, the haptomonads upon becoming free again
usually degenerate at once.

[188]

UNIV. CALIF. PUBL. ZOOL. VOL. 19

[McCULLOCHj PLATE 5

PLATE 6

Figs. 93-96. Haptomonads from mid-stomach. Haptomonads of this type
may be found attached to wall of the ' ' crop, ' ' posterior part of mid-stomach
or anterior part of pyloric expansion. Small, slender flagellates usually with
a vesicular type of nucleus are shown in figures 93-95. Figure 94. Nuclear
membrane distorted, karyosome excentric. Figure 96 may show degeneration
since there is no nuclear membrane and the chromatin is breaking up.

Figs. 97-124. Haptomonads from pyloric expansion (Figs. 97-106). Transi-
tion haptomonads lining mid-portion of the pyloric expansion. Bodies becom-
ing pear-shaped; flagella largely absorbed and intracellular throughout length.
Nucleus usually vesicular. Binary fission is common among these forms. Their
size is reduced in this way.

Figs. 107-124. Small haptomonads, found in the extreme posterior portions
of the pyloric expansion. They form a dense compact layer of parasites at-
tached to epithelial lining. Keduction in size shown, beginning with figure 107.
Degeneration is shown in the nuclear structure, as in figure 114, and in the
cytoplasm of figures 117, 118. Figures 118-124 are all haptomonads which show
advanced stages of degeneration; the cytoplasm is vacuolate and the nuclear
structure is no longer normal. Most of these have become detached from the
wall of the pyloric expansion.

Figs. 125-131. A series of final spore forms from rectum, showing thick
periplast, deeply stained cytoplasm, and two chromatin bodies. Nuclear rhizo-
plast not visible in these deeply stained spores.

[190]

UNIV, CALIF, PUBL. ZOOL. VOL. 19

[McCULLOCH] PLATE 6

93

100

94

101

107

108

110 115

IJ7

118 i\9

120

122

123 124

125 126

•

127

128

129 130 131

UNIVERSITY OF CALIFORNIA PUBLICATIONS— (Continued)

8. Osteological Relationships of Three Species of Beavers, by F. Harvey

Holden. Pp. 75-114, platea 6-12, 18 text figures. March, 1917 ... .40

9. Notes on the Systematic Status of the Toads and Frogs of California, by

Charles Lewis Camp. Pp. 115-125, 8 text figures. February, 1917 .10

10. A Distributional List of the Amphibians and Reptiles of California, by

Joseph Grinnell and Charles Lewis Camp. Pp. 127-208, 14 figures in text.
July, 1917 _ .85

11. A Study of the Races of the White-Fronted Goose (Anser albifrons) Occur-

ring in California, by H. S. Swarth and Harold C. Bryant. Pp. 209-222,

2 figures in text, plate 13. October, 1917 ._„ .15

12. A Synopsis of the Bats of California, by Hilda Wood Grinnell. Pp. 223-404,

plates 14-24, 24 text figures. January 31, 1918 _ 2.00

15. The Pacific Coast Jays of the Genus Aphelocoma, by H. S. Swarth. Pp.

405-422, 1 figure in text. February 23, 1918 ^. .20

14. Six New Mammals from the Mohave Desert and Inyo Regions of California,
by Joseph Grinnell. Pp. 423-430.

16. Notes on Some Bats from Alaska and British Columbia, by Hilda Wood

Grinnell. Pp. 431-433.

Nos. 14 and 15 in one cover. April, 1918 .15

16. Revision of the Rodent Genus Aplodontia, by Walter P. Taylor. Pp. 435-

504, plates 25-29, 16 text figures. May, 1918 75

17. The Subspecies of the Mountain Chickadee, by Joseph Grinnell. Pp. 605-

515, 3 text figures. May, 1918 15

18. Excavations of Burrows of the Rodent Aplodontia, with Observations on

the Habits of the Animal, by Charles Lewis Camp. Pp. 517-536, 6 figures

in text. June, 1918 .20

Index, pp. 537-545.

VoL 18. 1. Mitosis in Giardia microti, by William C. Boeck. Pp. 1-26, plate 1. Octo-
ber 1917 .35

***'*> •* •*'•* * .......-.«——.—•-...••-.•.*.»••.

2. An Unusual Extension of the Distribution of the Shipworm in San Fran-
cisco Bay, California, by Albert L. Barrows. Pp. 27-43. December, 1917. .20

8. Description of Some New Species of Polynoidae from the Coast of Cali-
fornia, by Christine Essenberg. Pp. 45-60, plates 23. October, 1917 — .20

4. New Species of AmpMnomidae from the Pacific Coast, by Christine Essen-

berg. Pp. 61-74, plates 4-5. October, 1917 — 15

5. Crithidia euryopMhalmi, sp. nov., from the Hemipteran Bug, Euryophthalmus

convivus Stal, by Irene McCulloch. Pp. 75-88, 35 text figures. Decem-
ber, 1917 .15

6. On the Orientation of Erythropsis, by Charles Atwood Kofoid and Olive

Swezy. Pp. 89-102, 12 figures in text. December, 1917 15

7. The Transmission of Nervous Impulses in Relation to Locomotion in the

Earthworm, by John F. Bovard. Pp. 103-134, 14 figures in text. January,
1918 „ „ .35

8. The Function of the Giant Fibers in Earthworms, by John F. Bovard. Pp.

135-144, 1 figure in text. January, 1918 - ._ — .10

9. A Rapid Method for the Detection of Protozoan Cysts in Mammalian

Faeces, by William C. Boeck. Pp. 145-149. December, 1917 ~ .05

10. The Musculature of Heptanchus maculatus, by Pirie Davidson... Pp. 151-170,

12 figures in text. March, 1918 _ — 25

11. The Factors Controlling the Distribution of the Polynoidae of the Pacific

Coast of North America, by Christine Essenberg. Pp. 171-238, plates 6-8,

2 figures in text. March, 1918-. ~ — .75

12. Differentials in Behavior of the Two Generations of Salpa dvmocratica

Relative to the Temperature of the Sea, by Ellis L. MichaeL Pp. 239-298,
plates 9-11, 1 figure in text. March, 1918 —

13. A Quantitative Analysis of the Molluscan Fauna of San Francisco Bay, by

E. L. Packard. Pp. 299-336, plates 12-13, 6 figs, in text. April, 1918 — .40

14. The Neuromotor Apparatus of Euplotes patella, by Harry B. Yocom. Pp.

337-396, plates 14-16. September, 1918 _ .70

15. The Significance of Skeletal Variations in the Genus Peridinium, by A. L.

Barrows. Pp. 397-478, plates 17-20, 19 figures in text. June, 1918 90

UNIVERSITY OF CALIFORNIA PUBLICATIONS— (Continued)

16. The Subclavian Vein and its Relations in Elasmobranch Fishes, by J.

Frank DanieL Pp. 479-484, 2 figures in text. August, 1918 .10

17. The Cercaria of the Japanese Blood Fluke, Schistosoma japonicum Kat-

surada, by William W. Cort. Pp. 485-507, 3 figures in text.

18. Notes on the Eggs and Miracidia of the Human Schistosomes, by William

W. Cort. Pp. 509-519, 7 figures in text.

Nos. 17 and 18 In one cover. January, 1919 _ 35

Index in preparation.

Vol.19. 1. Reaction of Various Plankton Animals with Reference to their Diurnal

Migrations, by Calvin O. Esterly. Pp. 1-83. April, 1919 85

2. The Pteropod Desmopterus pacificus (sp. nov.), by Christine Essenberg.

Pp. 85-88, 2 figures in text. May, 1919 05

3. Studies on Giardia microti, by William 0. Boeck. Pp. 85-136, plate 1, 19

figures in text 60

4. A Comparison of the Life Cycle of Crithidia With that of Trypanosoma in

the Invertebrate Host, by Irene McCulloch. Pp. 135-190, plates 2-6, 3
figures in text. October, 1919 60

5. A Muscid Larva of the San Francisco Bay Region which Sucks the Blood

of Nestling Birds, by O. E. Plath. Pp. 191-200. February, 1919 .10

6. Binary Fission in Collodictyon triciUatum Carter, by Robert Clinton Rhodes.

Pp. 201-274, plates 7-14, 4 figures in text (In press)

7. The Excretory System of a Stylet Cercaria, by William W. Cort. Pp. 275-

281, 1 figure in text. August, 1919 10

Vol.20. 1. Studies on the Parasites of the Termites I. On Streblomastix strix, a
Polymastigote Flagellate with a Linear Plasmodial Phase, by Charles
Atwood Kofoid and Olive Swezy. Pp. 1-20, plates 1-2, 1 figure in
text. July, 1919 25

2. Studies on the Parasites of the Termites II. On Trichomitus termitidis,

a Polymastigote Flagellate with a Highly Developed Neuromotor System,
by Charles Atwood Kofoid and Olive Swezy. Pp. 21-40, plates 3-4, 2
figures in text. July, 1919 - .25

3. Studies on the Parasites of the Termites III. On Trichonymplia campanula

Sp. Nov., by Charles Atwood Kofoid and Olive Swezy. Pp. 41-98, plates
5-12, 4 figures in text. July, 1919 :. 75

4. Studies on the Parasites of the Termites IV. On Leidyopsis sphaerica

Gen. Nov., Sp. Nov., by Charles Atwood Kofoid and Olive Swezy. Pp.
99-116, plates 13-14, 1 figure in text. July, 1919.._ „ .25

Vol. 21. 1. A Revision of the Microtus calif ornicus Group of Meadow Mice, by Rem-
ington Kellogg. Pp. 1-42, 1 figure in text. December, 1918 50

2. Five New Five-toed Kangaroo Rats from California, by Joseph Grinnell.

Pp. 43-47. March, 1919 05

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