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University of California Publications in

ZOOLOGY

‘Ansonlan
Oris ing
oe

VOLUME XX
1919-1923

WITH FORTY-TWO PLATES

EDITORS
CHARLES ATWOOD KOFOID
SAMUEL J. HOLMES
JOSEPH GRINNELL

UNIVERSITY OF CALIFORNIA PRESS
BERKELEY, CALIFORNIA

bo

abe

13.

14.

CONTENTS

. Studies on the Parasites of the Termites I. On Streblomastix striz,

a Polymastigote Flagellate with a Linear Plasmodial Phase, by
Charles Atwood Kofoid and Olive Swezy; with plates 1-2; 1
TAYE QING) THA, WER aon creecece spec Bae eee econ eee LE Eo ESE ROS SE SCE SacR Eco CESS

. Studies on the Parasites of the Termites Il. On Trichomitus ter-

mitidis, a Polymastigote Flagellate with a Highly Developed
Neuromotor System, by Charles Atwood Kofoid and Olive
Swezy; with plates 3-4; 2 figures in text —..-..------------------c-s-eeeeo-

. Studies on the Parasites of the Termites III. On Trichonympha

campanula sp. noy., by Charles Atwood Kofoid and Olive Swezy;
Vuln Hedespiayst GUI Ck save ehe. TON SD eee ee eee ree ee

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

sphaerica Gen. Nov., sp. nov., by Charles Atwood Kofoid and
Olive Swezy; with plates 13-14; 1 figure in text —............-............

. On the Morphology and Mitosis of Chilomastix mesnili (Wenyon).

A Common Flagellate of the Human Intestine, by Charles A.
Kofoid and Olive Swezy; with plates 15-17; 2 figures in text...

. A Critical Review of the Nomenclature of Human Intestinal Flagel-

lates, Cercomonas, Chilomastix, Trichomonas, Tetratrichomonas,
and Giardia, by Charles Atwood Kofoid; 9 figures in text -.........

. On the Free, Encysted, and Budding Stages of Cowncilmania lafleuri,

a Parasitic Amoeba of the Human Intestine, by Charles Atwood
Kofoid and Olive Swezy; with plates 18-22; 3 figures in text -.....

. Mitosis and Fission in the Active and Eneysted Phases of Giardia

enterica (Grassi) of Man, with a Discussion of the Method of
Origin of Bilateral Symmetry in the Polymastigote Flagellates,
by Charles Atwood Kofoid and Olive Swezy; with plates 23-26;
TTL, sae WET SUN SS ree ee meer eee SS See ee ocr

. The Micro-Injection of Paramaecium, by Chas. Wm. Rees ..................
10.

On Balantidium coli (Malmsten) and Balantidium suis (sp. nov.),
with an Account of their Neuromotor Apparatus, by J. Daley
McDonald; with plates 27-28; 15 figures in text -.......-....-.--.2.-.-----

Mitosis in Endamoeba dysenteriae in the Bone Marrow in Arth-
ritis Deformans, by Charles Atwood Kofoid and Olive Swezy;
2f SAAS UMASS. EAN NEB, eee ee eer een eee

. Endamoeba dysenteriae in the Lymph Glands of Man in Hodgkin’s

Disease, by Charles A. Kofoid, Luther M. Boyers, M.D., and
Olives Swiezyes 4 digumes! tm bextra c cece wc nee acre ce nna cee ener erereae=

Mitosis in the Eneysted Stages of Endamoeba coli (Loesch), by
Olives Swiezysewatheplates| 293i oo eereeescrnnrereeecnen es care eeny nee tneenneceeness

The Neuromotor Apparatus of Paramaecium, by Charles William
Rees; with plates 32-36; 5 figures im text -.....---...-22.sc--sesseeeceeeee

PAGES

21-40

41-98

99-116

117-144

145-168

199-234

235-242

301-3807

309-312

313-332

333-364

15.

16.

i

A Comparison of the Cysts of Endamoeba coli and Councilmania
lafleuri in Congo Red, by Nathaniel Bercovitz, M.D. .......-.-........-..-
On the Morphology and Behavior of Pentatrichomonas ardin deltetli
(Derrieu and Raynaud), by Charles Atwood Kofoid and Olive
Swezy,; wath plate 37; 1 figure am) text 2 oe ca eeceseereee eee
The Pseudopodial Method of Feeding by Trichonymphid Flagellates
Parasitic in Wood-eating Termites, by Olive Swezy; 14 figures
FLOSS: Urge eee se eS

. Methods of Obtaining Amoeba-free Rats for Experimental Infee-

tion with Intestinal Amoeba, by John F. Kessel -_._..--...----.---..

. Experimental Infection of Rats and Mice with the Common Intestinal

Amoeba of Man, by John F. Kessel; with plates 38-39 ...........

20. On the Genus Councilmania, Budding Intestinal Amoebae Parasitic

in Man and Rodents, by Charles A. Kofoid, Olive Swezy and
ohm) We Wessels 99 oii prae s tr e Xstiee ees eme ree cea

. Morphology and Binary Fission of Menoidium inecurvum (Fres.)

Klebs, by Richard P. Hall; with plates 40-41; 2 figures in text...

2. A Skin Reaction to Extracts of Leishmania tropica and Leishmania

infantum, by Edna Hannibal Wagener; with plate 42 -.........

23. The Distinguishing Characteristics of the Parasitic Amoebae of

Culture Rats and Mice, by John F. Kessel; with plates 43-48; 3
AUG UO SAM) OK) oe soe nas coeneeee mare nc rene men amet eer Seen ee

BO ge Re Ne PRR a PE a rs tare

365-871

373-390

391-400

401-408

409-430

431-445

447-476

477-488

489-544
545

UNIVERSITY OF CALIFORNIA PUBLICATIONS
IN 4

ZOOLOGY
Vol. 20, No. 1, pp. 1-20, plates 1-2, 1 figure in text July 14, 1919

STUDIES ON THE PARASITES OF THE TER-
MITES I. ON STREBLOMASTIX STRIX, A
POLYMASTIGOTE FLAGELLATE WITH A
LINEAR PLASMODIAL PHASE.

BY

~ CHARLES ATWOOD KOFOID anbD OLIVE SWEZY

ES Saget
Ces FOr

-BUG1 31915

UNIVERSITY OF CALIFORNIA PRESS
BERKELEY

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UNIVERSITY OF CALIFORNIA PUBLICATIONS
IN

ZOOLOGY
Vol. 20, No. 1, pp. 1-20, plates 1-2, 1 figure intext . July 14, 1919

STUDIES ON THE PARASITES OF THE TER-
MITES I. ON STREBLOMASTIX STRIX, A

POLYMASTIGOTE FLAGELLATE WITH ~\

A LINEAR PLASMODIAL PHASE / :

BN

CHARLES ATWOOD KOFOID anp OLIVE SWEZY

CONTENTS
PAGE
LUE PEEYS IOC NGA | se 2a se es eer eR ee Ce Sere eA eo Ree 1
Material _.. 2
CLE OL GAG ah ee Pee er eee 3
(OKANO. rac eet P nh Seal eA Mae Buh ae ea ai oA We AE rel sip en pe Pe 4

VOTH OO ayy seers eee eect
Size and shape of body
Gy foplasmay sesso
Neuromotor apparatus
Direction of torsion ....
DSC S11 en Se
Binary fission
Multiple fission ..
PANG ADEE ONSYaeesssreree nee A US Mies seen yo arene eh ey
VRST EERE IORAYSI SW OfSIe ocean pe ee eT eee
Summary
TUSSI TERS 0 CCUG Ye ene ee a Oe

INTRODUCTION

One of the most curious and unique faunal associations to be found
among the parasitic Protozoa is the group parasitic or commensal
in the intestinal tract of the social termites. These parasites are
remarkable not only for the vast numbers that may be found within
a single host, but also for the degree of development and specialization
which distinguishes many of the species. This is especially true of the

2 University of California Publications in Zoology [ Vo. 20

forms belonging to the family Trichonymphidae, such as Trichonympha
Leidy, which are among the most highly specialized members of the
Protozoa.

Along with these more complex forms are others which, while
simpler in structure, yet show certain peculiar morphological char-
acteristics that distinguish them as a group apart from other intes-
tinal flagellates. Among these we find Pyrsonympha vertens and
Dinenympha gracilis deseribed by Leidy in 1881.

Later investigators have added both to the number of genera and
of species of these peculiar flagellates.

MATERIAL

The material for these studies was obtained from one species of
termite which is abundant on the University campus at Berkeley.
This is Termopsis angusticollis Walker and was identified for us by
Dr. Nathan Banks of the Museum of Comparative Zoology at Cam-
bridge, Mass. Most of this material was obtained from the decayed
trunk of an oak tree in Strawberry Cafion. Many of the same species
were obtained during the swarming season from the piles on Meiggs
Wharf, San Franciseo, by Dr. A. D. Drew of the Public Health
Service.

These termites are large and show an infection of about one hun-
dred per cent, soldiers, workers and males of the colony being infected
alike. The amount of infection in a single individual is relatively
enormous. The abdomen is large and nearly filled by the greatly
swollen intestine. This distension is caused by the vast numbers of
parasitic and commensal protozoans which fill the lumen of the intes-
tine. When this is opened a thick milky fluid exudes. Under the
lens this is found to be composed of great quantities of these small
forms, thickly massed together, along with fragments of wood upon
which the host, as well as some of its commensals, feeds.

In Termopsis angusticollis four different species of large protozoans
are invariably present, sometimes about equal in number or with one
predominating over the others. In addition to these there are usually
present minute forms of two, sometimes three different species of
flagellates, the whole forming a complex of organisms wonderful both
for variety and amount. Of these forms the two largest species
belonging to the family Trichonymphidae-and a third species belong-
ing to the Polymastigidae, will be reserved for discussion in later

1919] Kofoid-Swezy: Streblomastix strix 3
papers. In the present paper the fourth member of this group, also
a polymastigote flagellate, will be considered, with a discussion of its
morphology, relationships and life history in so far as they have been
determined. 2

TECHNIQUE

The flagellates found in the termites are exceedingly delicate.
Great difficulty has been experienced in keeping them alive for con-
tinuous observation under the microscope for any length of time.
The use of distilled or tap water resulted in the complete dissolution
of the larger flagellates in a few minutes. The smaller ones would
survive a somewhat longer period. Various other eulture media were
tried, such as Ringers’ solution, normal salt solution, malted milk and
the white of egg. Of these the last one was the most successful, a few
flagellates surviving in the culture at the end of twenty-four hours.
These cultures were made with a hanging drop or with a greater
amount of fluid in a deep eulture slide.

Intra vitam stains, such as neutral red, Janus green and new
methylene blue G G, were used. Of these neutral red gave the best
results.

The methods of fixing and staining which have been found the most
satisfactory were those outlined by us in previous work on parasitic
flagellates (Kofoid and Swezy, 1915), that is, a modified Heidenhain’s
iron haematoxylin following fixation in hot Schaudinn’s fluid. Other
stains as well as various fixing agents were tried, both with smear
preparations and with sectioned material. In the latter cases two
methods were followed. In the first the entire abdomen of the termite
was used, fixed in Schaudinn’s or strong Flemming’s solution. In the
second the intestine was teased out in a drop of normal salt solution
and then placed in the fixing fluid. These sections were stained with
haematoxylin or with a modified Mallory’s connective tissue stain
(Yocom, 1918).

Considerable difficulty has been experienced in making good prepa-
rations of this material by the ordinary smear methods. The execeed-
ing delieaey of the various flagellates results in distortions of the body
and breaking down of its cytoplasmic organization. This is usually
confined to the posterior end, leaving the anterior end, nucleus and
motor organelles intact. This difficulty was partly overcome by using
albumen fixative on the cover slip and diluting the contents of the

4 University of California Publications in Zoology — (Vou. 20

intestine with a small drop of normal salt solution before making the
smear. The material thus treated may be spread with less mechanical
injury and the albumen prevents the great loss of organisms that would
otherwise occur when it is placed in the fixing fluid. The addition of
albumen, however, necessitates quick work in making the prepara-
tions, to prevent the death of the organisms through its action rather
than that of the fixing fluids.

OCCURRENCE

These flagellates are more restricted in their occurrence in the
intestine of the host than are the other forms which are present with
it. They are seldom found far away from the mucus of the epithe-
jium, usually attaching themselves to it (pl. 1, fig. 9) by means of
the holdfast-like anterior end of the body. They may be seen com-
pletely filling the folds of the wall of the intestine with the posterior
portion projecting into the lumen of the canal.

Near the anterior part of the posterior region of the intestine,
immediately behind the origin of the malpighian tubules, a slight
enlargement of the intestine may be noted, with one side marked by
two lines of constriction passing backward for a short distance. This
forms a rounded chamber marked off from the main portion of the
eanal. In eross-seetions this may be found eompletely filled with
Streblomastixz, a dense coating of the flagellates attached to the wall
and others filling the remainder of the cavity. Plate 2, figure 8 shows
a small portion of the wall in this region with a few only of the at-
tached flagellates.

These flagellates occur much less frequently in other parts of the
posterior and mid-regions of the intestine, but when present are always
restricted to the peripheral zone with the larger flagellates oeeupying
the remainder of the lumen. They have been found in nearly seventy
per cent of the hosts examined.

MORPHOLOGY

Streblomastix is profoundly a linear organism. Elongation dom-
inates all of its organelles in adaptation to its crowded grouping in
its parasitic habitat. This elongation affects not only the body as a
whole but also the nucleus, rhizoplast, and flagella, and pervades not
only the normal vegetative trophozoite, but also the gigantic over-

1919] Kofoid-Swezy: Streblomastixz strix 5

grown and possibly abnormal phases oceasionally found. The linear
form of body and also of nucleus continues not only during the
trophozoite phase but likewise, in so far as we have seen the stages,
during both binary and multiple fission. During multiple fission
itself the organism becomes a greatly elongated thread with its nuclei
stretched lengthwise as a constricting thread in the axis of the body.
All trace of rounding up or sphericity seems thus to have been
banished utterly from both body and nucleus at all stages of its life
eyele.

S1zzE AND SHAPE OF Bopy

The body is ordinarily elongate fusiform, tapering subequally at
the two ends. Either or both ends (pl. 1, figs. 1, 7) may be some-
what blunt but the usual form of the anterior end is a slender cone
while the posterior one may have a trifle more convexity. Its length
is generally from twelve to sixteen times its greatest diameter which
is found near the middle of the body. The shorter individuals
(pl. 1, fig. 5) may be only six times the diameter. These are evidently
recent schizonts. On the other hand, giant individuals, which are
possibly approaching multiple fission (pl. 2, fig. 13), may be thirty

‘

times their diameter in length, and the ‘‘plasmodial’’ stage of mul-
tiple fission (pl. 2, fig. 14) attains a length as much as seventy times
its own diameter. Measurements of two hundred individuals gave
a frequency curve with a marked left-hand skew with the mode at 40p
and the extreme range in length of from 20 to 530u. One-half of
the individuals were included between 20 and 80u. The longest indi-
viduals ineluded those in which multiple fission was in progress and
it is probable that the others were approaching that phase.

The contour of the body is not a smooth line, for the surface is
traversed by spiral ridges with furrows between, giving it the form,
except for its taper, of the shaft of a Norman Romanesque column.
These ridges are four in number, broadly convex and equidistant and
they wind about the body from the anterior end posteriorly from the
left over to the right. It is thus like a left-hand screw if the anterior
end is regarded as the tip. The steepness of the spiral varies with
the length of the organism, its contraction, and the phase of the life
eycle. In late stages of binary (pl. 2, fig. 17) and multiple fission
(pl. 2, fig. 14) much of the torsion is relaxed. In stages which may
be prior to binary fission (pl. 1, fig. 7; pl. 2, fig. 10) these may be

6 University of California Publications in Zoology — [Vou. 20

three to five turns, and a giant individual, presumably preceding
multiple fission (pl. 2, fig. 13), has eight turns. Normal vegetative
individuals (pl. 1, figs. 1, 6, 8) have one to two turns only.

The grooves between the ridges mark the location of eetoplasmie
lines of deeply stainmg material, possibly myonemes, or extensions

Fig. A. Semidiagrammatic figure of Streblomastia striz gen. nov., sp. Nov.
Black granules in cytoplasm show particles stained intra vitam with neutral red.

Abbreviations: bl., blepharoplast; cent., centrosome; /fl., flagella; my., myo-
nemes; ”., nucleus; rh., rhizoplast; st. gr., stained granules. 2000.

of the neuromotor system. They are feebly stained, if at all, in
binary and multiple fission (pl. 2, figs. 14, 17, 19) and are sometimes
marked by accumulations of minute granules staining black with
haematoxylin. They terminate anteriorly at or near the blepharoplast
and fade out posteriorly.

|

1919] Kofoid-—Swezy: Streblomastia strix

CYTOPLASM

The cell contents are undifferentiated. No separation of ectoplasm,
pellicle, and endoplasm is visible. There is no eytostome and no food
particles or food vacuoles have been detected. There is no centractile
vacuole visible. The only differentiated structures normally visible
in the organism are the nucleus and the neuromotor apparatus.
However, on treatment with neutral red (fig. A) certain granules
beeame stained in the axial eytoplasm in the posterior two-thirds of
the body, indicating an endoplasmic territory within which diges-
tion of the absorbed food was in progress.

Neuromotor APPARATUS

This neuromotor apparatus consists of a centrosome, rhizoplast,
blepharoplast, myonemes, and six flagella. The centrosome (fig. A,
cent.; pl. 1, fig. 9; pl. 2, fig. 10) is found at the anterior tip of the
elongated nucleus. It appears to be a spherical granule in or on the
nuclear membrane. It is temporary or evanescent and does not appear
to play any visible’ part in either binary or multiple fission. It is
quite possible that the granule thus interpreted is a mere temporary
accumulation of chromatoidal substance on the rhizoplast without
morphological meaning. Its position with reference to nucleus, rhizo-
plast and blepharoplast is similar to that of the centrosomes in Giardia
(Kofoid and Christiansen, 1915, and Boeck, 1917) hence the designa-
tion suggested. It may be that the extrusion of the rhizoplast serves
to bring the structure into view from a more intimate union with
the nuclear membrane.

The rhizoplast (fig. A, rh.) is a slender, deeply staining thread
running anteriorly from the centrosome or the anterior tip of the
nucleus to the blepharoplast. Its length ordinarily is about equal
to the greatest diameter of the body and the line of demareation
between the attenuate end of the nucleus and this thread is often
not readily determined. There is a probability that this is more or
less contractile, as is seen on a comparison of our figures. In one
instance (pl. 1, fig. 7) this strueture appears to be foreshortened
and thickened, by contraction, onto the anterior end of the nucleus.

One remarkable feature of this structure is its capacity of being
extended beyond the anterior end of the body as a long spike bear-
ing the blepharoplast and its attached flagella at its tip. A econ-

8 University of California Publications in Zoology — [Vou. 20

siderable increase in length to nearly three or even six times the
normal seems possible (pl. 1, fig. 3). No evidence of an extension
of the protoplasmic pellicle to form a sheath for this remarkable
organ has been found. It may also be so foreshortened that the
blepharoplast and centrosome are brought into close juxtaposition
(joll, al, ines, 2c

In view of the fact that the individuals lie closely packed against
the digestive epithelium with the blepharoplast thrust against the
epithelial cell of the host, it appears that this protrusible organ serves
in some way as a part of a somewhat adjustable holdfast. We have
no evidence that it can be or is ever thrust into the body of the cell,
though such a mode of attachment seems possible.

The blepharoplast (fig. A, bl.)is a sphere at the anterior end of
the rhizoplast about 0.5 in diameter. The six flagella spring directly
from it. It lies normally in the extreme anterior end of the body and
is carried out with the extruded rhizoplast. The possibility of its
being drawn out in detaching the parasite from its adhesion to the
cells of the host is not precluded, but the numbers of such cases and
the retention of normal symmetry of both body and rhizoplast does
not support the suggestion of foreeable extraction:

In one instance (pl. 1, fig. 4) a terminal blob of eytoplasm with
a deeply staining terminal cap is attached to the side of the blepharo-
plast, and in another a considerable mass of protoplasm hes about
the extruded blepharoplast. While these may be abnormalities it is
possible that under certain conditions the eytoplasm assists locally
in the holdfast funetion of the blepharoplast by forming an enlarged
mass about it.

The six flagella are equal, habitually trailed posteriorly and about
half as long as the body. They serve to keep up the circulation of
the fluid contents of the digestive tract as they le parallel to the
closely packed bodies of the parasites (pl. 1, fig. 8) in the folds of
the digestive epithelium.

The four peripheral spiral threads (fig. A, my.) which terminate
at or near the blepharoplast must be regarded as a part of the neuro-
motor apparatus. Their relations to the blepharoplast and their
stainability as well as the homology suggest this. Their function,
if contractile (and their spiral course indieates this), appears to be to
foree the blepharoplast into intimate contact with the cells of the host.
They persist at cytolysis and individuals are often found (pl. 1,
fiz. 10) in which these myonemes are frayed out as distinct lines.

1919 | Kofoid-Swezy: Streblomastix stria 9

These myonemes are evidently quite firm fibers, somewhat elastic and
more or less rigid. They often stain very deeply especially in dis-
integrating individuals (pl. 1, fig. 1).

’ DirEcTION OF TORSION

The direction of torsion of these elements of the neuromotor appa-
ratus is not without a deep significance. It is the same as that of the
undulating membrane or attached flagellum of Trichomonas. Tricho-
mitus, Tetratrichomonas and Eutrichomastix (Kofoid and Swezy,
1915), other polymastigotes in which torsion finds some structural
expression. This same direction of torsion appears in the myonemes
of Pyrsonympha and Dinenympha (eidy, 1881). Grassi and San-
dias (1893) reverse the direction of Pyrsonympha and of Holomasti-
gotes in their figures, while Porter (1897) figures both directions. It
is perhaps significant that his figures from life have the reversed diree-
tion while those from preparations, and therefore presumably accu-
rate, have the normal leiotropic, or to be expected, direction. The
reversals figured by Grassi and Sandias require confirmation before
acceptanee. The Pyrsonympha of this contribution (1893) is later
designated as Spirotrichonympha by Grassi and Foa (1911) but with-
out note of the differences in torsion. Zulueta (1915) figures the leio-
tropic direction in what appears to be Grassi’s Spirotrichonympha.
While it is quite possible that both leiotropie and dexiotropie genera
or species exist, or that functional reversals of torsion occur in the
individual it is even more evident that critical observations are essen-
tial to establish these diametrically opposed conditions. Pending such
investigations the preponderance of the evidence favors the view that
the torsion of the more primitive Trichonymphidae is leiotropic, that
is from right over to left posteriorly, as it is in Streblomastiz.

This is also the fundamental direction of the girdle and of the
encircling transverse flagellum of the Dinoflagellata, and also of the
attached collar-forming, ribbon-like flagellum of the Craspedomonadina
(Burck, 1909). These facts are suggestive of an extensive and deep-
seated leiotropism in the organization of the Mastigophora which finds
expression in both externally attached flagella and internal contrac-
tile myonemes. That it may be conditioned by some equally per-
vasive stereometriec properties of certain compounds of the living
substance seems plausible.

10 University of California Publications in Zoology —[Vou. 20

NucLeus

The nueleus (fig. A, ».) shares the elongation which affects the
body and appears to be pulled far anterior by the holdfast function
of the blepharoplast so that in comparison with other polymastigotes
its location is exceptionally far anterior. Its length is from 0.3 to 0.5
that of the body itself and its shape is fusiform but much more slender
than the body, its length being fifteen to twenty-five times its diameter.
It tapers about equally at both ends and appears in most of our
preparations as a solid black axial strand in the anterior part of the
body. Unless very strongly decolorized no internal structures can
be made out. It appears to be composed of almost solid chromatin.
When sufficiently decolorized (pl. 1, figs. 7, 9) a distinet nuclear
membrane is evident within which a single row of black chromatin
spherules, decreasing in size towards each end, can be detected. These
are not uniform in size or arrangement and are about twenty-five in
number. They are not unlike the chromomeres which we have found
in the chromosomes of 7'richonympha.

BINARY FISSION

The life history of Streblomastirx presents those phases of develop-
ment which we (Kofoid and Swezy, 1915 and Kofoid and Christiansen,
.1915) have previously described for other polymastigotes, namely,
binary and multiple fission. As yet no eneystment has been detected
and no indications of sexual reproduction. The differences in size
which we find would doubtless some years ago have afforded a basis
for the speculative designation of microgametes and macrogametes
and the corresponding gametocytes as well as for the predication of
sex, as Hartmann (1910) did in the case of Trichonympha. However,
in the absence of evidence of sexual behavior and observed fusion of
gametie nuclei, the free swing of such speculation is wisely held in
abeyance.

Binary fission occurs in the trophozoite stage. There is some
evidence that it is eyelic since many individuals in approximately
the same stage of mitosis will be found in a single host. It is not,
however, restricted wholly to such eyeles since isolated cases of fission
have been found and not all individuals parasitic in one host are in
fission at one time. Successive infections and diverse stocks of the
parasites doubtless ‘exist in the host and may afford the occasion for
this diversity. ‘

1919] Kofoid-Swezy: Streblomastix strix 11

Jnfortunately our material, though extensive, has not given us
all of the stages of nuclear behavior during fission so that we are
unable to trace wholly, the successive phases of mitosis. We have
found no clear evidence of chromosome formation, beyond the twenty-
five or more spherical aggregates of chromatin in the hnear nucleus.
We have found no spherical stage of the nucleus, no skein, and have
not detected the division of the blepharoplast which doubtless occurs,
neither have we been able to find the paradesmose spun out between
the daughter blepharoplasts (pl. 2, figs. 12, 13).

The process of binary fission, in so far as our partial evidence
goes, takes place without any. rounding-up of the elongated body.
The anteriorly located blepharoplast divides, new flagella arise from
one or both of the daughters, and one migrates to the opposite end
of the body (pl. 2, fig. 11). In the meantime the nucleus has become
greatly elongated, reaching from end to end of the body. It then
constricts at the middle (pl. 2, fig. 12), finally parts there (pl. 2,
fig. 19) and the two schizonts separate. To all appearances this is
transverse division. Longitudinal division is, however, the funda-
mental and universal method of binary fission in the Euflagellata as
compared with the Ciliata in which transverse division oceurs. This
seeming departure from the normal is, however, more apparent than
real, for if the anterior blepharoplast divides and one daughter
migrates to the posterior end we will have such an arrangement of schi-
zonts as in Trichomonas after mitosis but before plasmotomy (Kofoid
and Swezy, 1915, pl. 4, fig. 39). This is a temporary relation in such
a metabolie form as Trichomonas but a more lasting one in Streblo-
mastic. The mode of division is therefore still morphologically longi-
tudinal though almost the last vestige of the appearance of that type
of division has been submerged by the dominating elongation of the
body in Streblomastix. While it is possible that there is a series of
skein-chromosome changes in the nucleus which has escaped us, our
present evidence indicates that these are also suppressed or hidden
in the dense chromatin threads which part by simple median constric-
tion (pl. 2, fig. 12). This parting is delayed until the posterior
daughter blepharoplast is in its final position, as in other polymasti-
gotes (Kofoid and Swezy, 1915). The frequent oceurrence of stages
in sehizogony with the nucleus as yet undivided or dividing, but the
neuromotor organelles in duplicate, indicates that both nuclear con-
striction and plasmotomy following thereon are «prolonged processes.

12 University of California Publications in Zoology — [Vou. 20

MULTIPLE FISSION

Multiple fission in Streblomastix is a eyelic process occurring in
many individuals in a single host at one time. A few only may be
found in this stage, or, in some instances, at least, the majority of
individuals may be in the multinucleate phase.

This condition is preceded by the growth of the schizont from the
small size resulting from multiple (pl. 2, fig. 13) or binary (fig. 17)
fission to a much larger or even giant stage (pl. 2, fig. 13), which
may be as much as twenty-six times the length of the smallest schizont.
The body may have as much as sixty times the mass of the smallest
stages. The nucleus, however, does not inerease proportionately,
remaining, in fact, at least in many instances, almost unchanged
(pl. 2; fig. 13).

At some period during this increase in size multiple fission sets
in. Not all trophozoites entering upon it attain the maximum size
as will be seen on a comparison of figures 15 and 16 on plate 2.
It is possible that figure 16 represents only a detached section of a
larger plasmodium which is fragmenting, or it may be a small tropho-
zoite in the initial stages of multiple fission.

Contrary to the behavior of the blepharoplast-flagella complex
in binary fission where it leads in division, preceding the nucleus,
we find that in multiple fission nuclear division by transverse cen-
striction is taking place prior to the division of the blepharoplast
(pl. 2, figs. 14, 15). The type of nuclear division is the same as in
binary fission. We have not seen stages of multiplication of the ble-
pharoplast or of plasmotomy.

The linear form persists during the period of multiple fission and
the nucleus becomes an elongated axial chromatin thread which
becomes attenuate locally and parts transversely (pl. 2, figs. 13-16).
The number of nuclear segments varies, probably in a 2-4-8 sequence,
although irregularities in this are apparent. The largest number
observed is eight. This accords with multiple fission in other poly-
mastigotes (Kofoid and Swezy, 1915; Kofoid and Christiansen, 1915).

There is some evidence that this stage is contractile and that when
foreshortened the nuclei slip by one another. This is not a common
condition and probably does not represent a rounded-up condition
obligatory for multiple fission but rather a passing response to
stimulus resulting in contraction.

1919] Kofoid-Swezy: Streblomastix strix 13

Multiple fission stages which we have seen provide for eight
schizonts when the plasmodium parts by plasmotomy into its con-
stituent zooids. From irregularities in the number of nuclei in prep-
arations in which multiple fission is common, it seems probable that
plasmotomy is an irregular dropping off of individuals or groups of
individuals from the common mass as in Trichomonas. We have no
evidence as to the presence of a centrosome during multiple fission
and none as to the origin of the new blepharoplast-flagella complexes
of the daughter schizonts.

ADAPTATIONS

Although seemingly simple in structure Streblomastix presents a
series of structural adaptations which in the light of its parasitic mode
of life become significant of intimate correlations with the conditions
under which it exists and its habits. The entire loss of the cytostome
is associated with feeding by osmosis and results in the disappearance
of the bilateral asymmetry characteristic of polvmastigotes such as
Trichomonas. The absence of large food particles makes possible the
elimination or reduction of cyclosis of the endoplasm and facilitates
the change of form to a long and relatively very narrow spindle
within which such movement would be impeded. The spiral course
of the myonemes or spiral striae provides a most effective form of
mechanism for an energetic thrust of the holdfast blepharoplast
against or into the cells of the host. It is also a form of contraction
which would disturb but slightly the closely packed grouping of the
parasites. The spirally fluted surface combined with the action of
the posteriorly direeted flagella would give rise to vortex currents
of the cireumambient digestive fluids and thus provide the circulation
essential to the metabolism of the parasite while at the same time
permitting their segregation apart from the other organisms of the
digestive tract of the host. The contractile extrusible rhizoplast-
blepharoplast complex with its blob of cytoplasm affords an efficient
structural holdfast. The elongation of the nucleus provides a spa-
tially advantageous grouping for the nucleo-cytoplasmic interchanges
in the absence of marked eyclosis.

The neuromotor apparatus is so arranged as to give well distributed
contact with the surrounding medium by means of flagella and striae
and with the host by means of the blepharoplast-rhizoplast, while this
in turn is connected with the nucleus, thus establishing the structural
essentials for efficient codrdination of functions.

14 University of California Publications in Zoology — [Vou. 20

Even the reproductive phases of binary and multiple fission
retain the elongated form characteristic of the trophozoite, thus per-
mitting those stages to retain their position among the segregated
parasites of their own kind. In other polymastigotes, such as Tricho-
monas, the stage of multiple fission is an amoeboid plasmodium, a
rounded-up, somewhat shapeless amoeboid mass (Kofoid and Swezy,
1915). In Streblomastix, however, the linear form persists through-
out this phase, in so far as we have observed it, although it shows
greater laxity of form and less torsion (pl. 2, figs. 14, 15) than do
the vegetative trophozoites. Thus in every feature of its structure and
phase of its life history Streblomastix is intimately adapted to its
peculiar parasitic mode of fe notwithstanding its seeming simplicity
of structure.

RELATIONSHIPS

The presence of six flagella definitely allocates Streblomastix in the
Order Polymastigina. Its relative simplicity of structure as com-
pared with most genera of this order is shown by the undifferentiated
condition of the flagella. There is no single specialized trailer at-
tached as an undulating membrane, and none intracytoplasmic as an
axostyle.

The presence of the four longitudinal spiral ectoplasmie ‘‘myon-
emes’’ or extensions of the neuromotor apparatus is very suggestive of
a relationship to the Trichonymphidae in most of which such lines
arise from the blepharoplast and are the stems from which spring the
many so-called cilia. This relationship will be more evident on
detailed comparison. There are eight such lines in Dinenympha
gracilis (Leidy, 1881) along the course of each of which small cilia
take their origin, but there are no developed anterior flagella. Zulueta
(1915) has shown that these extend posteriorly as free flagella and
that they are grouped four and four on the daughter centrosomes at
the poles of the spindle at mitosis. The species upon which Zulueta
worked appears to be the same as that figured by Grassi and Sandias
(1893, pl. 5, figs. 18-20) which Grassi and Foa (1911) later distinguish
from Leidy’s species as Spirotrichonympha. Pyrsonympha vertens
(Leidy, 1881 and Porter, 1897) likewise has eight such lnes arising
from the blepharoplast with small lateral cilia arising frem them.
Anteriorly there is a blepharoplast from which a free slender thread
extends anteriorly into the host cell not unlike the rhizoplast-blepharo-

1919] Kofoid-Swezy: Streblomastix striz 15

plast of Streblomastix in superficial appearance, but possibly homo-
logous with flagella and derived by modification from one or more of
them. There are four such lines in Holomastigotes (Grassi and
Sandias, 1893) giving rise to lateral cilia and pursuing a spiral course
posteriorly from what is probably an anterior blepharoplast. There
are, however, no large anterior flagella arising from this point.

The form most nearly allied to Streblomastix appears to be
Pyrsonympha by reason of the persistenee of an anterior outgrowth
from the blepharoplast which may be homologized with flagella. How-
ever, it has lateral cilia arising from its spiral lines. These S/reblo-
mastix entirely lacks. This absence of the lateral ciliary coat justifies
the exclusion of Streblomastix from the Trichonymphidae and renders
its retention in the Polymastigina necessary. However, if may be
regarded as closely related to that branch of the polymastigote stock
from which the Trichonymphidae originated. Streblomastix thus
forms a living link between the Polymastigina and the Trichonym-
phidae, linking the latter seemingly aberrant forms more closely and
definitely than heretofore to the Flagellata as its most highly speeial-
ized order.

It is obvious that Streblomastiz ean not be allocated in the family

*Hexamitidae and that its relationships with the Polymastigidae are
relatively remote. Even its inclusion in the Polymastigina is some-
what problematical. Its transfer to the Trichonymphidae is defensible
but requires a profound modification in the definition of that order.
To set forth more strongly its intermediate position we have left it in
the Polymastigina and propose for it a new family, the Streblomasti-
gidae, as follows:

Family Streblomastigidae fam. nov.
Polymastigina with spiral myonemes and anterior flagella.
Streblomastiz gen. nov.
Streblomastigidae with six anterior flagella and four leiotropie
myonemes. Type species Streblomastix striz sp. noy. from
Termopsis angusticolis Walker.

16 University of California Publications in Zoology [Vou. 20

SUMMARY

1. Streblomastix strix occurs as an intestinal parasite of the ter-
mite Termopsis angusticollis and is usually found attached to the
epithelium of the intestine posterior to the malpighian tubules, seg-
regated from the other parasites in the lumen.

2. It is a linear organism with the nucleus elongated to conform
to the shape of the body. Its neuromotor apparatus consists of
centrosome, blepharoplast, four myonemes and six flagella, connected
with the nucleus by the rhizoplast.

3. Binary fission apparently occurs without spindle formation.
The nucleus elongates and becomes constricted prior to the econ-
striction of the protoplasmic body.

4. Multiple fission is a eyelic process occurring In many individuals
in a single host at one time. It may be preceded by the formation of
giant individuals. The body retains its linear formation throughout
the process as in binary fission. The nucleus elongates and constricts
into daughter nuclei. The greatest number observed is eight.

5. The shape of the body and the arrangement of its neuromotor
apparatus show striking adaptation to the habitat in which the flagel-
late is found and to its parasitic mode of life.

6. Streblomastiz forms a living lnk between the Polymastigina
and the Trichonymphidae but without close relations in either group.
We therefore propose for it a new family, Streblomastigidae, which
we place in the Polymastigina.

Zoological Laboratory, University of California,
Berkeley, California.
Transmitted September 5, 1918.

1919] Kofoid-Swezy: Streblomastix strix ali

LITERATURE CITED

Boeck, W. C.
1917. Mitosis in Giardia microti. Univ. Calif. Publ. Zool., 18, 1-26, pl. 1.

BurcK, C.
1909. Zur Kenntnis der Histologie einiger Hornschwimme, sowie Studien
liber einige Choanoflagellaten. (Heidelberg, Rossler), 61 pp., 2 pls.

GRASSI, B., and FoA, A.
1911. Intorno ai Protozoi dei Termitidi. Nota preliminare. Rend. R. Accad.
dei Lineei, Cl. Sci. Fis. Mat. e Nat., Rome, (5), 20, 725-741.

GRASSI, B., and SAnprAs, A.
1893. Costituzione e sviluppo della societa dei Termitidi. (Catania Galatola),
150 pp., 4 pls.

HARTMANN, M.
1910. Untersuchungen tber Bau und Entwicklung der Trichonymphiden
(Lrichonympha hertwigi n. sp.). Festschr. z. Hertwigs, 1, 349-396,
pls. 27-30, 3 figs. in text.

Koromw, C. A., and CHRISTIANSEN, E. B.
1915. On binary and multiple fission in Giardia muris (Grassi). Univ. Calif.
Publ. Zool., 16, 30-34, pls. 5-8, 1 fig. in text.

Kororp, C. A.,;and SweEzy, O.
1915. Mitosis and multiple fission in trichomonad flagellates. Proc. Amer.
Acad. Arts. Sci., Boston, 51, 289-378, pls. 1-8, 7 figs. in text.
LEIpy, J.
1881. The parasites of the termites. Jour, Acad. Nat. Sci., Philadelphia, (2),
8, 425-450, pls. 51, 52.

Porter, J. F.
1897. Trichonympha and other parasites of Termes flavipes. Bull. Mus.
Comp. Zool., Cambridge, 31, 48-68, pls. 1-6.
Yocom, H. B.
1918. The neuromotor apparatus of Huplotes patella. Univ. Calif. Publ. Zool.,
18, 337-396, pls. 14-16.
ZULUETA, A. DE
1918. Sobre la reproduction de Dinenympha gracilis Leidy. Trab. Mus. Nae.
Cienc. Nat., Madrid, ser. Zool., 23, 15 pp., 1 pl., 6 figs. in text.

EXPLANATION OF PLATES

All drawings of Streblomastix strix were made with camera lucida from
material stained with iron haematoxylin, with a magnification of 2500 unless
otherwise stated. ‘

PLATE 1

Fig. 1. Ordinary trophozoite showing few turns in torsion of the body.

Fig. 2. Contracted specimen showing centrosome and blepharoplast near
together. .

Fig. 3. Giant individual not fully drawn. Note elongated rhizoplast with
blob of cytoplasm surrounding blepharoplast.

Fig. 4. Individual showing definite nuclear membrane and blob of proto-
plasm with chromatin cap attached to the blepharoplast.

Fig. 5. Body slightly contracted with little torsion.

Fig. 6. Myonemes showing heavily stained lines.

Fig. 7. Trophozoite with considerable torsion of body.

Fig. 8. Position of parasites in villi of intestine; attached to the mucous
lining but not to the cells of the wall.

Fig. 9. Trophozoite with greatly elongate rhizoplast.

[18]

ZOOL

ALIF. PUBL

cC

Fig. 10.
Fig. 11.
groundwork.
Fig. 12.
Fig. 13.
Fig. 14.

PLATE 2

Division form. Note length and structure of nucleus.

Individual with myonemes fraying out from the protoplasmic

Binary fission with the nucleus constricting.
Giant individual.

Multiple fission with nucleus constricted in the formation of seven

or eight schizonts.

. 15.
A UGE
be los
Gales
b Ales

Multiple fission form preceding constriction of nucleus.
Multiple fission form with three schizonts.

Telophase of binary fission.

Individual in process of fission.

Final stage of binary fission; parting of schizonts.

[20]

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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, I figurein=text.. duly, 19292 cn So ee ne
1. A Revision of the Microtus Californicus Group of Meadow Mice, by Rem-_ ee

ington Kellogg, Pp. 1-42, 1 figure in text. December, 1918 02...

2. Five New Five-toed Kangaroo Rats from Callioreia, by Joseph Grinnell.
~Ppr 43476 March, 1919 eee cea stgpt Sevens etcvags vn sAnpsn soe anreneevawupances

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UNIVERSITY OF CALIFORNIA PUBLICATIONS

IN
ZOOLOGY :
Vol. 20, No. 2; pp. 21-40, plates 3-4, 2 figures in text July 14,1919

STUDIES ON THE PARASITES OF THE TER-
MITES Il. ON TRICHOMITUS TERMITIDIS, A
POLYMASTIGOTE FLAGELLATE WITH A
HIGHLY DEVELOPED NEUROMOTOR

_ SYSTEM. i

BY

Spaeers Sea
Sy aly Ob G05 aa
S Ry

AUGI31919

Se eas,
ith sonian pepoe

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UNIVERSITY OF CALIFORNIA PUBLICATIONS
IN

ZOOLOGY
Vol. 20, No. 2, pp. 21-40, plates 3-4, 2 figures in text July 14, 1919

STUDIES ON THE PARASITES OF THE TER-
MITES II. ON TRICHOMITUS TERMITIDIS,
A POLYMASTIGOTE FLAGELLATE _
WITH A HIGHLY DEVELOPED /
NEUROMOTOR SYSTEM bere

BY Quy
if
CHARLES ATWOOD KOFOID anp OLIVE SWEZY “Sonia

CONTENTS

PAGE

TERS COUN Se a ea eee RR ee a ERO eo ESE 21
(OSLER aN INE) eS EE ED ee ee 7)
JNUUG ETON AY OGY 25 ee

Shape and size of body

CON AIO OY ENS AT ete ce Ro re eae Ea ea ee eS 24

@yhOSt Ome eae aerate coe ate ee Ses Pi Fiera esa oe AE (etn MMe SSE 2 antl --.. 26

Neuromotor system -...

TSG UKE SUES SR SRE a eee
VIS HOSTS Tee resect e nas ce Sen ca oe nt Sa sae ena sue coat cass nacaes act nce cawostpeesSsavacauets omesneesesi Ne é
Binary and multiple fission .. 35
J BYAVOYARLTNGTN GD «conse sep recone oe ee cee oie ae Se a ee ee re Bee eer een eee 36
TRHCEN ENED OS FS) Oe Ee ee OE 36
Summary .........- s
Literature cited
Pexplamatrony (Ots (Plates! ces.cse-escsetccenc sees wat < cece 2s eereece asters oe ncasets caste csnesbesabtiesteeecseaeeebedes-2 OS

INTRODUCTION

The occurrence in polymastigote flagellates of a structurally inte-
erated fibrillar complex consisting of centrosome, rhizoplast, blepharo-
plast, axostyle, undulating membrane, parabasal body, and flagella in
Trichomonas was deseribed by us (1915) and the complex designated
as an extra-nuclear motor apparatus. Its analogy to the neuromotor
apparatus of the ciliate Diplodinium was noted.

22 Unwersity of California Publications in Zoology [| Vou. 20

In the following year the more highly specialized and intimately
integrated fibrillar apparatus of the binucleate diplozooie Giardia
was definitely designated (Kofoid and Christiansen, 1916) as the
neuromotor apparatus. In a paper read before the Second Pan-
American Scientific Congress at Washington, January 7, 1916, the
senior author extended the neuromotor conception to the flagellates
generally to include the centrosome-blepharoplast and its external and
internal fibrillar derivatives and connections under the name of the
neuromotor apparatus.

It is the purpose of this paper to deseribe the neuromotor appa-
ratus or system of one of the simpler trichomonads in which there is no
axostyle but in which there occurs in response to the parasitie habit
an exceptionally massive development and structural continuity of
the several elements of this codrdinating organ system. The organism
also presents a prophetic prolongation of the period of existence of
the paradesmose and of the incipient stage of mitosis,—features which
are strongly suggestive of a tendeney which, if continued, might well
culminate in the evolution of the diplozooie flagellates, such as
Giardia. The poteney of the biochemical environment of parasites
in bringing to expression latent possibilities of the organization of
the living substance is once again demonstrated in this flagellate of
those extraordinarily parasitized insects, the termites.

OCCURRENCE

This flagellate has been found abundantly in Termopsis angusti-
collis Walk., a large termite commonly found in decayed oak trees
on the University campus. The flagellate infests the posterior and
midregions of the intestinal tract of the termite with only a slight
infection or none at all of the anterior region. It is found in the
lumen of the canal with no attachment to the wall. Associated with it
is a large Trichonympha, and in eross-section of the entire intestinal
tract it is found that these two flagellates, with the latter usually
predominating, completely fill the lumen of the canal.

Almost every individual of this species of termite which has been
examined has been found to harbor these flagellates. The number
in a single host may vary greatly as it or the trichonymph may be
the dominant form. In some instances the latter species may be rare
with Trichomitus termitidis present in vast quantities. These are
sporadic cases, however, with no indications of a rhythmical cycle

1919] Kofoid-Swezy: Trichomitus ternutidis 23

that is seasonal in its occurrence, as shown by examinations of the
host which have been made throughout the year.

Trichomitus greatly resembles its near relative Trichomonas in its
activities. It is, however, difficult to keep these flagellates alive in
cultures, hence observations on the active forms have been limited.
The extreme fragility of the cytoplasmic body as contrasted with the
stout, persistent parabasal body, is particularly striking in prepara-
tions of living material. A few seconds usually suffices, in ordinary
tap water, for the dissolution of the protoplasm, leaving the neuro-
motor system still intact.

Nutrition in Trichomitus is holozoic. It, like Trichonympha, is
evidently only a commensal, or at least is not truly parasitic, i.e., liv-
ing on the tissues or fluids of the host. The food particles found
within the cytoplasm consist principally of woody fibers upon which
the termite feeds.

MORPHOLOGY

The morphology of this species of T'richomitus is of especial signifi-
cance not only in view of the distinetness with which the neuromotor
organ system is developed and integrated but also in the unquestion-
able certainty with which the relationship of the centrosome to the
blepharoplast is established, as will be shown later. The relatively
large size of the organisms (75 to 150”) and their abundance have
made possible an analysis of these structures not obtainable with the
smaller trichomonads of our earlier studies (Kofoid and Swezy, 1915).

SHAPE AND SIZE oF Bopy

The body of Trichomitus termitidis is exceedingly amoeboid and
protean in hfe, having neither constaney of form nor resistance to
deformation on contact with other organisms or objects. Its periplast
is unusually thin and delicate and in the larger forms especially is not
infrequently ruptured in the making of smear preparations. It has
nevertheless a certain characteristic range of forms within which, in
free movement, it is seen or preserved on fixation. These vary from
the asymmetrical pyriform contour, with the large end anterior and
the posterior tapering to a blind point (pl. 3, fig. 1), to the ellipsoidal
(pl. 3, fig. 14) or subspheroidal shape (pl. 3, fig. 5), with the slightly
greater diameter posterior to the center.

24 University of California Publications in Zoology — [Vou. 20

The factors conducing to these changes in form are the stages of
general contraction of the body, the mass of food vacuoles which is
usually greater in the more rounded forms, and the proportional
length of the parabasal body and undulating membrane. As a result
of multiple fission (pl. 4, figs. 28, 30) one of the daughters receives
the ancestral parabasal and two of its associated flagella, both of which
are disproportionally large for the cytoplasmic mass of this schizont.
Regulative resorbtion, in (pl. 4, fig. 31) or out of a cyst, or rapid
cytoplasmic growth, would be necessary to readjust the volumetric
relation of the neuromotor system and the cytoplasmic mass.

The range in size in this species is very considerable (fig. A, 1-7).
The smallest schizonts we have recorded (pl. 4, fig. 32) are but 16u
in length while the largest exceed 200u. These giant individuals are
probably approaching multiple fission. They rarely survive the
smearing operation intact. Not infrequently the nucleus and _ its
attached neuromotor system of such giant individuals will be found
intact and still active after the loss of the eytoplasm indicating that
this stage 1s particularly susceptible to destruction under normal con-
ditions in the host. Most of the individuals seen range from 75 to
125, in length.

The organs of Trichomitus (fig. A, 5) consist of the eytostome
(eyt.), nucleus (n.), food vacuoles (f. vac.), and the neuromotor organ
system. We will now consider these organs with the eytoplasm in
detail.

CyTOPLASM

The cytoplasm of Trichomitus termitidis is reasonably labile, finely
granular, and somewhat alveolar in structure. This lability may be
the cause of such abnormal proportions (such as are seen in plate 4,
figure 30) rather than the inheritance of the ancestral parabasal sug-
gested above. The dropping off by plasmotomy of the labile eyto-
plasm has been observed by us in Trichomonas augusta (Kofoid and
Swezy, 1915). No contractile vacuole is present but food vacuoles
(fig. A, 5, f. vac.) are found everywhere within the body except about
the nucleus. These contain fragments of cellulose from the digestive
tract of the termite or coccoid bodies, possibly bacterial (pl. 3, fig. 5).
Defecation of undigested fragments has not been seen.

Upon treatment with neutral red a large number of food particles
or metaplasmie droplets stain deeply. They le scattered throughout
the cytoplasm and are larger near the center of the body (fig. A, 8).

1919] Kofoid-Swezy: Trichomitus termitidis 25

cee ee ey (arf

Fig. A. <A series showing variations in size in Trichomitus termitidis.
8 is drawn from individual stained with neutral red.

Abbreviations: ant. fi., anteror flagella; bl., blepharoplast; cent., centrosome ;
cent. rh., centrosome-rhizoplast; cyt., eytostome; f.vac., food vacuole; m. fl., mar-
ginal flagellum; n., nucleus; ». rh., nuclear-rhizoplast; par. b., parabasal body;
parad., paradesmose; und. mem., undulating membrane. X 700.

Figure

26 University of California Publications in Zoology — [ Vou. 20

CyTOSTOME

The cytostome lies on the anterior ventral surface at the extreme
anterior end of the body. It is a large elongated asymmetrical pocket,
slender pyriform in outline but curved on its right side against the
nucleus. Its length is about 0.3 that of the body and its width 0.3 to
0.2 its own length. It leads into the cytoplasm near the center of the
body. Its large size, great flexibility and its slight projection ante-
riorly in a prominent lip all indicate its efficiency as a food-grasping
and enveloping organ. We have found it during the later stages of
binary fission, in the plasmodium of multiple fission, but not in the
eneysted condition. We have not been able to determine the exact
mode of origin of new cytosomes. Its location immediately adjacent
to the blepharoplast and nucleus necessitates a high degree of elas-
ticity, integrity and resistance on the part of these organelles and the
rhizoplasts arising from them.

NEUROMOTOR SYSTEM

The use of the term organ system to designate the complex, struc-
turally integrated apparatus which links together the nucleus and
motor organs and plays such a distinctive réle at mitosis, seems jus-
tified by the canons of comparative morphology, unless it be that the
dogma of the Cell Theory blights such morphological license. This
organ system includes the blepharoplast (fig. A, 5, bl.) from which
spring directly the three anterior flagella (ant. fl.), the attached, pos-
teriorly directed undulating membrane (und. mem.) with its marginal
flagellum (m. fl.), and the deeper lying parabasal body (par. b.), and
a nuclear rhizoplast (n.rh.). The centrosome (cent.) les within the
centroblepharoplast, emerging at mitosis with its own independent
centrosomal rhizoplasts (cent. rh.) joining the ends of the paradesmose
(parad.) to the parent blepharoplast.

It is noteworthy from the standpoint of comparative cytology that
the motor organelles, flagella, and undulating membrane terminate
in and originate from the centroblepharoplast. The nucleus never
loses its connection, by one or more rhizoplasts, with this structure,
which also plays a dominant role in the drama of mitosis for at this
time there springs from it the centrosome, which later divides, forming
the paradesmose and its connecting rhizoplasts. To it also is attached
the enormously large parabasal body, a reservoir of chromatoidal sub-

1919] Kofoid-Swezy: Trichomitus termitidis 27

stance. This centroblepharoplast is thus most truly a morphological
center intimately associated with the motor organs of this cell and
their activities during the vegetative phase. At mitosis it dominates
not only the extra-nuclear neuromotor system but also the polarization
and subsequent movements of the chromosomes within the nucleus
(fig. B, and pl. 3, figs. 5-21) as well. It will be difficult to find in
any metazoan cell so continuous and complete a control, so pervasive
an influence upon cell activities by the cell organ there known as the
centrosome, as we find by the centroblepharoplast in T’richomitus.

The centroblepharoplast, during the vegetative phase of Tricho-
mitus (fig. A, 6), is a minute granule about a micron in diameter
anterior to the nucleus and attached to the anterior end of a single
nuclear rhizoplast (fig. A, 5, n. rh.; fig. A, 2). This rhizoplast is a
delicate thread easily overlooked. The centroblepharoplast itself is
imbedded in the end of the deeply staining parabasal body, and may
likewise readily escape detection.

In view of its later history it seems advisable to designate this
granule at this period as the centroblepharoplast, since from it emerges
the parent centrosome at mitosis. There is, however, no duplicity of
structure evident, and there is no granule at any time at the point
where the nuclear rhizoplast passing from the centroblepharoplast
(fig. B, 1) and later from the blepharoplast proper (fig. B, 5) meets
the nuclear membrane. After the centrosome withdraws from the
larger granule (fig. B; pl. 3, figs. 6-21), the latter becomes a ble-
pharoplast in the restricted sense of a basal granule from which the
flagella originate, having no other function in mitosis, whereas the
centrosome emerging from it divides and its daughters form the
paradesmose between them, assume a polar position thereon and move
to the nucleus (fig. B; pl. 3, fig. 21).

In our investigations of mitosis in the trichomonads (1915, pl. 2:
figs. 21, 23; pl. 3, figs. 24, 29) there appeared to be a separation of
the polar centrosome-blepharoplast into two granules, one of which,
the centrosome, remained in the polar position on the nucleus, and
the other, the blepharoplast, usually with the flagella attached, was
removed a short distance therefrom. The conditions which we have
found in Trichomitus where there is a general, more complete and
perfectly distinet separation of these two organelles is thus the full
accomplishment of the segregation imperfectly realized in Tricho-
monas.

The flagella are four in number, the three undifferentiated, equal,
anterior ones (fig. A, 5, ant. fl.) and the attached posteriorly directed

28 University of California Publications in Zoology (Vou. 20

one included within the undulating membrane as its marginal fiber
(m. fl.) and carried out beyond the projecting tip of the parabasal
body as a bit of free flagellum. The anterior flagella usually exceed
the body in length. The posteriorly directed location in our figures
is merely for spatial accommodation, an anterior direction being usual
in life.

The undulating membrane (fig. A, 5, und. mem.) is attached to the
left side of the body (fig. A, 1) in a sweeping C- or S-shaped curve
reaching to the posterior end of the body (fig. A, 4, 6; pl. 3, figs. 1, 14).
It exceeds the length of the body two to three times in some small
schizonts (pl. 4, fig. 30). The coiling into the S-shaped forms appears
to be an accommodation of the somewhat rigid but elastic parabasal,
when longer than the body, to its location within the cytoplasm.
The membrane always follows the course of the parabasal and remains
adherent to it upon cytolysis (pl. 3, fig. 11). In one ease (pl. 3, fig. 4)
a detached membrane consisting only of the marginal flagellum and
the fold of the protoplasmic pellicle running from the parabasal around
the flagellum, was found free in a smear preparation. The membrane
and flagellum are thrown into twelve to twenty subequal, subequi-
distant waves of contraction which fade out in the distalmost end.

The parabasal body (fig. A, 5, par. b.) is a rigid, elastic, deeply
staining, chromatoidal rod lying at the base of the undulating mem-
brane in the peripheral plasma of the body. Its C- or S-shaped course
appears to determine the direction of that membrane. Its length
usually exceeds that of the body by 10 to 25% and its diameter,
2 to 3.54, 1s greatest somewhat anterior to its middle. From this
region it tapers gradually toward either end, terminating anteriorly
at the centroblepharoplast (fig. A, 6), or in mitosis at the blepharo-
plast proper (fig. A, 5), and posteriorly at its junction with the
marginal flagellum which projects beyond their union for a short dis-
tance as a free lash. It stains densely with haematoxylin and con-
stitutes the dominating feature of the organism in all preparations
and in life. It shows in stained sections (pl. 3, figs. 3 and 3a) a
differentiated structure consisting of an outer deeply staining shell
less than a fifth of its diameter in thickness and a less deeply stained
core. This core is traversed by wedgelike dises of the cortical sub-
stance, which arise principally on the concave face and fade away
towards the opposite side.

We have elsewhere (Kofoid and Swezy, 1915; Kofoid, 1917) inter-
preted the parabasal body as a reservoir of substances utilized by the
neuromotor system in motor activities. It is obvious on observation

1919] Kofoid-Swezy: Trichomitus termitidis 29

that locomotion by Trichomitus in the midst of the seething mass of
parasites in the digestive tract of ZYermopsis, involves not a little
expenditure of energy. It is also conceivable that the conditions of
life therein are subject to marked variations incident not only to the
food and feeding of the host but also to the varying constituents of
the enormous mass of parasitic associates and their changing meta-
bolism due to phases of their reproductive activity. Biochemical
changes of no small import are consequently a feature of this crea-
ture’s environment. That some of these are peculiarly fatal to
Trichomitus is evident from the unusual numbers of moribund or
eytolized individuals, each represented by a more or less decadent
nucleus and its attached neuromotor apparatus, which may be found
in most smear preparations.

Considerable changes in extent and volume of this structure are
apparent upon an inspection of our figures, and even more so in our
preparations. These are indicative of changes resulting from meta-
bolism, or multiple fission, or both. In addition to the storage or
reservoir function it is apparent that the parabasal in J'richomitus
serves also as a somewhat rigid organ of attachment for the undulat-
ing membrane.

The other organs of the neuromotor system, the paradesmose and
its rhizoplasts will be discussed in connection with mitosis. The
paradesmose is a more or less temporary organ in most trichomonads,
but in Trichomitus the organism appears to pass a much greater part
of its existence in what is comparable to the prophase stage of tricho-
monad mitosis, so that the paradesmose is actually present, suspended
by rhizoplasts from the blepharoplasts (fig. A, 5), and the whole neuro-
motor system is in some phase of duplication in many of the individuals
which we have seen. The stage with a single centroblepharoplast and
rhizoplast (fig. A, 1, 2, 6) is relatively much less common in this
species than in other trichomonads. This prolongation of the pro-
phase is the first step towards diplozoie organization such as we find
realized in Giardia, where nuclear division is added to that of the
duplication of the neuromotor system with the resulting formation
of a coordinating system for multicellular organization.

NuciLEeus

The nucleus (fig. A, n.) is a symmetrical ellipsoidal structure,
or even ovoidal or pyriform, with the wider end posterior (fig. B, 1).
The longer axis is two to three times the shorter one in length. It

30 University of California Publications in Zoology [ Vou. 20

lies in or near and parallel to the major axis of the’ body on the left
side of the eytostome within a short distance of the anterior end of the
body. It shows distinetly a peripheral clear zone which is somewhat
regularly chambered (fig. A, 1, 4, 5; pl. 3, fig. 9) as we have found it
also in Trichonympha. This zone surrounds the dark, dense and often
seemingly undifferentiated central chromatin mass. On heavy destain-
ing this central mass is at times resolved into fairly uniform rounded
granules (pl. 3, fig. 2) which appear to have some special relation
to the elements of the chambered zone, indicating the possibility of a
persistent organization of the nucleus. In some instances resting
nuclei (fig. B, 1; pl. 3, fig. 5) show large deeply staining granules
resembling nucleoli but these are as a rule absent. It is quite possible
that these may be end knobs of emerging chromosomes.

The size of the nucleus ranges from 10 to 20 in length whereas
that of the body ranges from 16 to 200” or even more. Although
larger individuals have larger nuclei the increase in the volume of the
cytoplasm is many-fold greater than is the increase of the nucleus in
these giant forms.

One of the most significant and striking features of the life his-
tory of Trichomitus in the digestive tract of Termopsis is the repeated
and seemingly constant occurrence of large numbers of isolated neu-
romotor systems with the nucleus attached but no enveloping eyto-
plasm. It is evident that the delicate pellicle is easily destroyed, and
the labile cytoplasm escapes. Such an isolated structure in late pro-
phase with duplicated neuromotor systems but degenerate nucleus is
seen in plate 3, figure 11. These occurrences afford indisputable
evidence of the organic continuity and structural integration of the
neuromotor system of Trichomitus and of its direct and efficient
physical connection with the nucleus. One of us (Swezy, 1915a) has
noted a similar phenomenon in Hexamitus, a diplozooie polymastigote.

Still more significant is the fact that such isolated systems are
still capable of flagellar activity and locomotion after the destructive
process of cytolysis of the cytoplasm. They continue to move for some
time in smears of the contents of the digestive tract mounted in tap
water. The very large number of isolated systems found in some
smears is indicative of a considerable period of persistence of the
isolated neuromotor system and nucleus after the loss of cytoplasm.
It is obvious that grave limitations on such activities must arise as a
result of the loss of the cytoplasm. The nucleo-cytoplasmie reactions
are suspended, nutrition is impeded if not wholly suspended, and
rapid exhaustion is accelerated by the loss.

1919] Kofoid-Swezy: Trichomitus termitidis 31

MITOSIS

Owing to this prolongation of the prophase of mitosis in T'richo-
mitus termitidis an exceptional opportunity is afforded for a detailed
study of the behavior of the neuromotor system during mitosis. This
is made possible by reason of the fact that the centroblepharoplast is
the center of the neuromotor system and the point of origin of struc-
tures and processes playing the main réle in mitosis.

The phases of mitosis recognizable in the division of Trichonitus
are those of the metazoan cell, but, as shown in our (1915) discussion
on mitosis in trichomonads, considerably differentiated by the associa-
tion of the extra-nuclear organelles of the cell in the protozoan from
that in the usually simpler metazoan unit. In Trichonitus these
differences, in consequence of the massive development of the neuro-
motor system, are even more developed than in the other trichomonads.
They consist mainly in the sharp separation of centrosome and ble-
pharoplast and the excessive prolongation of the prophase.

The resting stage of Trichomitus has a single nuclear rhizoplast
running from the centroblepharoplast to the anterior end of the
nucleus where it is attached to the membrane without evidence of
enlargement into a centrosome on the nuclear membrane. This rhizo-
plast is often very short (fig. A, 1) and is never very long. The
nucleus has in this period a dense, coarsely and uniformly granular
central chromatin mass in which no polarization or evidence of chro-
mosome formation is present.

The prophase (fig. B; pl. 3, figs. 1, 2, 5-15) is a prolonged one
and in it the duplication of the entire neuromotor system takes place
by division and outgrowth from the centroblepharoplast. The initial
step is the splitting of the centroblepharoplast and the nuclear rhizo-
plast (fig. B, 1). It appears in some eases (pl. 3, fig. 1) that the
rhizoplast may divide at the nuclear membrane first and split distally
towards the centroblepharoplast as though these were directly upon
the nuclear membrane at the anterior end of the nucleus. At the
same time the centroblepharoplast separates into its constituent centro-
some and blepharoplast, with the latter immediately dividing, one
granule taking a single flagellum, the new parabasal body and one
of the two nuclear rhizoplasts (fig. B, 1). The granule remaining
attached to the old parabasal body takes the remainder of the flagella
and the second nuclear rhizoplast. As these two blepharoplasts sepa-
rate a thread is drawn out from each attaching them to the centro-

32 University of California Publications in Zoology [Vou. 20

some (fig. B, 1). This is followed by a division of the centrosome.
As the two new centrosomes move apart a darkly staining line or bar
is drawn out between them, the paradesmose (fig. A, 5, parad.; fig. B).

The rhizoplasts connecting the centrosomes with the blepharo-
plasts gradually elongate with the paradesmose also increasing in both
length and thickness. The latter structure with its connected centro-
somes moves down until it comes to rest upon the nuclear membrane
(fig. B). Asa result of this the centrosome-rhizoplasts come to have
the same length as the nuclear rhizoplasts. All four of these rhizo-
plasts are exceedingly delicate, particularly the nuclear ones and the
latter especially long escaped our notice. Since the two are rather close
together (fig. B, 5) their distinctiveness may be easily overlooked.
The centrosomes (fig. A, 5, cent.) are minute knobs on the ends of the
paradesmose, which is a stout, heavy, sometimes granular, deeply
staining bar. They are not always visible as expansions of the bar and
are never seen detached from it.

In the meantime the new undulating membrane and parabasal
body have reached sizes equal to the ancestral ones (pl. 3, fig. 11;
fig. B, 2-6). The parabasal body first appears as a slender dark
thread growing posteriorly subparallel to the old parabasal in the
peripheral cytoplasm (pl. 3, fig. 5). With the beginning of the
formation of the paradesmose (fig. B, 2) the new flagella have all
formed by outgrowth from the daughter blepharoplasts, two anterior
ones and a posteriorly directed one as a marginal filament from the
blepharoplast attached to the new parabasal, and only one anterior
one from that attached to the old parabasal. At first (fig. B, 2) the
new undulating membrane is very narrow but it soon attains full
structural size and functional efficiency (pl. 3, fig. 11). With the
completion of these structures by growth the duplication of the neuro-
motor system is accomplished.

Up to this time the changes visible within the nuclear membrane
have been very shght. The chromatin granules or chromomeres grow
larger and darker, and evidences of polarization appear in the linear
grouping of the granules (fig. B, 6; pl. 3, figs. 6-10) which ecul-
minates in the emergence of linear V-shaped chromosomes. During
this process a deeply staining cone-shaped extension of the central
chromatin mass projects anteriorly until it comes in contact with
the paradesmose (fig. B, 6; pl. 3, figs. 6-8). As this disappears the
V-shaped chromosomes become more evident in the central mass, and

1919] Kofoid-Swezy: Trichomitus termitidis 33
gradually spread out below the paradesmose as though hung across
a string suspended from its ends (pl. 3, figs. 10, 12, 15).

The number of chromosomes is rather obscure since the loops are
at all times rather closely entangled. It appears to be twelve or
thereabouts. In the earlier phases each is composed (pl. 3, fig. 10)
of a line of distinct granules, like chromomeres, but these fade out as
the metaphase approaches.

Fig. B. Development of paradesmose in Trichomitus termitidis. 1. Out-
growth of new parabasal body after separation of centrosome and blepharoplast
and division of latter. 2. Centrosome divided and paradesmose forming be-
tween them; new flagella and undulating membrane formed. 3. Elongation of
paradesmose as it moves down to the nucleus with the lengthening of the
centrosome-rhizoplasts. 4. Later stage of same, condensing chromatin in nucleus
with cone-shaped projection starting towards the nuclear membrane and parades-
mose. 5. Paradesmose attached to nuclear membrane. 6. Attachment of central
chromatin mass to paradesmose and formation of spindle; beginning of formation
of chromosomes. X 1575.

The metaphase is obscured by the fact that there appears to be
no arrangement of the chromosomes in an equatorial plate and no
amphiaster phase of the nucleus. The nuclear structures appear to
conform their arrangement to the stout bar-shaped paradesmose and
not the latter mould itself to the configuration of the nucleus as in
Trichomonas and Eutrichomastix (Kofoid and Swezy, 1915).

34 University of California Publications in Zoology (Vou. 20

The loops or V-shaped chromosomes are gradually drawn by the
angle of the V towards the two ends of the paradesmose (pl. 3,
figs. 15, 17, 18). It is possible that each original loop is split length-
wise during this movement but the evidence for it is by no means
clear. During this process the loops shorten, thicken, and stain more
deeply, so that when they have finally parted (pl. 3, figs. 16, 20, 21)
they form chrysanthemum-shaped rosettes.

The anaphase is brief and is also dominated by the stout parades-
mose, which continues to produce a one-sided, asymmetrical grouping
of the two groups of parting chromosomes and to modify the constric-
tion of the nuclear membrane so that it is also one-sided. The nuclear
membrane remains intact throughout the whole process of mitosis.
In the late anaphase constriction is completed, the nuclei separate
and move apart (pl. 4, fig. 22), stretching out the paradesmose be-
tween them as a result of the uncodrdinated activities of the two
daughter neuromotor systems, which are attached to the nuclei by
their rhizoplasts.

The telophase (pl. 4, fig. 36) ensues before plasmotomy. In it
the chromatin of the massed chromosomes rounds up in the central
mass and the clear zone reappears and the ellipsoidal form is resumed.
The paradesmose also fades away, and the centrosome merges with
the daughter blepharoplast which, by the shortening of the nuclear
rhizoplast, comes to lie closer to the nuclear membrane, thus bringing
the schizont back to the nuclear condition prior to mitosis.

The process of mitosis in Trichomitus is similar to that in other
trichomonads in that the nuclear membrane remains intact through-
out the process, the extra-nuclear paradesmose arises between the
daughter centrosomes, and the dupleation of the neuromotor system
proceeds from the centroblepharoplast and takes place prior to divi-
sion of the chromosomes. It differs in having a distinct separation
of centrosome and blepharoplast for a long period, in haying a rigid
bar-shaped paradesmose, and in the large size and greater elongation
of the chromosomes. This higher specialization is eonditioned by
crowded conditions of parasitie life in association with other parasites
of relatively complex organization. The conditions of locomotion in
this association and the excessive amount of stimulation consequent
thereon are causes conducive to the extraordinary development of the
neuromotor apparatus in Trichomitus and the resulting modifications
in mitosis.

1919] Kofoid-Swezy: Trichomitus termitidis 35

BINARY AND MULTIPLE FISSION

Both of these processes take place frequently in Trichomitus.
There is much evidence of a high death rate in this species within the
digestive tract of its host. This is compensated for by rapid multi-
plication. For this also there is abundant evidence in our material.

The distinction between stages of binary and multiple fission is
not readily made in all eases in early stages. The earliest phases of
both are obviously the same in mitotic phenomenon. Binucleate plas-
modia may lead on to further division when prophases appear in their
nuclei (pl. 4, fig. 23). When, however, no later prophase phenomena
are evident and the organism is not unusually large (pl. 4, fig. 36)
binary fission only may be expected. It also oceurs in the small cysts
(pl. 4, fig. 26) and small free forms ( pl. 4, fig. 29).

Multiple fission, on the other hand, occurs in large individuals and
leads to the formation of, presumably, eight-celled plasmodia.

One such large plasmodium with six constituent zooids is seen in
plate 4, fig. 28. It is possibly in plasmotomy and has lost two of its
members. Not all multiple fission plasmodia are as large as this.
Smaller ones, in which the first division has been completed and the
second initiated, are frequently found (pl. 4, figs. 23, 25, 35).

The process is one of three repeated divisions prior to plasmotomy
with the formation of an eight-nucleate somatella and its subsequent
disintegration into its constituent zooids by their detachment singly
or in groups. These somatella or plasmodial stages are exceedingly
mobile and the constituent individuals shift about without seeming
order of arrangement. The uncodrdinated movements of the powerful
neuromotor apparatus of the individual zooids finally result in their
separation. The connecting paradesmoses are lost long before this
separation.

No trace of unequivocal sexual phenomena has been detected.
Large and small individuals simulating macrogametes and micro-
gametes and gametocytes are present. Binucleate individuals without
evidence of recent division occur (pl. 4, fig. 36), simulating zygotes,
and similar associations are found in eysts (pl. 4, fig. 26). There is,
however, no evidence of maturation divisions leading to gamete forma-
tion, no sexual behavior detected, and no evidence of the fusion of
gametie nuclei. In the absence of such evidence any conclusions as
to the possibility of sexual reproduction in this organism must be held
in abeyance.

36 University of California Publications in Zoology — [Vou. 20
Y 0] J

ENCYSTMENT

Associated with the vegetative and fission stages of Trichomitus
in a few hosts but not all, we have found many small individuals
(pl. 4, figs. 24, 29-33) in which binary fission is occurring and in
which there is a tendeney for the body to round up into a spheroidal
or ellipsoidal mass. These small sizes may result from rapid fission
without compensating growth or from plasmotomy of part of the eyto-
plasm. In these same hosts occur also numerous ellipsoidal cysts
about 13 by 20 with a deeply stained network with thickenings at
the nodes spread over the surface (pl. 4, figs. 26, 27). The cyst wall
is double and the network is due to the accumulations of some stainable
substance between the walls. Within the cyst is a single individual
(pl. 4, fig. 31) with a very long parabasal and undulating membrane
making nearly two complete coils, such as might result from the coiling
up of an individual with abnormally large neuromotor system (pl. 4,
fig. 30). In other cases two such individuals (pl. 4, fig. 26) are found
within the cyst. This might result from eneystment after or during
mitosis but prior to plasmotomy. Such cysts may facilitate the carry-
ing over of infection from one individual host to another. They have
all the indications of being resistant stages.

RELATIONSHIPS

This is a species of Trichomitus, a genus founded by one of us
(Swezy, 1915b) for the reception of a minute and simple trichomonad
from amphibians. In its vegetative phases the form here described
has the morphological features of Trichomitus parvus Swezy, namely,
three anterior flagella, undulating membrane, parabasal, and no axo-
style. It differs greatly in size, in the massive development of the
parabasal, and at mitosis in the distinct separation of centrosome and
blepharoplast. Binary and multiple fission were followed in the species
from amphibians but no trace of such separation was detected.

Such a difference as this might justify generic separation but it
might be impracticable to apply it in future diagnosis of any species
of the genus which may come to light. The difference is, however, of
such morphological import as to justify subgeneric separation. We
accordingly assign it to

Trichomitopsis subgen. nov.
Trichomitus with centrosome separated from blepharoplast at
mitosis. Type species Trichomitus termitidis sp. nov. from
Termopsis angusticollis Walker.

1919] Kofoid-Swezy: Trichomitus termitidis 37

SUMMARY

1. Trichomitus termitidis sp. nov. occurs in the intestinal tract of
Termopsis angusticollis. It is apparently not pathological to its host,
is never attached to the wall and feeds on the débris of the intestinal
contents.

2. It has a highly developed neuromotor system with parabasal
body, undulating membrane, centroblepharoplast and flagella attached
by a rhizoplast to the nucleus.

3. Binary fission occurs frequently. Mitosis is marked by the de-
velopment of a large paradesmose following the separation of the
centrosome from the blepharoplast. One schizont retains the old para-
basal body and membrane, while new ones are formed for the other.

4. Multiple fission results in the formation of an eight-zooid soma-
tella followed by plasmotomy.

5. Owing to the great differences in the process of mitosis between
Trichomitus parvus and the new species, 7’. termitidis, subgenerie dis-
tinetion is given to the latter, as we assign it to the new subgenus
Trichomitopsis.

Zoological Laboratory, University of California,
Berkeley, California.
Transmitted September 6, 1918,

LITERATURE CITED

Koro, C. A.
1917. The biological and medical significance of the intestinal flagellates.
Proc. Second Pan Amer. Sci. Cong., 1915-16, 10, 546-565.

Koroip, C. A., and CHRISTIANSEN, E. B.
1915. On binary and multiple fission in Giardia muris (Grassi). Univ. Calif.
Publ. Zool., 16, 30-54, pls. 5-8, 1 fig. in text.

Korom, C. A., and Swezy, O.
1915. Mitosis and multiple fission in trichomonad flagellates. Proc. Amer.
Acad. Arts Sei., 51, 289-378, pls. 1-8, 7 figs. in text.
Swezy, O.
1915a. Binary and multiple fission in Hexamitus. Univ. Calif. Publ. Zool.,
16, 71-88, pls. 9-11.
1915b. On a new trichomonad flagellate, Trichomitus parvus, from the intes-
tine of amphibians. Jbid., 16, 89-94, pl. 12.

EXPLANATION OF PLATES

All figures of Trichomitus termitidis sp. noy., from material stained with iron
alum haematoxylin. Magnification 625, unless otherwise stated.

PLATE 3
Fig. 1. Trophozoite in early prophase of division with nuclear rhizoplast
divided.

Fig. 2. Isolated neuromotor apparatus of the same stage with nucleus
attached.

Fig. 3. Sagittal section of the parabasal showing the outer deeply staining
shell and the inner core. X 1250.

Fig. 3a. Cross-section of the same. X 1250.

Fig. 4. Isolated undulating membrane with darkly staining marginal
flagellum. X 1250.

Fig. 5. Early prophase with outgrowing of new parabasal body.

Fig. 6-10. Early prophase stages showing gradual condensation of chro-
matin into definite chromosomes. New parabasals, undulating membranes and
flagella complete.

Fig. 11. Isolated neuromotor system in prophase stage of division.

Figs. 12, 13. Spindle forming with chromosomes attached to it by the angle
of the V. X 1200.

Fig. 14. Prophase. Note relative lengths of parabasal bodies.
Figs. 15, 17. Separation of chromosomes into two groups. X 1250.
Figs. 16, 18-20. Anaphase with paradesmose elongating. X 1250.

Fig. 21. Beginning of constriction of the nuclear membrane. X 1250.

[38]

at

Fig.
length o
Fig.
Fig.
Fig.
Fig.
x 1250.
Fig.
Fig.

2.

27.

28.

of flagella,

Fig.

Fig.

29.
30.

parabasal.

Fig.

Fig.
Fig.
Fig.

Fig.
Fig.

2

36.

PLATE 4

Late anaphase, showing formation of new cytostomes. Note

2

f paradesmose.
23.

24,
25.
26.

Multiple fission with beginning of the third division of nucleus.
Small individual apparently about to encyst.

Multiple fission with nuclei in preparation for second division.
Encysted form which has divided. Note structure of eyst wall.

Encysted form showing outer surface of cyst only. X 1250.

Somatella of six zooids formed by multiple fission, Full complement
parabasals, undulating membranes and nuclei.

Dividing individuals just escaped from cyst.

Product of multiple fission. Note relatively enormous length of

Encysted form with single nucleus.

Individual following excystment.

Small form dividing. Note relative lengths of parabasals.
Late anaphase of binary fission.

Second division of nucleus in multiple fission.

Telophase of binary fission.

[ 40 ]

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UNIVERSITY OF CALIFORNIA PUBLICATIONS
IN

ZOOLOGY
Vol. 20, No. 3, pp. 41-98, plates 5-12, 4 figures in text July 14, 1919

STUDIES ON THE PARASITES OF THE TERM-
ITES III. ON TRICHONYMPHA /

CAMPANULA SP. NOV. >
BY
CHARLES ATWOOD KOFOID anp OLIVE SWEZY 33 W/
“sonlan De”
CONTENTS
PAGE
TOTS, cee eee SEP ea 42
(QyeceqemnaeSnats) WANE ENCARTA GAIN S| pe a 43
BUS EN 05 ue oe aut
SPOS WL [SEAT ee ce EE SESS 45
DN [COU EAK Oe T OY HO hE AS] 1 Ce eS ee 47
TBAT VTA: FEMALES) espera ee ee 49
ESATERINOD, TENOKETENS ooo oe Ne hee oe se Gna iet pee Se at ener So Ree PEEP eo Ag
Locomotor organelles 50
Oblique fibers —... 52
Centroblepharoplast - . 53
PNA eS TUE SO rece ee Se ee . d4
ARTAMSVELSE HI ONCM CS) maa acetate econ eee csecencecacsecescneteseen sae ==) 00
SETI lO pea SRN CRS UT CLD © eee nen ne ce eae once 56
Mom pai burdiinrallsanay Om CM CS aces as ceca cn NE cs nase so oc nccecenaneesaseceee 57
TROUT) noe een ene eee ct Eee Ee Ae 57
(Che see ral) GINS CLES OT ee ee ee EE aR 59
TESTO SSE, ee SS Fe ea ge ee 61
Division of the neuromotor apparatus and ectoplasmic¢ structures -........... 62
INETHIOYSISL eet a a a 65
Prophase ..... 65
Metaphase - 68
Anaphase 68
Telophase G9)
DY ASCUISS OTOL NL GOSUS jecsces tse ere eaes ee ra en a ae See ere eas Nc alr 69
TRUE FO EUO Rox wecteeectrecestcce beeen Rr et 77
PSST NRT TY ATG oe ea acc Sos Sn ww cn waa nee Faec tsb sac besesseunet nassonseeevieutbecctepedceueitusosass 81
TORMENT) CAD ees cc sete a ce a re 82

42 University of California Publications in Zoology [ Von. 20

INTRODUCTION

Among the instances of parasitism by the Protozoa in the alimen-
tary tracts of the Metazoa, none exceeds that found in the termites
as to the relative volume of the invading parasites, the diversity of
organization found among them, and the degree of specialization
which they attain. In the vaneuard of this evolutionary development
stands the genus Trichonympha discovered by that pioneer American
investigator, Dr. Joseph Leidy, who in 1877 and 1881 first revealed
this teeming menagerie of termite parasites to scientific view.

The extent of specialization attained by Trichonympha and its
allies has obscured their relationships, allied them at first with the
ciliates, and obliterated their true affinities with the flagellates. The
purpose of the present paper is in part to place the flagellate origin
and relationships of the Trichonymphidae on a firm cytological foun-
dation. It is also our aim to set forth the most complicated neuro-
motor system thus far known among the Protozoa, to analyse its
elements, relate them to the elaborate motor activities of the indi-
vidual, and trace their behavior during the mitosis of this highly
specialized cell.

The degree of structural complexity, the extent of the coordinated
mechanism, the number of its constituent elements, and the striking
similarity of their interrelations in Trichonympha to those obtaiming
in multicellular organisms, is illuminating as to the biological sig-
nificance of cellular organization. This organism is a single cell, with
one nucleus, yet it has attained a degree of structural complexity and
functional diversity in respect to one organ system comparable almost
with that of its host and surpassing that of many of the lower Meta-
zoa. The multicellular state is plainly not an essential condition for
evolutionary specialization and functional efficiency, except as it places
limits on the size of organisms and on developmental processes arising
therefrom. The differentiation of organs within the confines of a
single cell is here accomplished with a perfection comparable with that
where the organ is a complex of diverse cells instead of one of many
parts within a single cell. The organism and not its cells is the
fundamental basis of differentiation.

1919 | Kofoid-Swezy: Trichonympha campanula 43

OCCURRENCE AND ACTIVITIES

The posterior and mid-regions of the intestinal tract of Termopsis
angusticollis Walker are usually found to be greatly distended, often
filling the entire cavity of the body. “It is in these regions that Tricho-
nympha campanula is found in great abundance, filling the entire
lumen of the canal but never attached to its walls. When other
flagellates, as Streblomastix strix and two other smaller forms, are
present with it in any numbers, these are found occupying the region
near the walls, with Trichonympha filling the central portion of the
canal.

The intestinal contents resemble a thick milky fluid, the great con-
sistency of which is due to the vast numbers of protozoans which it
contains, along with minute débris of woody particles. Through this
mass the trichonymphs move with considerable rapidity, using the
mobile anterior portion of the body-to clear a pathway. This is done
by quick sidewise movements, such as those shown in figure C, 2,
bending the anterior cone first to one side and then to the other
without halting in its progress. Its path is usually straight ahead
with little or no rotation of the body on its longitudinal axis.

The group of long anterior flagella seems to be its main propelling
organelle. These are thrown forward and sidewise somewhat as other
flagellates use their flagella, with, however, less of the forward motion
than is usual with anteriorly attached flagella. The flagella or cila
on the remainder of the body are uniformly directed posteriorly.
These are found to have a characteristic motion, both during loco-
motion of the organism and when it is at “‘rest.’” Waves of contrac-
tion pass constantly from the anterior end backward to the tips of
the flagella, affecting all on any given plane alike. The motion of the
eroup of longer, anterior flagella, particularly when they are directed
posteriorly, as is frequently the ease, may coincide with these waves
of contraction, which then include all the ciliary covering of the body.
These waves continue during observation on the slide until the round-
ing up of the body in the culture fluid prior to dissolution.

These flagellates are extremely susceptible to environmental
changes. When placed in eulture slides with tap water, distilled
water, normal salt solution, or Ringer’s fluid, the results have been
disastrous in a very short time, varying from a few minutes to
half an hour. The body rounds up with considerable distension,
becomes transparent with an almost complete loss of organelles, the
nucleus only remaining visible until the final dissolution of the body.

44 University of California Publications in Zoology [ Vox. 20

The flagella continue moving as long as they are visible at the begin-
ning of this process but soon disappear.

Dilute egg albumen has been found to be the most satisfactory
culture medium but even with this these flagellates have been kept
alive only a few hours, with the rounding-up process soon visible in
a majority of the forms.

As has been earlier noted for trichonomad flagellates (Kofoid and
Swezy, 1915), binary fission in Trichonympha campanula is cyclic in
its occurrence, being found abundantly in occasional individual hosts.

Tn order to secure all stages of the division eycle, preparations were
made every day continuously for thirty days, the number of individual
hosts used each time varying from three to ten or more. For several
days also these preparations were made at two-hour intervals during
the day. In this way we have been able to secure a fairly complete
series of figures of all stages of the mitotic process. A few isolated
division forms may be present in almost any host, but in general it has
been found that where more than these occur in a single host, that
from one-third to one-half or even more of the individuals of a single
parasitic species observed, will present some signs of division and
usually of a single stage of mitosis. This is especially true of the
early prophase stages which, on the whole, have been most abundant
in our material. A few slides have been found, however, on which
almost all stages of the division process may be found. This seems
to be the exception and not the rule.

MORPHOLOGY

Every observer of the parasites of the digestive tract of the
termites is amazed, on his first glance at its seething contents, at the
locomotor activities displayed by its constituent organisms. Foremost
among these in constancy, agility, and variety of its movements is
Trichonympha. It is signficeant that Leidy (1877) used the specific
name agilis for the first trichonymph discovered. This capacity for
motor activities is based on structural features of corresponding com-
plexity. Hence it is that any discussion of the morphology of this
animal is mainly occupied with the neuromotor system. The only
other differentiated structure found in the body is the nucleus. There
is no apparent mouth, no exeretory system, no food-taking organs.
The food vacuoles are temporary in a seemingly undifferentiated
endoplasm. The structural specialization of this parasite thus affects
mainly one organ system only, the neuromotor system.

1919] Kofoid-Swezy: Trichonympha campanula 45

SHAPE AND SIZE
The shape of the body is companulate with the posterior end
broadly rounded (fig. B), almost spheroidal, while anteriorly it is a
tapering, slightly convex cone of 20°. The anterior half of the body

Fig. A. Trichonympha campanula, drawn from life, showing metabolic
changes in the posterior portion of the body of the normal trophozoite. X 186,
has a transdiameter of about one-half or less than that of the posterior
part. It is contracted to a slender acuminate point at the anterior end
which is surmounted by a rounded, transparent, caplike structure
(fig. B, oper.) which we eall the operculum. This covers a shallow
depression at the base of which is a small pit surrounded by a darkly
staining ring (fig. C, 5, 9). The base of this pitlike depression may

46 University of California Publications in Zoology [ VoL. 20

be protruded a short distance (fig. C, 1, 2, 8) or may be deeply with-
drawn (fig. C, 5). The membrane forming the eaplike covering is
thin, remarkably transparent, and does not stain with any of the
reagents used in the preparations of the material.

The body is radially symmetrical with graceful outlines. All
trace of the characteristic asymmetry of the flagellates is lost. Its
length is two or three times its greatest transdiameter which is in the
posterior third of the body. The shape of the body may vary consid-
erably from the campanulate form. The early prophases of division
are marked by a characteristic rounding up of the entire body (pl. 7,
fig. 31). The variation in shape in the ordinary trophozoite stage are
less marked. Some of these are shown in figure A. The anterior half
of the body is more stable in its outlines than is the posterior half
and shows fewer metabolic changes. It may become thickened, losing
the graceful curved lines, becoming conical in shape (fig. A, 4), or
with a transdiameter equal to that of the posterior part (fig. A, 1).

It is in the posterior half of the body that the most striking varia-
tions are found. This is covered only with a thin periplast in contrast
with the thick ectoplasm of the anterior part (fig. B). As a result of
this condition it is the most delicate part and the one which most
frequently shows the effects of injury in the manipulation of the
material and its preparation for microscopical examination. In addi-
tion to this individuals are sometimes found which show metabolic
changes in the posterior part of the organism, which are not the
result of injury but may possibly be due to some chemical changes in
the surrounding medium. These changes are shown in figures A, 2-4.

The posterior part may be drawn out into a slender cylinder
with a consequent shortening of its transdiameter, and may some-
times equal or eyen exceed the length of the remainder of the body
(fig. A, 2). Individuals thus affected have been observed in living
material and appear normal in other respects.

In size this is the largest known species in the genus T'richo-
nympha. In general length it varies from 250 to 460u and in width
from 110 to 200un. The average length is about 350p. A frequency
curve plotted from 121 measurements had its mode at 360, and a slight
right-hand skew. Trichonympha agilis, as figured by Leidy (1881)
and Porter (1897), varies in length from 60 to about 100y, and for
T. hertwigi Hartmann (1910) gives the length as between 260 and
330u. The latter species equals in length many individuals of 7.
campanula, but falls short of the larger specimens, as well as having

1919] Kofoid—Swezy: Trichonympha campanula 47

a more slender body, its width varying from 40 to 60u. In shape
T. campanula differs but little from 7. agilis, the latter having a
somewhat broader transdiameter anteriorly with a noticeable con-
striction near the middle of the body or slightly anterior to it. This
constriction is entirely absent in 7. campanula. Its divergence from
T. hertwigi is still more pronounced, varying greatly in proportions
from any of the three distinct forms which Hartmann (1910) has
described under that name, being more slender anteriorly and broader
posteriorly.

NevurRoMoTOR SYSTEM

Included in this system is the entire set of fibers concerned in
movement of all parts of the body, both in the ectoplasm and the
endoplasm, the external coat of cilia, the three zones of flagella, and
the centroblepharoplast from which the other fibrils radiate, take
their origin, or with which they have some more or less direct con-
nection. In view of its many elements, their diversification into
various groups, and their structural coordination we apply the con-
ception of an organ system to their complex and designate it as the
neuromotor system.

This system consists of two distinct parts, lying respectively in the
ectoplasm and endoplasm, and differing in their staining reactions
and in their relationships to the process of mitosis. The first part is
composed of the flagella, their basal granules and connections, the
anastomosing sheet of oblique fibers, the centroblepharoplast and
paradesmose. It appears to be more highly specialized as conductile
organelles, although the flagella may be sensory and are certainly
contractile. This part of the system shares in mitosis, forms the polar
centrosomes, the structures radiating therefrom, and the paradesmose.
These organs lie in or project from the ectoplasm. The other part
consists of two antagonistic sets of fibers, the outer circular and
inner longitudinal myonemes. These lie against if not in the endo-
plasm, and take no direct part in mitosis. Their connections with
the centroblepharoplast are problematical. They are primarily con-
tractile.

The use of the term neuromotor to designate the system is based
on morphological grounds and observations on the behavior of the
animal. It responds to stimuli, contracts, and moves as it might be
expected to do with such a structurally coérdinated mechanism. It
must, however, be evident that the distinction between strictly neural

48 University of California Publications in Zoology [ Vou. 20

and strictly motor functions can not be sharply drawn and that the
two functions are in all probability not wholly separated and carried
on by distinet organs, but are rather in most, if not all parts of the
system, served to some extent by the same structures. It is possible
that the basal ciliary lines (b. cil., fig. B) are wholly conductile rather
than motor, and that the centroblepharoplast has little if any motor
function. It is likewise not improbable that the longitudinal and
transverse myonemes are mainly motor and that the flagella and cilia
are sensory, conductile, and motor, while the enveloping undiffer-
entiated cytoplasm in which all these structures lie, except cilia and
flagella, has doubtless preserved some of its primitive neuromotor
capacities. The oblique fibers are by their staining reaction allied
to the neural system and function rather than to the motor. These
overlapping conditions, however, do not preclude the use of the term
neuromotor system to designate the complex integrated fibrillar
structure of Trichonympha.

The discussion of this system will for convenience include also that
of the surrounding cytoplasmic structures, such as the surface ridges,
and the alveolar layer, although these are not strictly parts of the
system.

The neuromotor system (fig. B) may be divided into two very
unequal parts according to its location in the two fundamental sub-
divisions of the cytoplasm, the ectoplasmic and endoplasmic. The
ectoplasmie portion consists of the anteriorly located centroble-
pharoplast (centrobleph.) from which spring the anterior flagella
(ant. fl.) and from which radiate posteriorly the longitudinal basal
ciliary lines (b. cil.) which give rise laterally to the lateral cilia
(lat. cil.) and posteriorly to the posterior cilia (post. cil.). These
lines are in the axes of the longitudinal ridges (surf. rdg.) which cover
the surface above the equator of the posterior region. From the
centroblepharoplast arise also the spirally directed, opposing sets of
oblique fibers (obl. f.).

One set of fibers, the circular transverse myonemes (tr. my.), lies
in the innermost zone of the ectoplasm, or outermost zone of endo-
plasm. Their course is such that their connection, if any exists, with
the centroblepharoplast can not be traced. In the peripheral layer
of endoplasm the stout longitudinal myonemes run posteriorly from
the centroblepharoplast to the margin of the thick ectoplasmie zone.
No trace of any other part of the neuromotor apparatus can be found
within the labile endoplasm. The juxtaposition of nucleus and cen-

1919] Kofoid—Swezy: Trichonympha campanula 49

troblepharoplast at mitosis is suggestive of some structural relation
such as is represented by the nuclear rhizoplast in the Polymastigina,
but no rhizoplast has as yet been found by us in Trichonympha.

ECTOPLASMIC STRUCTURES

A superficial examination of T. campanula reveals the fact that
the anterior two-thirds of the body is marked off by a thick ectoplasm.
This is thickest anteriorly and as its extreme posterior limit becomes
thin, disappearing distally in the frail pellicle of the posterior region
of the body (fig. B; pl. 5, fig. 6). Under the low powers of the micro-
scope this appears as a nearly clear zone in the living flagellate, dis-
tinetly marked off from the granular endoplasm. Higher magnifica-
tions bring out the fact that it is divided into three distinct zones
which are traversed by fine lines and that one layer or zone contains
alveoli closely massed together. These are, (1) the outer projecting
ridge, (2) the alveolar layer, and (3) the inner ectoplasmic layer
(fig. B; pl. 12, fig. 80). In stained material the structure of the ecto-
plasmie region can be more clearly differentiated, and reveals a high
degree of complexity. These regions will now be described, beginning
with the outer zone and proceeding inward.

Surrace Ripges: The outer surface of the body is raised in rela-
tively high, narrow, longitudinal ridges (fig. B, surf. rdg.) which are
best observed in a transverse section (pl. 5, fig. 4; pl. 12, fig. 80).
The cilia or flagella which cover the surface of the body spring from
the erest of each ridge. These ridges extend from the anterior end
posteriorly to the equator. The spaces separating them become nar-
rower anteriorly and the ridges fewer in number, finally converging
around the base of the operculum-like structure at the extreme
anterior tip of the body (pl. 5, fig. 1). Posteriorly they fade out
at the point where the differentiated ectoplasm and flagella disappear,
giving place to the thin periplast of that region of the body. The
crests, or tops, of the ridges are narrow or knifelike. The base of each
ridge is usually somewhat compressed (pl. 5, fig. 4). Farther
posteriorly the base becomes broader (pl. 12, fig. 80) and the ridges
disappear so that the entire surface becomes smooth. The ridges have
been well illustrated by Porter (1897, p. 2, fig. 17) for 7. agilis, cross-
sections of the body being nearly identical in that species and 7.
campanula. Their course is longitudinal, not spiral, and is not
changed to a spiral on contraction.

50 University of Califorma Publications in Zoology [ VoL. 20

Locomotor ORGANELLES: The surface of the anterior two-thirds
or more of the body is covered with cilia or flagella. Both terms are
not inappropriate here, since three distinct lengths of this hairlike
covering is found in this species, in three distinet zones or locations.
At the anterior end of the body is a zone of long, threadlike flagella
(fig. B, ant. fl.; pl. 5, fig. 1), that may have a length equal to one-
third that of the body. These arise from the narrow portion imme-
diately behind the operculum, directly from the centroblepharoplast
or its immediate branches. Posterior to this group and extending
backward for slightly more than half the length of the body is a thick
covering of short, cilia-like hairs (fig. B, cil.; pl. 5, fig. 1) of uniform
length. These have a leneth of one-fourth to one-fifth or less of the
anterior flagella. In apparent texture and thickness they seem to be
similar to the longer flagella of both the anterior and posterior regions,
differmg from them only in length.

The third zone of long flagella is found between the distal limits
of the short cilia and the posterior end of the ectoplasmic differentia-
tion of the surface of the body (fig. B, post. fl.; pl. 5, fig. 1). These
are considerably longer than the flagella of the anterior zone, often
having a length equal to that of the entire body (fig. A). They extend
posteriorly, trailing after the body when in motion, and often inter-
secting (fig. A, 1) or twisting around in a loose spiral (figs. A, 2, 4).

The anterior group of flagella seems to be the chief means of
locomotion. When the organism is at rest constant vibratory waves
pass through the entire coating of cilia and flagella, beginning at the
anterior end and passing posteriorly to the tip of the longest flagella,
_ but dying out in intensity distally. These vibrations continue in the
cilia and posterior flagella when the flagellate is in motion but the rate
of movement of the anterior flagella is greatly accelerated. They are
thrown out in longer and stronger vibrations, resulting in a rapid
movement forward of the body.

Flagella intermediate in length between these three groups are not
infrequent (pl. 5, fig. 3) near the margin of the areas. In 7. agilis,
as figured by both Leidy (1881) and Porter (1897), the entire area
of short cilia is replaced by flagella intermediate in length between
the short anterior group and the longer posterior flagella. This results
in a thick coat of long flagella for nearly the entire body in that
species.

Each flagellum or cilium in 7. campanula arises from a minute
basal granule below the ridges of the surface of the body (pl. 12,

SSS =

Z- fi
We LAs
Bail
Sy i
Be ee ee ee = i

_--surf..rdg.

| eee ts

Fig. B. Diagrammatic figure of T'richonympha campanula. Sections of the body show
the structures found at different levels. Surface ridges form the outer layer with their
rows of flagella; beneath are successively the oblique fibers, alveolar layer and trans-
verse myonemes. In the endoplasm are the longitudinal myonemes.

Abbreviations: alv. l., alveolar layer; ant. fl., anterior zone of flagella; bas. gr., basal
granules; centrobleph., centroblepharoplast; ect., eetoplasm; end., endoplasm; f.b., food
bodies; heterchr., heterochromosome; lat. fl., lateral zone of flagella; long. my., longitudinal
myonemes; n., nucleus; obl. f., oblique fibers; ope?., operulum; per., periplast; post. fi.,
posterior zone of flagella; surf. rdg., surface ridges; tr. my., transverse myonemes. X 600.

52 University of California Publications in Zoology [ Von. 20

fig. 80). Extending out from each granule is a slender thread or
rhizoplast, the basal part of the flagellum. This passes up through
the ridge and leaves the crest as the single flagellum. The flagella
are placed closely together so that the rows of basal granules form
continuous lines extending from the anterior end of the organism
posteriorly (fig. B, b. gr.). No connection could be found between
the separate granules in the same rows or in successive rows, except
that afforded by the oblique fibers which will be described below. No
differences could be detected between the basal portions of the three
different groups of flagella.

OBLiQuE Frsers: The most superficial examination of this species
of Trichonympha reveals a wonderful development of myoneme-like
fibers which cross and intercross in an intricate pattern over the entire
two-thirds or more of the surface of the body. A closer examination
brings to light three different sets of these fibers lying at different
levels, and all more or less visible in the living organism. The outer-
most layer of these is composed of oblique myoneme-like fibers and
will be described first.

These lie immediately below the surface ridges and cover the same
portions of the body as do the other ectoplasmie structures (fig. B,
obl. f.). On plate 12, figures 79, 84, and 85, these fibers are shown
in dividing forms with the other ectoplasmie structures omitted. The
number of fibers or separate strands is somewhat reduced in the
drawing to obtain clearness, both in these figures and in others which
appear elsewhere on the plates.

The oblique fibers arise from the darkly staining masses, the cen-
troblepharoplast (fig. B, centrobleph.; pl. 6, figs. 7, 10), at the base
of the short, narrowed anterior part of the body. Figure 7, plate 6,
gives a vertical view of this region in an individual which had become
rounded up, preparatory to division. This condition results in a
spreading apart of the fibers, giving a clearer picture of these
structures than may be obtained in the ordinary trophozoite. Thick
strands stream out in all directions from the irregular borders of the
centroblepharoplast, which soon break up into small threadlike
branches. At first longitudinal as they leave their place of origin,
this direction is lost with the first branching, the threads extending
obliquely and crossing and intererossing with one another in a com-
plex, anastomosing network. Each intersection of two branches seems
to anastomose completely so that the course of a single branch is soon
lost. In addition to this network of fibers, very slender, minute

1919] Kofoid—Swezy: Trichonympha campanula 53

branches are being continuously given off which pass to the surface
along the ridges (pl. 6, fig. 9). The basal granules of the flagella
seem to be connected with these minute branches, as in optical section
the flagella are seen to be continuous with the branches that are given
off by the oblique fibers. In cross-sections of the body this connection
could not be followed, owing apparently to decolorization in the
staining methods used.

The structure of these fibers is scarcely granular and presents a
more nearly homogenous appearance than is the case with either the
transverse or the longitudinal myonemes. In the living organism they
may be seen as very slender refractive lines. In preparations stained
with iron haematoxylin they are very distinct as greyish lines, darker
in the anterior region. Owing to their affinity for this stain a con-
siderable degree of decolorization is required before they lose the
black color. With Mallory’s connective-tissue stain these fibers
usually, though not invariably, show a clear red color similar to that
found in the neuromotor apparatus of ciliates.

CENTROBLEPHAROPLAST: Intimately related to these fibers is an-
other structure at the anterior end of the body which we have called
the centroblepharoplast, an organelle homologous with the blepharo-
plast or centroblepharoplast found in other flagellates (Kofoid and
Swezy, 1915), and suggestively like the motorium in ciliates (Sharp,
1914, Yocom, 1918), although that organ has no proven relations to
mitosis as has this structure. Owing to the staining reactions and
apparent structure of the oblique fibers first described, it seems prob-
able that they are, of all the neuromotor apparatus, most intimately
associated with the centroblepharoplast as well as with the flagella.

The anterior end of the body becomes narrow, sometimes with a
slightly constricted appearance (pl. 6, fig. 6), but usually subconical
in outline (pl. 6, figs. 1, 2). The ectoplasmie zone is here much
thicker than in other regions of the body. In the center of the ter-
minal cone is a slender cone-shaped structure composed of several
strands of darkly staining material surrounding a central core which
does not stain (fig. B, core; pl. 6, figs. 1, 6). This reaches the tip
of the cone, where it may terminate in two ways. The first of these
presents a ringlike appearance in vertical view, with the central core
of endoplasm showing in the center as a light area (pl. 6, figs. 3, 7).
This is a circular band around the core, to which the radiating
strands of the oblique fibers are attached at their anterior ends. The
second method is generally found in division stages and shows the

54 University of California Publications in Zoology [ VoL. 20

sides with their darkly staining strands drawn out beyond the central
core and terminating in a point (pl. 6, fig. 10) with a complete obliter-
ation of the core. Some curious modifications of this are sometimes
seen in which a considerable amount of the darkly staining material
has accumulated at the tip and is thrown out sideways into hornlike
processes (pl. 6, fig. 9; pl. 8, fig. 34; fig. C, 6).

This axial structure extends backward for a short distance from
the apex of the cone, tubular in shape or slightly larger posteriorly
(pl. 5, figs. 1, 6). Near its base the separate strands, which are
usually quite distinct, become enlarged into broad, irregular, darkly
staining masses (fig. C) which appear to fray out around their distal
margins. These may sometimes be separated (pl. 6, figs. 9, 13) into
ropelike and brushlike masses, or an almost continuous band may be
formed around the base of the tubular part of the neuromotor appa-
ratus (pl. 6, fig. 7). Distally these masses break up into the oblique
fibers which have been described above. The two structures seem to
be continuous and composed of the same material. They connect the
centroblepharoplast with the external motor organs, the flagella, by
their intimate association with their basal granules.

ALVEOLAR Layer: Closely filling the same regions traversed by the
oblique fibers is a layer of alveoli (fig. B, alv.; pl. 7, fig. 24). This
is continuous in extent with the other ectoplasmic structures, but
along the posterior border it may sometimes seem to merge into the
larger endoplasmic alveoli which often fill the posterior portion of the
body (pl. 5, fig. 6).

In cross-section the alveoli seem fairly regular (pl. 5, fig. 4), and
are placed closely together. In surface view they appear less regular
with larger alveoli in the posterior region. In the rounded dividing
forms this layer is often very striking, the alevoli having the appear-
ance of clear globules much larger than those usually present in the
normal vegetative forms (pl. 7, fig. 24; pl. 12, fig. 76), possibly as a
result of pressure. The intra-alveolar spaces are traversed by
branches from the oblique fibers (pl. 6, fig. 9) and by the basal
fibrils of the flagella, the basal granules lying in the zone immedi-
ately beneath the alveoli, which is also occupied by the major portion
of the oblique fibers (pl. 5, fig. 4). The zone lying between the
alveolar layer and the surface is also traversed by these basal fibrils
which give to that region a striated appearance (pl. 5, fig. 5). Optical
sections from the posterior border of the ectoplasm in a dividing
trichonymph (pl. 12, fig. 80) show the basal granules lying close

ph
1

)

1919] Kofoid-Swezy: Trichonympha campanula

to the inner border of the alveoli. The outer zone between the alveoli
and the surface ridges has here almost disappeared, the alveoli abut-
ting on the bases of the ridges, from which they are rather widely
separated anteriorly (pl. 5, fig. 4).

A distinet alveolar zone lying in the ectoplasm is a ciliate rather
than a flagellate characteristic. It is not, however, entirely unknown

Fig. C. Trichonympha campanula. Sketches of the anterior end of the body
to show its great mobility. Figure 2 illustrates the successive movements used in
moving forward through the thick intestinal contents. X 200.

in the latter group. In the subgenus Pachydiniwn of the genus
Gymnodinium, among the dinoflagellates, the different species are
provided with a differentiated ectoplasm the outer layer of which
consists of rather large alveoli (Kofoid and Swezy, 1919d).
TRANSVERSE MyonemEs: The innermost layer of ectoplasm, be-
neath the alveolar and oblique fiber zone, is occupied by the transverse
myonemes (fig. B, tr. my.). These differ in structure from the oblique

56 University of Califorma Publications in Zoology [ Vox. 20

fibers, being granular in appearance as are the longitudinal ones.
They are circular, passing around the body in nearly transverse
planes, and may be found throughout the entire region of thickened
ectoplasm. Each myoneme is a slender, granular band, not more than
one or two microns in thickness. These he in parallel rows which give
a finely striate appearance to the innermost borders of the ectoplasm.

These transverse myonemes are only faintly outlined in the stained
specimens and are usually obscured by the wealth of oblique fibers
which overlie them.

ENDOPLASMIC STRUCTURES

In the living organism the body is marked off into three distinct
regions, the outer clear ectoplasmie zone, the anterior endoplasmic
region of rather dense homogeneous appearance, and the posterior
endoplasm filled with coarse alveoli and food particles. The two endo-
plasmic regions are always more or less clearly differentiated, but
without the granular layer which separates these two portions of the
body in Trichonympha agilis (Porter, 1897). The transition from
one region to the other is distinguished by a change of structure rather
than by a delimiting layer of differentiated ectoplasm.

The anterior region extends from the anterior end backward for
about two-thirds or slightly more of the total length of the body
(pl. 5, fig. 6). Proximally a slender plug or core of endoplasm
extends out through the narrow neck formed by the centroblepharo-
plast to the periphery of the body. This region of endoplasm is
coarsely granular without distinct alveoli. The distal portion, extend-
ing up to and around the nucleus, is usually more dense, often taking
a darker stain with iron haematoxylin, showing greater metabolic
activity than in the anterior portion. It is also frequently filled with
minute flecks of a dark color (pl. 5, fig. 6; pl. 7, fig. 23). These are
often present in the posterior region of endoplasm and are possibly
remnants of bacteria which have been ingested with other food particles.
The possibility of their being chromidia is not excluded, however.

The posterior portion of endoplasm is filled with large alveolar
spaces with the interstices occupied by coarsely granular plasma
(pl. 5, fig. 6; pl. 7, fig. 28). This part of the body usually contains
an abundance of food particles. As most of the termites examined
were obtained from decayed wood, this was the only foreign material
found in the intestine. It also seems to form at least a part of the

“]

1919] Kofoid-Swezy: Trichonympha campanula 5

food of the trichonymphs, since few specimens were noted which did
not contain small particles of wood in the endoplasm. The entire
posterior portion of the body is often found densely filled with this
material which is light in color in the unstained preparations. The
particles seem to be confined exclusively to the posterior region of
the endoplasm.

LonerrupiInaL Myonemes: The longitudinal myonemes are found
in the outer layer of endoplasm (fig. B, long. my.), a short distance
below the inner layer of ectoplasm containing the transverse myonemes.
These extend from the base of the tubular portion of the neuromotor
apparatus posteriorly to near the end of the differentiated ectoplasmic
region (fig. B, long. my.). They are subparallel for most of their
length, spreading apart posteriorly and converging anteriorly until
they meet in the region of the lobes of the centroblepharoplast. . The
connection between these myonemes and this portion of the neuro-
motor system is one difficult to determine. The myonemes stain only
faintly or not at all in the ordinary smear preparations. In sections
they may be seen as dark granular masses near the outer border of
endoplasm. It is in the living flagellates that they are best observed.
Here they may be seen as slender bands of homogeneous appearance,
somewhat refractive, and extending in an anteroposterior direction
and vibrating with the movements of the body. In stained material
they appear as strands of rather coarse granules which are somewhat
denser and slightly darker than the surrounding endoplasm. Their
structure seems to be entirely changed by the processes of fixing and
staining.

The longitudinal myonemes seem to be the chief organelles con-
cerned in flexions of the anterior end of the body. This is extremely
mobile, turning easily from side to side (fig. C, 2), sometimes reaching
backward upon itself until it touches the posterior portion of the body.
With such movements in the living organism somewhat slowed down,
these strands may be seen to sway slightly with each movement.

Nucuevus: The nucleus is a rotund ellipsoid lying in the middle
third of the body. Its position varies from one-third of the total
length of the body from the posterior end (pl. 7, fig. 23) to about
the mid-region (pl. 5, fig. 6). In a cross-section of the body at its
level it lies near the center of the plane (pl. 5, fig. 4). A thin, dis-
tinct nuclear membrane separates it from the surrounding plasma. In
its internal structure it presents some unusual features which differ-
entiate it from other flagellate nuclei.

58 University of California Publications in Zoology [ Von. 20

,

Tn the ‘‘resting’’ nucleus four parts may be distinguished (pl. 6,
fig. 11). The center is occupied by a linin reticulum closely filled by
a mass of chromatin granules. These are sometimes large and closely
massed together, or they may be small and show a definite linear
formation (pl. 6, fig. 14). In the predivision stages the latter
arrangement becomes the common one and results in the formation
of the chromosomes (pl. 6, figs. 14-18). Near the outer border of
this central mass and often slightly imbedded within it, is a small
vesicle surrounded by a very thin membrane. This contains a single
small, coiled or twisted rod of chromatin (fig. B, heterochr.; pl. 6,
figs. 11, 14, 16), surrounded by a clear area which does not stain.
The position of this vesicle varies somewhat in different individuals.
It is most frequently found near the end of the longer axis (pl. 6,
fies. 11, 14, 19), but may lie at the side of the nucleus near the end
of its shorter axis (pl. 6, fig. 16). Its size also varies somewhat as
well as that of the chromatin rod contained within it which we have
designated heterochromosome for convenience. In plate 6, figure 11,
the vesicle is relatively small and nearly filled by the chromatin rod.
In figure 16 of the same plate, both the vesicle and the chromatin
rod are relatively large.

Outside of these two nuclear regions is a zone of large, clear
alveoli (pl. 6, fig. 11). The walls of the alveoli seem, in some cases,
to be continuous with the linin reticulum of the central area, but
otherwise the two regions are distinct. The alveoli are rounded out-
wardly and pressed close together on their imner faces. They are
filled with a clear fluid which does not stain. Outside of this alveolar
zone and separating it from the outer membrane is a granular area,
the rather coarse granules of which stain lightly with iron haema-
toxylin. This granular portion may have a width equal to half that
of the alveolar zone (pl. 6, fig. 11), or it may narrow down to a thin
line (pl. 6, fig. 14). Im many individuals both the granular and
alveolar regions may be almost indistinguishable, the central chroma-
tin mass nearly filling the entire nuclear spaces.

In its structure the nucleus of this flagellate recalls that of Gyro-
‘dinium corallinum (Kofoid and Swezy, 1919d, pl. 10, fig. 117); the
latter, however, has been observed only in the living condition. In
this it presents a similar alveolar zone surrounding a central mass
of granules. The presence of a small vesicle with its chromatin rod,
or heterochromosome, has not been observed in this species.

1919] Kofoid-Swezy: Trichonympha campanula 59

GENERAL DISCUSSION

The entire absence of a cytostome in this flagellate has proven a
source of some difficulty in explaining its methods of food taking.
It is distinetly holozoic in its mode of nutrition, as the abundance
of food particles found in the endoplasm testify. These often fill the
posterior region and consist of particles of wood and bacteria, and
even eneysted forms of Trichomitus termitidis. The anterior region
of endoplasm has, in all individuals observed, been entirely free from
food bodies or vacuoles, with the exception of small, darkly staining
rodjlets which may be bacteria or possibly chromidia. ‘The particles
of wood found in the posterior region of endoplasm are often rela-
tively huge and may be contained in a distinet food vacuole, but are
usually found lying free in the plasma, without evident vacuoles.

The method of ingestion of these particles is a complete mystery.
Leidy (1881), in his account of these flagellates, called attention to
the presence of food bodies and the lack of any visible channel for
their entrance into the body. Kent (1884), in his studies on the
forms from the Tasmanian ants, decided that there was an oral aper-
ture at one side of the body a short distance from the apical extremity.
From this he traced a narrow oesophageal tract which opened into the
digestive cavity at the posterior region of the body. He further
states that in a medium of thinly diluted milk both the pharynx and
digestive tract were frequently found filled with the milk corpuscles.

Porter (1897) attempted to confirm these observations of Kent’s,
both in the living animals and by means of sections of the body, but
was unable to find any trace of an oral aperture. He does, however,
offer another solution to this problem, that is, that the food particles
are drawn to the posterior part of the body by the cilia and there
ingested through the thin pellicle. Unfortunately the evidences for
this are unconvincing.

Other investigators working on these forms, e.g. Hartmann and
Grassi, have been equally unsuccessful in solving this mystery, nor
has our own work afforded any hght upon the subject. It is mani-
festly impossible to consider that food may be taken in at any point
of the surface covered by the highly differentiated ectoplasm possessed
by this flagellate. That the food is taken in at the extreme posterior
portion of the body seems to be in direct contradiction to all known
methods of feeding among Protozoa or elsewhere. There remains,
then, the anterior end of the body to consider.

60 University of California Publications in Zoology [ VoL. 20

The extreme anterior tip of the cone-shaped end or head of the
body reveals, in surface view (pl. 6, figs. 7, 12), a central core
of endoplasm surrounded by a dark ring at the base of a pitlike
depression. This central core of endoplasm extends backward through
the tubular part of this darkly staining structure, the centroblepharo-
plast complex (pl. 5, fig. 6), and connects with the endoplasm of
the body. As shown in figure 3, plate 5, this structure presents the
requisites for functioning as a eytopharynx leading into the body.
Its size as compared with that of the ingested food particles, would
not militate against such a supposition, since the great flexibility of
these parts might also be correlated with a considerable degree of
elasticity permitting distension. That the centrosome should form
part of the mouth structures, however, seems hardly plausible, but
scarcely less so than that its food should be taken in at the posterior
end of the body. The fact that the operculum appears always to be
intact and to cover over the anterior tip of the body militates against
this interpretation.

An analogous condition, in case the core is the gullet of Tricho-
nympha, is found in Diplodinium (Sharp, 1914), where a ring of
neuromotor material and connections surrounds the guliet. There is
no evidence, however, that this ring has the remotest relation to any
centrosome of this ciliate. Ciliates are, moreover, not mononucleate
as 1s Trichonympha.

Porter (1897) has deseribed for Trichonympha agilis a peculiarity
in the structure of the anterior part of the body, which might afford
some basis for the view of Kent that a cytostome existed in this
region. He described the cone-shaped end or ‘‘nipple,’’ as he terms
it, as separated from the remainder of the body by a deep constriction,
the central axial rod forming the only means of union between the
two parts of the body. This appearance is shown in our own material
also (pl. 5, fig. 5) but Porter’s explanation of the structure of the
body at this point does not agree with the actual conditions as we
find them. Our own interpretation follows.

At the base of the tubular portion of the centroblepharoplast and
abutting upon its lobes, is a clear area that forms a ring completely
surrounding the tube. This is not traversed by the fibers that give
to the remainder of the ectoplasm a striate appearance, nor is it granu-
lar in its composition. In some individuals this lack of myonemes
and fibrils may be seen to extend to the outer surface which then shows
a zone devoid of flagella covering this region. Usually the outer zones

1919] Kofoid-Swezy: Trichonympha campanula 61

are completely filled by the wealth of fibrils which crowd this part
of the ectoplasm. In no ease, however, have the lines bordering the
different layers of the ectoplasm, which here is unusually thick, been
found to be broken, or otherwise to present any indications of a con-
striction in the surface of the body at this place. Were this the case,
flexions of the head or cone, such as are common in many individuals
on every slide, would betray the lack of continuity on one side at least,
and no instance of this has been observed, though the flagellates
have been thrown into every conceivable attitude in making smear
preparations. The surface lines or ridges on the cone also seem to
be continuous with those of the body without a break in their con-
tinuity. We find no evidence of an anterolateral cytostome.

What purpose this circular vacuole may subserve, finds no
explanation in observations we have been able to make. It is almost
or quite obliterated in many individuals and always disappears at
the time of division. Its conspicuousness when present in stained
material, with its complete lack of structural differentiation in the
midst of a highly differentiated zone, would suggest that it is a fluid-
filled vacuole of non-stainable substance but gives no further aid in
explaining it. That it might function as a cytostome seems impossible.

Evidences of the complex ectoplasmic and neuromotor structures
which we have deseribed above, are to be found in the figures of
Porter (1897) for Trichonympha agilis, and in those of Hartmann
(1910) for two, at least, of the species he assigns to 7. hertwigi. In
the former the alveolar zone of ectoplasm with the surface ridges,
and something of the fibrillar system with its centroblepharoplast, are
shown. In two species which Hartmann has figured, the centro-
blepharoplast and suggestions of the complex of myonemes may be
found. The complete minute structure has in no case been worked
out heretofore, neither has the presence of an integrated system been
noted nor its relation to mitosis demonstrated.

BINARY FISSION

Binary fission and mitosis in Trichonympha campanula present
some interesting phases, both in regard to cytoplasmic structures and
mitotic phenomena, the latter bearing some striking resemblances to
certain stages in the mitosis of the metazoan germ cells. These
processes will be discussed separately, beginning with the division of
the centroblepharoplast and related ectoplasmic structures.

62 University of California Publications in Zoology [ Vo. 20

Division oF THE NEuROMOTOR APPARATUS AND HEcCTOPLASMIC
STRUCTURES

The onset of division is marked by certain nuclear changes that
will be discussed later. The first evidences of it in the grosser struc-
tures of the organism are found in the change from a bell-shape to
a spherical contour in the body as a whole (pl. 7, figs. 24, 31). With
this change the nucleus migrates anteriorly until it comes to he
immediately below the centroblepharoplast (fig. 31). No trace of a
connecting rhizoplast has been found. ‘This change in the form of
the body results in a spreading apart of the myonemes and fibrils of
the ectoplasmice layers, with an apparent enlargement of the alveoli,
so that these structures are more easily studied in this stage than
in the more usual but contracted form of the normal vegetative
trophozoite.

The next step in the division process is found in the partition
into two parts of the lobes at the base of the tubular portion of the
neuromotor system, the centroblepharoplast. With this occurs a sep-
aration of the entire ectoplasmic layer into two parts, with a small,
spindle-shaped portion of endoplasm appearing in the chasm thus
made (pl. 7, fig. 30; pl. 8, fig. 35). At about the same time or slightly
earlier, the opereculum-like, transparent cap with the cup-shaped
depression which it covered, disappears, and the tip of the tubular
neuromotor apparatus reaches quite to or near the outer surface of
the anterior end of the body.

The splitting or division of the tubular part of the neuromotor
apparatus or centroblepharoplast proceeds from the base anteriorly
to the tip (pl. 8, figs. 33, 34), and with the splitting each half draws
together its parted edges until they meet and each moiety forms a
new tube. The attachment to each other at the tips may persist for
some time, as such stages are more common than are the intermediate
stages shown in plate 8, figure 33. As the two halves of the tube
separate, strands of darkly staining material are found joining the
two inner surfaces near their bases (pl. 8, fig. 34). This is the
paradesmose which functions in the formation of the spindle, and is
apparently drawn out from the material of the centroblepharoplast
itself. With the final separation of the tips of the new daughter
tubes the paradesmose remains.as the only connecting link between
the two structures (pl. 8, fig. 36).

1919] Kofoid—Swezy: Trichonympha campanula 63

As the two parts of the centroblepharoplast separate, the alveoli
frequently form a rosette at the base of each (pl. 8, fig. 38; pl. 9,
fig. 42), presenting, especially in a focus showing the oblique fibers,
striking resemblances to well developed asters in optical section.
With the further separation, the angle formed by the splitting ecto-
plasmic structures becomes greater and extends farther out towards
the periphery of the ectoplasmie region (pl. 8, fig. 38). This region
is usually conspicuous since the flagella as well as surface ridges,
myonemes and alveoli have been drawn aside, leaving only the granu-
lar endoplasm beneath the thin ectoplasmic layer. The paradesmose
becomes thicker and broader, generally forming a heavy band that
persists throughout the entire process of division, and may oceasion-
ally be found for some time after the final separation of the daughter
nuelei (pl. 11, fig. 75).

No other changes in the ectoplasmiec structures during these stages
have been detected. The staining reactions of the neuromotor appa-
ratus seem to vary slightly as the different phases follow each other.
At the beginning of its division it often presents a greater affinity for
iron haematoxylin than during the later phases. This, however, may
possibly be due to changes in the preparation of the material, as
decolorization of slides containing mitotic figures was carried on with
the view of obtaining the best possible results for the chromosomes,
without regard to the remainder of the cell.

In the final stages of division, when the complete separation of the
divided organelles has taken place, an enveloping movement of the
ectoplasmic zone may be seen. ‘This begins with a gradual creeping
out of the margins of the divided area (pl. 12, fig. 76), partly as a
result of the spreading of structures already formed and partly as
new outgrowths over the undifferentiated intermediate zones. As
the two daughter organisms separate, pulling out a long protoplasmic
bridge between them, the rounding-up of the body which results aids
in closing the gap between the margins of ectoplasm (pl. 12, fig. 79).
This process may, however, be hastened or retarded in some indi-
viduals, and is not always synchronous in the two daughter organisms.
This possibly results from contractions of the intermediate zones or
of the old differentiated zones. In plate 12, figure 85, an almost
complete union of the borders of the ectoplasm has taken place in one
individual of the dividing body, while the other still shows a rather
broad gap between the margins of ectoplasm. In figure 84 of the
same plate the final separation of the two daughter organisms has

64 University of California Publications in Zoology [ Vox. 20

taken place with only a comparatively small amount of new ectoplasm
formed in one individual.

The behavior of the neuromotor apparatus during mitosis is
significant in its intimate association with the nucleus. The entire
extra-nuclear mechanism which consists of polar centrosome, the
astral rays attached thereto and the paradesmose which is stretched
out between them as an extranuclear band of large size, is all a
direct transformation of the most intimately connected parts of the
neuromotor apparatus, the centroblepharoplast, the oblique anasto-
mosing fibrils, the lines of basal granules and the attached flagella
and cilia. The longitudinal and transverse myonemes simply part
at the zone of bipartition of the ectoplasmie territory, without forming
an integral part, with structural modifications, of the nuclear figure
of mitosis. The comprehensive fashion in which the sum total of
the neuromotor system, excluding myonemes, forms the extra-nuclear
mechanism of mitosis, is instructive in the matter of the unity of the
system, and its integration into an organic complex which survives
the shock and readjustments contingent upon mitosis without
dedifferentiation and reorganization. This is in marked contrast
with the extent of such dedifferentiation and reorganization in the
multinucleate Ciliata, such as Hwplotes (Yocom, 1918).

Another feature of cytological significance is the derivation of the
astral rays of the nuclear spindle from what are structurally dis-
tinetly fibrillar organs, the anastomosing oblique fibers. The ciliary
lines which are more granular in appearance in stained material, more
homogeneous and distinctly fibrillar in life, also form radiating lines
in semicircle from the poles of the paradesmose. The resemblance to
the aster of dividing metazoan cells is so striking that one is inclined
to regard them as homologous structures. If so, Chambers’ con-
clusions (1917) that the asters are sol phases of the surrounding
cytoplasmic gel present considerable difficulties, as does likewise the
fact that these fibers in Trichonympha le in one superficial plane in
the surface of the organism, while the asters of the Metazoa are
infiltrated through the mass of the cytoplasm in three dimensions to
a much wider extent. The latter difficulty is not insurmountable
since it is a logical consequence of the structural specialization of the
cell which is the whole undivided trichonymph. It may also be true
that with so universal a phenomenon as mitosis pervading all types
of living substance, we should expect to find the bipolar organization
which appears in the nuclear figure utilizing not one but many diverse

1919] Kofoid-—Swezy: Trichonympha campanula 65

strnetural units in its make-up, according to the nature of the cell in
which it occurs—even so diverse as the neuromotor system of a
flagellate and the sol phase of reversible cytoplasm. It is the organ-
ization rather than the state of the substance that is significant.

Mrrosis

Evidences of the approach of division may be looked for in the
nucleus before any changes may be apparent in either the ectoplasmie
structures or the external form of the body. The changes that take
place in the nucleus relate to the formation of the chromosomes, and
are significant both from the standpoint of their later history and
of the question of the continuity of the chromosomes. Their develop-
ment will now be taken up in detail.

PropHase: The structure of the vegetative nucleus shown in
figures 11 and 14 of plate 6, has already been described. The central
masses of chromatin seem to lie at the intersections of the linin
reticulum, in some cases (fig. 11) the individual masses being large
enough to completely fill the interstices also. In the early prophase
the outer alveoli disappear, leaving a clear space with a granular
region near the membrane (pl. 6, fig. 15). This granular material
later becomes diffused through the entire intranuclear spaces and
persists throughout mitosis. The chromatin moves out from the
rounded particles along the lines of the reticulum, sometimes before
the alveolar zone has disappeared (fig. 14). This movement becomes
more evident with the change in the outer region of the nucleus, and
a slight expansion takes place, with the chromatin filling a larger area
than is usual in the vegetative stage.

The outpushing of chromatin from the central masses is not equal
in all directions. This may be due to a lack of continuity in the linin
reticulum or to its breaking up. The latter seems the more probable
explanation, as threadlike ends are frequently seen in these stages
(fig. 17). This breaking-up appearance begins at one side of the
nucleus while the remainder still shows a close reticulum thickly
encrusted with chromatin (pl. 6, fig. 15; pl. 7, fig. 26). As the
breaking up proceeds further the rounded masses gradually disappear,
the chromatin apparently moving out along the threads which assume
a thicker, more compact appearance.

As these threads become differentiated some evidences may be
found of a longitudinal split in each one (pl. 7, figs. 25, 27, 28).
This may occur in some threads while the remainder are still emerging

66 Unwersity of California Publications in Zoology [ Vox. 20

from the undifferentiated mass of chromatin-encrusted network, hence
the number of threads originally formed can not be determined. The
separation of the two parts thus formed seems to take place immedi-
ately, since in slightly later stages the number of threads appears
much greater, with no evidences of the splitting of a single thread
(pl. 6, figs. 20-22; pl. 7, figs. 28, 29). In these stages also many
of the threads or chromosomes are arranged in pairs which are nearly
equal in size and length. Ina later stage, but still one which precedes
the rounding up of the body, this pairing of the chromosomes becomes
more pronounced (pl. 6, fig. 18). The threads thus sorted out in
pairs are evidently the products of the splitting of the original threads
or chromosomes.

During the time these changes in the central nuclear mass are
taking place, the small vesicle with its single coiled, chromatin thread
or chromosome remains intact, with no apparent change beyond an
enlargement of the vesicle itself. This forms a large clear area with
the chromosome contained within it loosely coiled or V-shaped (pl. 7,
fie. 29; pl. 6, figs. 16, 20). In common with the remainder of the
chromatin of the nucleus, it seems to increase somewhat in bulk dur-
ing the early prophase, though this is not invariably the case (pl. 9,
fig. 46). The vesicle disappears before the end of the prophase,
leaving the single chromosome lying in a clear space apart and
detached from the remainder of the chromatin threads (pl. 6, fig. 22.;
pl. 7, fig. 29). This isolation is apparently retained throughout the
subsequent stages, though this can be satisfactorily demonstrated only
when the nucleus is oriented so that it is seen near the lateral margin
(pl. 9, figs. 46, 47). In other positions its relations are obscured by
the great number of chromosomes.

The occurrence of a definite, continuous spireme stage is not
certain, the short chromatin threads or chromosomes apparenfly being
formed directly from the breaking up of the reticulum (pl. 6, figs.
14-22), before the body of the flagellate has begun to round up or
give other evidences of the approach of division (pl. 7, fig. 23). The
exact number of the threads thus formed seems to be fifty-two. In
the stages represented in figures 18 to 22, plate 6, these could not be
counted, but in the later stages, represented by figures 44, 47 and
49, plate 9, with the chromosomes more fully organized, this eould
be done with a considerable degree of accuracy, and the number of
chromosomes given is based on counts made on fifteen different
individuals.

1919] Kofoid—Swezy: Trichonympha campanula 67

Each chromosome consists of a long thread composed of chromo-
meres closely strung together (pl. 6, figs. 20-22; pl. 9, fig. 44).
In earlier stages these appear diffuse but later become more compact,
at the same time drawing together at the ends to form a loop (pl. 9,
figs. 46-49). These loops may appear coiled together (fig. 46) or
may preserve a V-shape. Both of these appearances may be seen
in the same nucleus (fig. 49).

Soon after the appearance of definite chromosomes the flagellate
begins to round up, with an anterior migration of the nucleus (pl. 7,
fig. 31). This is followed by the splitting of the centroblepharoplast
and the separation of the two halves which remain connected by the
darkly staining paradesmose (pl. 8, fig. 33).

The nucleus at this stage is found a short distance below the base
of the dividing neuromotor system (pl. 7, fig. 30). The chronological
relation of the changes occurring in the nucleus and those of the
neuromotor apparatus vary considerably. The stages shown in figures
20 to 22, plate 6, usually occur before the centroblepharoplast divides,
yet occasionally the paradesmose may be fully formed before definite
chromosomes appear. The formation of the spindle fibers im-
mediately below the paradesmose does not occur until the nucleus
approaches the paradesmose with the nuclear membrane apparently
touching it. The spindle fibers are stretched between the dark masses
or centroblepharoplasts at either end of the paradesmose (pl. 9, figs.
41, 48), but inside the nuclear membrane. When these are fully
formed the nuclear membrane is drawn out to a spindle shape with
the ends reaching the ends of the paradesmose (pl. 10, figs. 55-59).
The latter structure remains outside of the membrane but closely
pressed against it, usually partly imbedded within a fold, which in
many views gives it the appearance of occupying the center of the
spindle and chromosomes (pl. 10, fig. 55). In reality, however, the
chromosomes and spindle fibers are at all times completely separated
from it by the nuclear membrane. Its position thus approaches that
of the centrodesmose or central spindle of the metazoan cell. Since
it is outside of the nuclear membrane it is a paradesmose.

With the beginning of the formation of spindle fibers or somewhat
earlier, another change takes place in the chromosomes, the loops
straightening out so that the chromosomes come to lie parallel to the
paradesmose. This process may be followed in figures 56 to 59,
plate 10, with the chromosomes in various stages of unbending. The
completion of this gives the equatorial plate phase (pl. 10, fig. 59),

68 University of California Publications in Zoology [ Von. 20

with the chromosomes still joined by an end to end union in the
equatorial plane. The behavior of the heterochromosome is not always
easy to determine in this stage. In figure 56, plate 10, it is found
lying near one end of the mass of chromosomes. Its attachment to
distinet spindle fibers could not be demonstrated. In figure 59,
plate 10, it has moved nearer the equatorial plane but is still outside
the main mass of chromosomes.

MerapHase: The equatorial plate is wide and usually heavily
stained, with the chromosomes closely massed together. It is appar-
ently of shorter duration than either the prophase or later stages.
With the elongation of the nucleus the chromosomes separate at the
middle point, 1.e., at the apex of the looped thread or V of the chromo-
some before it became attached to the spindle (pl. 10, fig. 60; pl. 11,
fig. 62).

During the later prophase the small chromatin thread or hetero-
chromosome is often obscured, particularly in the formation of the
equatorial plate. With the separation of the chromosomes in the
metaphase, however, this again becomes prominent. It is found that
the vesicle has disappeared and the single thread has divided (pl. 10,
fig. 61). The separation of the two new heterochromosomes in the
metaphase lags somewhat behind that of the other chromosomes, hence
these usually may be seen between the two groups as they pass towards
the poles (pl. 11, figs. 62, 64). The attachment of these chromosomes
to spindle fibers in these stages, as in the earlier ones, has not been
determined.

ANAPHASE: The separation of the chromosomes after the final
parting seems to take place by reason of an elongation of the entire
nucleus in the equatorial region, rather than by a shortening of the
spindle fibers, since the chromosomes have in no ease been found
closely attached to the poles. The spindle fibers remain approxi-
mately the same length throughout the anaphase until they disappear
in the telophase (pl. 11, figs. 61-65, 73, 74). The elongation of the
nucleus in the equatorial region is accompanied by a constriction of
the nuclear membrane. It is usually drawn out into a long, slender
strand before the final break occurs which separates the two daughter
nuclei (pl. 11, figs. 71, 72). At the same time the paradesmose also
lengthens as the centroblepharoplast complexes of the newly forming
daughter cells move farther apart (pl. 11, figs. 68, 72, 75).

The connecting nuclear thread thus formed soon breaks and the
elongated portion of each daughter nucleus is gradually withdrawn

1919] Kofoid—Swezy: Trichonympha campanula 69

(pl. 11, figs. 70, 73, 74), the nuclei becoming rounded or nearly so,
with a separation from the paradesmose and the centroblepharoplast
(fig. 75). The paradesmose loses its staining reactions and soon fades
out, apparently being resorbed, either in the cytoplasm or by the
centroblepharoplast complexes. The chromosomes in the meanwhile
undergo few or no changes, retaining a position at some distance from
the poles after the disappearance of the spindle fibers, which may
not occur until the nucleus has begun to round up.

TrLOPHASE: The reorganization of the nucleus may take place
before the constriction of the eytoplasmie body (pl. 12, figs. 76, 79),
or it may be delayed until after separation of the two daughter cells
(fig. 84). The heterochromosome may be found at this period lying
near the ends of the chromosomes opposite to their point of attach-
ment to the spindle fibers (pl. 11, figs. 73-75).

A vesicle is formed about this (pl. 12, fig. 83), and the other
chromosomes become aggregated in the central part of the nucleus.
These gradually lose their parallel positions and become mingled in
a coarse network (figs. 83, 78, 81) which resembles in many respects
a similar stage of the prophase nucleus (pl. 6, fig. 16). The nucleus
may begin its migration away from the centroblepharoplast to the
posterior region of the body even before final constriction of the cell
(pl. 12, fig. 76), though this is usually delayed until the daughter
flagellates begin to assume their clongate, campanulate form.

Discussion oF Mrrosis

Observations on the division of the members of this unique genus
have been scanty heretofore. Foa (1904) describes and figures some
stages of this process in two species which she designates as Tricho-
nympha agilis forma minore and T. agilis forma maggiore. The stages
she has figured are strikingly similar to the same stages found in our
own material.

In both of these species division is preceded by a rounding up of
the body and an anterior migration of the nucleus, followed by divi-
sion of the anterior, narrowed portion of the body, the ‘‘tubolo,’’ and
the ectoplasmiec zone of flagella. As these structures separate they
spin out between them a stout band, the external spindle (fuso
esterno). The internal spindle (fuso interno) is formed by fibers
arising from the ends of this, passing through the nuclear membrane
and attaching themselves to the chromosomes. The remaining steps

70 University of California Publications in Zoology [ Vor. 20

in the process, so far as Foa has described them, are identical with
those of Trichonympha campanula. She does not, however, give
further details of the process, the formation of the chromosomes or
their number. She did not record division of the chromosomes in
the small form but figures longitudinal splitting in those of the larger
species.

In the nearly allied form, Holomastigotes (Trichonympha hert-
wigt) Hartmann (1910, pl. 28, figs. 23-29) has figured certain stages
of the prophase nucleus, leading up to the formation of the chromo-
somes, which are nearly identical with phases found in our own
material (pl. 6, figs. 14-16, 20-22). He also figures division of the
structure which we eall the centroblepharoplast and the anterior tip
of the body, processes which also closely parallel those found in our
own material. Further details of the mitotic phenomena he did not
record. ;

The work of earlier investigators of these flagellates, such as Leidy
(1881) and Porter (1897), also fails to give any clue to the details
of the division processes.

The abundance of division stages in our own material has given
us an unusual opportunity of determining the flagellate type of
mitosis in T'richonympha and to follow out the details of the mitotic
phenomena. The relatively large size of the nucleus of Trichonympha
and the structures connected with its division, renders it a favorable
object for study. In the foregoing outline of these various processes,
certain points have been omitted or briefly touched upon, which will
be discussed more fully in the following paragraphs, along with an
explanation of some of the terms used in this paper.

The union of blepharoplast and centrosome in one structure which
may or may not become separated at the time of division, is a
condition quite common throughout the flagellates generally. The
occasional separation of these in trichomonad flagellates (Kofoid and
Swezy, 1915), becomes a permanent condition in the mitosis of
Trichomitus termitidis (Kofoid and Swezy, 1919b). This relation-
ship makes the term centroblepharoplast an appropriate one for
this structure. The application of it to the more complex organelle
of Trichonympha is also based on these same relations. The enormous
increase In the number of flagella in this organism necessitates a
coordinating mechanism related to each flagellum individually. This
is found in the intricate system of fibrils radiating from the central
mass at the anterior cone-shaped portion of the body, which thus

1919] Kofoid—Swezy: Trichonympha campanula 71

becomes a huge blepharoplast complex. At the time of division the
entire structure divides into two parts, taking the réle of centrosomes
in the succeeding mitotie figures, while continuing its intimate rela-
tions with the flagella. The term centroblepharoplast thus designates
the dual functions of this organelle complex.

Equally distinctive and typical of the phenomena of mitosis in
flagellates is the formation of a paradesmose conecting the divided
centroblepharoplasts. In T'richomonas (Kofoid and Swezy, 1915)
at the occasional separation of the blepharoplasts and centrosomes
this structure appears to be connected with the blepharoplasts and
not with the centrosomes. In Trichomitus (Kofoid and Swezy,
1918), however, the paradesmose is found connecting the centro-
somes to which the blepharoplasts are attached by a slender rhizo-
plast. In this form these structures have a longer lease of life, the
majority of individuals noted showing the prophase stage with the
completion of the formation of the paradesmose. In both these
flagellates, as in Trichonympha, it subserves the same function in
mitosis with the same relative position owtside the nuclear membrane.

The heavy band connecting the two parts of the divided centro-
blepharoplast in Trichonympha, to which Foa (1904) has given the
name external spindle (fuso esterno), we consider homologous with
the paradesmose of the trichomonad and other flagellates, and have
so designated it. We have given a fuller discussion of this subject
in an earlier paper (1919c).

The similarity of the paradesmose and the ‘‘sphere’’ of Noctiluca
points to an homology between them, which thus links what has been
considered a peeuhar type of mitosis (Calkins, 1899) in the latter
form with conditions found among other flagellates. Whether the
nuclear membrane dissolves at the points of contact with the parades-
mose in Jrichonympha, as in Noctiluca, has not been definitely
ascertained, but no evidences to support such a conclusion have been
found. The ultimate fate of the paradesmose in both Noctiluca and
Trichonympha seems to be the same, that is, it fades out in the middle
and is absorbed or drawn up into the central mass of the centrosomes.

In the small chromatin rod, isolated from the remaining chromatin
contents of the nucleus, we have a structure that is unique among the
Protozoa and finds its nearest counterpart in the Metazoa in the
““sex’’ chromosomes of the germ cells. Its resemblances to the latter
are particularly striking during the different phases of division.

72 University of California Publications in Zoology [ Vor. 20

Its significance is problematical. No evidences of sex or sexual
behavior have thus far been found in these flagellates. Hartmann
(1910), it is true, has described both male and female forms in his
Trichonympha hertwigi. His observations, however, do not bear out
this assumption. No critical evidences of conjugation were found
by him, neither in the behavior of conjugating gametes nor in the
nuclear changes which precede this process. Indeed, it is evident
to anyone familiar with taxonomic conditions in the Protozoa, that
his male and female forms belong to different genera, as has already
been pointed out by Grassi (1911). In his ““junge, minnliche, and
weibliche’’? forms he has confused three distinct species and even
genera, with a fourth species added to this confusion in the ““oametes’’
which are minute oval flagellates such as are frequently abundant in
the intestinal contents of many termites. His elaborate life eycle of
this form is thus seen to be without adequate foundation, the produet
of an overwrought creative imagination.

This lack of evidence of sexual behavior in these organisms renders
doubly difficult any explanation of the significance of the function
of this peculiar structure. It remains distinct throughout both the
vegetative and division cycles, dividing in the metaphase, its position
that of a lagging chromosome in the anaphase, with one part going
to each daughter nucleus. At some stages it bears a strong resem-
blance to the chromatoid body described by Wilson (1913) in the
sperm cells of Pentatoma and some other insects. Its further behavior,
however, clearly distinguishes it from that body, which is cytoplasmic
in origin and does not divide. In Trichonympha this body has never
been found outside of the nucleus and behaves as do other chromo-
somes at the time of division. As a convenient designation for this
body and one which leaves its specific function still open to investi-
gation, we have used the term heterochromosome, since that word,
though originally used for the sex chromosome, has come to be
applied to other forms of chromosomes as well (Wilson, 1911). The
possibility is still open that further investigations may find undoubted
evidences of sexual behavior in these flagellates.

Certain other aspects of mitosis in Trichonympha show striking
resemblances to the mode of procedure found in the division of
metazoan germ cells. The most remarkable of these is found in the
““pairing’’ of the chromosomes with a reduction of their number from
fifty-two to twenty-six (pl. 9, figs. 44-5la). This pseudosynapsis,
however, may be explained on grounds other than that of sexual
behavior and our interpretation of it follows.

1919] Kofoid—Swezy: Trichonympha campanula 73

The formation of distinet chromosomes takes place in the nucleus
some time before any signs of division may be detected in the remain-
ing structures of the body (pl. 7, fig. 23). The nuclei shown in
figures 14 to 22, plate 6, and figures 25 and 26, plate 7, show
different steps in this process. The number of threads or chromo-
somes that result has not been made out clearly at this stage. With

Fig. D. Diagram illustrating the phases of nuclear mitosis in Trichonympha
campanula. One-half the number of chromosomes is shown. 1-7. Prophase. 1.
Vegetative phase of chromatin-encrusted network. 2. Splitting of the chromo-
somes. 3. Separation of chromosomes resulting from splitting but paired arrange-
ment noticeable. 4. Paradesmose formed between daughter centroblepharoplasts.
5. Formation of loops; nucleus approaching elongated paradesmose. 6. Tangled
stage in which pseudosynapsis occurs. 7. Number of chromosomes reduced one-
half. 8. Metaphase; looped chromosomes unfolding on the spindle. 9. Late
anaphase; paradesmose still connecting centroblepharoplasts. Chromosome marked
A is splitting in figure 2, appears as two chromosomes in 3 to 5, is reunited in 7
and 8, and separated into two distinct chromosomes in 9. The small coiled
chromosome is the heterochromosome.

their first appearance, however, signs of splits in the threads may be
detected (pl. 7, fig. 25; text fig. D). In the following stages these
threads become clearer and their number may be ascertained. The
nucleus is then found to contain fifty-two chromosomes arranged
somewhat in pairs (pl. 9, fig. 44; text fig. D), that is, the end of one

74 University of California Publications in Zoology [ VoL. 20

chromosome will be found near or attached to the end of another,
though the opposite ends may be widely separated. This is shown
more clearly in the diagrammatic scheme in figure D, where one-
half only of the actual number of chromosomes has been drawn in
each nucleus. Each of these groups of two chromosomes is probably
formed by the splitting of a single thread in the earlier stage
(fig. D, 2).

Following this, each chromosome becomes looped or doubled upon
itself (fig. D, 5). In this stage also it is found that there is some
suggestion of a grouping of the chromosomes in pairs (pl. 9, figs. 46,
49), though the ends may be more widely separated than in the pre-
vious stage. Following this the chromosomes condense into a tangled
mass of threads or contraction stage (fig. D, 6; pl. 9, figs. 42, 43,
45, 50), from which emerge twenty-six looped or V-shaped chromo-
somes (fig. D, 7; pl. 9, fig. 51a; pl. 10, figs. 52, 53). What happens
in this contracted condition can only be conjectured, since thus far
the details have escaped detection.

When the chromosomes divide in the metaphase the point of
separation occurs at the apex of the V or loop. If division here is
longitudinal, and the evidences of the earlier stages (fig. D) confirm
this view, the line of separation must be at one end of the original
split found in the chromosomes when first formed. This would postu-
late the supposition that the two halves of the original chromosome
are reunited at the time the change in the number of units from
fifty-two to twenty-six occurs. To illustrate this, let us follow the
course of the chromosomes marked A in figure D. In figure 2 the
chromosome A is a single thread that has partially split. This is com-
pleted in figure 4 but the ends of the chromosomes are still connected.
In figure 3 this connection has apparently been lost, for the ends
are separated by a considerable space. The course of these two threads
is lost in the contraction stage in figure 6 but they emerge again in
figure 7 as a single split chromosome which parts ‘‘transversely’’ on
the spindle in the following figure, the two threads having apparently
become united end to end into one, in the stage represented by figure 6.
The seemingly transverse division is in reality the final step in longi-
tudinal splitting of the original chromosome.

The only difficulty in this explanation lies in the lack of a visible
mechanism by means of which two threads which are more or less
widely separated from each other, are reunited, with their relative
positions those of the original split. This difficulty is, however, not

1919] Kofoid-Swezy: Trichonympha campanula 75

so formidable as would seem at first glance. A single chromosome
is evidently composed of a ground substance, framework, or thread
of linin in which the chromomeres are imbedded and which gives
such great consistency to the entire structure. This does not stain
and its presence is difficult to demonstrate satisfactorily except as it
is outlined by the stained chromatin.

At the time of division of the chromosomes in T'richonympha
(fig. D, 2; pl. 7, fig. 25), a physical continuity may still be retained
between the two threads thus formed, by the incomplete division of
this framework. This invisible link, whose existence is suggested by
the behavior of the ends of the chromosomes, would serve to keep one
end of each chromosome near the corresponding end of its fellow, and
would explain the apparent pairing of chromosomes in these stages
(pl. 9, figs. 46-49). These stages, however, have not been observed
in the living cell and it is possible that the spreading apart of such
pairs may be due to the manipulations of the operator in making
smears in the preparation of the material. It is conceivable, however,
that considerable separation or strain may occur in the chromosomes
of the normal cell without reaching the point of complete separation.

Given this physical continuity the drawing together of the two
threads and their condensation into two shorter parallel threads
joined at the apex, becomes a simple matter. Such precocious split-
ting and separation and their subsequent union before going on the
spindle, have been found to oceur in the chromosomes of the tricho-
monad flagellates (Kofoid and Swezy, 1915) and in Giardia (Boeck,
1917), a procedure which would seem to support an explanation simi-
lar to that given above. In view of our present knowledge of cytology,
the alternative explanation would be that we have here a synapsis of
chromosomes occurring in the ordinary vegetative division cycles,
since it is highly improbable, even were these flagellates found to be
sexually differentiated, that all the division cycles found within a
single year, would be only those of gametes and not the ordinary
trophozoite division, where such a reduction in the number of the
chromosomes would not be expected to oceur. The possibility that
the behavior of pregametie chromosomes in the Metazoa is a special-
ization of a more widely prevalent phase of mitosis in the Protozoa
is not precluded.

Evidences for the precocious splitting of the chromosomes immedi-
ately following the end of a division period, have been carefully
searched for, but thus far have not been found. At what period in

76 University of California Publications in Zoology [ Vor. 20

’

phase of the nucleus this occurs has not been deter-
mined. The abundance of such stages in what appear to be normal

the ‘‘resting’

vegetative forms, without other signs of the approach of division,
would suggest that it occurs very early in the between-division periods
of the life of the organism.

That we may have here a continuity of chromosomes from one
division period to the next is suggested by certain appearances of the
resting phases of the nucleus. This is best seen in figures 14, 15,
and 17 of plate 6, and figures 26 to 28 of plate 7. These are the
nuclear figures which are most frequently met with in the ordinary
trophozoite. They present a broken, ragged network with distinctly
marked ends of chromatin threads scattered through it. Occupying
the nodes of the network or sometimes at the ends of the threads, are
chromatin granules. The process of changing from this condition
to that of the distinctly marked chromosomes seems to consist In an
outmoving of the contents of the granules along the threads to which
they are attached (pl. 6, figs. 17, 19). On the completion of this,
the fully formed chromosomes become apparent, with an entire
absence of large chromatin granules. Differentiation of individual
chromosomes other than the heterochromosomes has not been detected.

In the late prophase of division the chromosomes retain distinct
outlines for a considerable period. With the disappearance of the
spindle fibers the chromosomes move out to near the center of the
nucleus, with the threads lying parallel or nearly so (pl. 12, figs. 77,
82). Without apparently losing their individuality, these separate
threads begin to change their position (fig. 78), and form a loose
network by the interweaving of the separate strands (figs. 78, 81, 83).
Later granules appear at the intersection of the threads which then
may become thinner and we have the same nuclear structure as that
shown in figures 27 and 28 of plate 7, and figures 14 and 15 of
plate 6. Distinct chromomeres cannot be detected in these stages
of the nucleus, but they appear later following the formation of
distinet chromosomes.

The type of division of the chromosomes is apparently transverse
in the later stages, but is in reality longitudinal, as shown by the
longitudinal splitting of the single threads (fig. D; pl. 7, fig. 25).
The contraction of the two, visibly separated halves of the chromo-
somes (pl. 9, fig. 49) into single short, thick threads with a V-shape
(fig. 51a), produces the units which are unfolded on the spindle as
long, single threads (pl. 10, figs. 56, 59). These part in the middle,

1919] Kofoid-—Swezy: Trichonympha campanula

at the apex of the original V, and thus finally complete the original
splitting. Division of the chromosomes is thus in T'richonympha, as
in other flagellates, a fundamentally longitudinal process.

In the division of the protoplasmie body the same longitudinal
type of division also holds true. In the rounding up of the body the
anteroposterior relations are somewhat obscured. The beginning of
the division process is found to be a longitudinal splitting of the
eentroblepharoplast and the cone-shaped anterior portion of the body
(pl. 8, figs. 33-36). This is followed by a splitting of the entire
ectoplasmie surface of the body in the same plane, which is funda-
mentally longitudinal (pl. 9, fig. 51). This relation is maintained
through the various stages of division to the early telophase (pl. 12,
fig. 76). At this point, however, the activity of the motor organelles
of the two attached daughter cells becomes operative in different
directions, resulting in a change in the orientation of the two parts.
With the continued opposing activities of the flagella the daughter
flagellates are found attached to each other at the posterior regions
only, giving an apparent transverse direction to the plane of division
(pl. 12, fig. 79). This is apparent only and not the real direction
which is fundamentally longitudinal. The morphological plane in
which the chromosomes finally part at the metaphase coincides with
that in which the highly organized neuromotor system is divided in
plasmotomy. The plane of division of the chromomeres and chromo-
somes, and of the organized structures of the cytoplasm whose
behavior at mitosis can be determined, is thus one and the same
morphologically longitudinal plane.

RELATIONSHIPS

The relationships of these peculiar and highly evolved organisms
has proven a source of some confusion. On the one hand ciliate
affinities have been claimed for them, and, on the other, they have
been listed as flagellates. Stein (1878), with the meager description
given in Leidy’s first paper in 1877 as his only basis of classification,
correctly placed them among the flagellates. In this he has been
followed by most later taxonomists. Leidy, however, with his fuller
account of their structure in 1881, considered them intermediate
between the gregarines and ciliates but more nearly related to the
former. Kent (1882) followed this by placing them among the
holotrichous ciliates in the faimly Trichonymphidae, a family he

78 University of California Publications in Zoology [ Vox. 20

formed for these parasites of the termites and included in it Pyrsonema
and Dinenympha.

The flagellate affinities of Trichonympha were recognized by
Grassi and Sandias (1893) who placed it in the family Lophomona-
didae in the Flagellata. Biitschl (1889), however, reverts to the
classification of Kent and recognized them as belonging to the ciliates.
Senn (1900), with some doubt as to their actual position, placed them
in the order Trichonymphida as an appendix to the Flagellata,
but later (1911) allocated them in the Euflagellata. Hickson (1903)
in Lankester’s Treatise on Zoology, added the family Trichonymphidae
as an appendix to the Ciliata. Doflein (1911) followed Senn in his
classification of these puzzling forms, and, as had been earlier done
by Butschh, attributed the formation of the family Trichonymphidae
to Leidy, overlooking the fact that Leidy nowhere attempted to classify
the forms he described, while the family in question had been formed
by Kent for these parasites of the termites.

The first complete systematic review of this subject is that given
by Grassi (1911), in which he presents some changes in the previous
taxonomic groupings. He formed a new order, the Hypermastigina,
closely following the order Polymastigina in the Flagellata. This
contained a single family, the Lophomonadidae Grassi. In this family
he placed all the forms possessing many flagella, as Trichonympha,
Lophomonas, and Joenia. Other changes that were made in the
taxonomic position of other members of this group of parasites will
be noted in a later paper discussing this subject.

In the genus Trichonympha he recognized two species, 7. agilis
Leidy and 7. minor Grassi. The species described by Hartmann in
1910 as T. hertwigi, he rejects as defined by the deseriber, dividing
the forms he has figured among three different genera, that is, the

9?

“‘yvoung form’’ is referred to Pyrsonympha, and two new genera are
created for the others. The ‘‘male’’ (Hartmann, 1910, pl. 28) he
placed in the genus Holomastigotoides and the ‘‘female’’ (pl. 30) in
the genus Pseudotrichonympha.

Poche, in 1913, added still further to the confusion already existing
in this group by creating a new order, the Trichonymphida, which
he placed in the Euflagellata. This contained four famliies, Dine-
nymphidae, Devescovinidae, Calonymphidae, and Trichonymphidae.
The family Devescovinidae he formed for a single genus, Devescovina
Foa, having but four flagella and in nowise related to the remainder
of this group of many-flagellated organisms. This genus we consider

1919] Kofoid-Swezy: Trichonympha campanula 79

belongs in the order Polymastigina near the genus Trichomonas, to
which its axostyle, parabasal body and four flagella ally it.

In a later paper we propose to give a fuller discussion of the
systematic relations of the parasites of the termites and will here
confine our attention to the genus Trichonympha, with the order and
family to which it belongs.

The question of flagellate or ciliate relationships of Trichonympha
is one the solution of which finds little difficulty in the light of facts
concerning its morphology and division presented in this paper. We
have already discussed some aspects of its relationships elsewhere
(1919c) and will here only point out a few additional considerations.
The relation between the motor organelles and the centroblepharo-
plast is distinctly flagellate in character. The nearest approach to
this relationship among the ciliates is perhaps that found in the
ciate Huplotes (Yocom, 1918). Here the ciliary components of
part of the cirri and the oral membranelles are bound together by
their connection with the motorium. The latter structure, however,
plays an entirely different rdle during division than does the centro-
blepharoplast of the flagellates. In Huplotes the motorium is passive
or apparently disappears at the time of division and is formed anew
in the daughter cells. In no ease does it play an active part in
mitosis. In the flagellates, on the other hand, this structure becomes
the dominant, guiding figure in mitosis, dividing and acting as centro-
somes in the formation of the mitotie spindle.

The occurrence of a highly differentiated ectoplasm is here corre-
lated with the development of the complex myonemes, which in turn
are probably directly correlated with the great crease in the number
of flagella. A comparable degree of ectoplasmic differentiation is
found in one other group of flagellates, the members of the subgenus
Pachydinium in the genus Gymnodinium among the dinoflagellates
(Kofoid and Swezy, 1919d), but without myonemes or flagella.

It is thus seen that in its morphology as well as division, Tricho-
nympha possesses distinetly flagellate characteristics with none that
are exclusively ciliate in character. The complexity of its structures
is the result of a high degree of specialization and parallel evolution,
and in no way connects it with the ciliates.

The possibility of these flagellates forming a connecting link
between the Flagellata and the Ciliata, is also one for which we can
find no adequate basis. Thus far no near relatives of Trichonympha
have been found as free-living forms. They are confined exclusively

80 University of California Publications in Zoology [ Vou. 20

to the réle of parasites or commensals of the termites and related
insects. This fact in itself would throw them outside the line of
evolution along which the present group of the presumably much
earlier evolved, free-living ciliates were developed.

For these reasons we accept the allocation given by Grassi for
these peculiar organisms, placing them among the true flagellates and,
as shown by a study of the various stages of division as well as
morphology, not far removed from certain members of the Poly-
mastigina.

The utilization of the order Hypermastigina, proposed by Grassi
(1911) for this group of flagellates possessmg numerous flagella, is
more appropriate as a descriptive term than either Trichonymphida
Poche or Lophomonadina Lankester. It is further desirable as at
once connoting its relation to the order Polymastigina near which
it stands in the Flagellata.

For the family designation of the group to which Trichonympha
belongs, we retain the term proposed by Kent (1882), Tricho-
nymphidae. The remaining constituent members of this family we
need not further specify at this time.

The genus Trichonympha contains three species previously de-
seribed: 7. agilis Leidy, from American termites; 7. Leidyi Kent,
from Tasmanian termites; and 7. minor Grassi, from Italian termites.
No figures have been given for the last two species, the description
of both being imperfect without dimensions or other exact data. To
these we add a fourth species, 7. campanula.

Key TO THE GENUS Trichonympha

Hypermastigina with flagella of various lengths disposed over about two-thirds
of the surface of the body; nucleus submedian; specialized ectoplasm; complex
neuromotor system; holozoie nutrition.

1. Large, 250y-460n
— Small, 100, or less
. Flagella of three distinct lengths, posterior endoplasm not separated from

anterior region by distinct line campanula sp. nov.

3. Flagella short, almost cilia-like, elongate or pyriform body ............ leidyi Kent
— Distinct line separating two regions of endoplasm, body constricted at the

point of separation, cilia long agilis Leidy
— Small form, flagella short, lines separating two regions of endoplasm long,
crossing each other behind nucleus .................---------s.-100-0---e---= minor Grassi

aie

bo

1919] Kofoid—Swezy: Trichonympha campanula 81

SUMMARY

1. This organism has a highly specialized flagellate type of
structure with a highly developed neuromotor system, the centro
blepharoplast connected by a complex system of oblique fibers with
the numerous flagella which cover two-thirds of the surface of the
body. Besides these fibers the ectoplasm contains an alveolar layer
and one of transverse myonemes. Immediately below it in the endo-
plasm are the longitudinal myonemes.

2. The nucleus is submedian in position and part of its chromatin
contents is permanently separated as a ‘‘heterochromosome’’ con-
tained within a small vesicle.

3. Nutrition is holozoic but its method of feeding is unknown. No
cytostome is present. The endoplasm is divided into two regions,
anterior and posterior. The latter region, which is covered only by a
thin pellicle and not the thick ectoplasm of the remainder of the body,
is usually filled with food particles. These are entirely absent from
the anterior region of endoplasm.

4. The body rounds up at the time of division and the centro-
blepharoplast divides, forming a paradesmose, the entire ectoplasm
splitting into two parts with it. These act as the centrosome in the
succeeding mitotic figures, the spindle fibers arising from the ends
of the paradesmose or the centroblepharoplasts.

5. Precocious splitting of the chromosomes takes place previous to
the prophase, forming fifty-two, V-shaped threads. In a pseudo-
telosynapsis this number is reduced to twenty-six. These part on the
spindle along the line of the original split.

6. Division of the chromosomes as well as the body is funda-
mentally longitudinal.

7. Trichonympha campanula is fundamentally flagellate in its
morphology and method of division and is in nowise related to the
ciliates. It shows a high degree of specialization and development
of its neuromotor system which is the most complex one thus far
described among the Protozoa.

8. It is a member of the family Trichonymphidae Kent in the
order Hypermastigina Grassi. This order stands near the Poly-
mastigina, to the members of which Trichonympha is nearly related
both morphologically and in its development.

Zoological Laboratory, University of California,

Berkeley, California.
Transmitted October 25, 1918.

82 University of California Publications in Zoology [ Vor. 20

LITERATURE CITED
Boeck, W.
1917. Mitosis in Giardia microti. Univ. Calif. Publ. Zool., 18, 1-26, pl. 1.
Butscu1i, O.

1889. Trichonymphidae Leidy 1877 emend., in Protozoa in Bronn, Klass. u.

Ordn. des Thierreichs, 1, part 3, pp. 1774-78, pl. 76, figs. 1, 3-6.
CaLKINs, G. N.

1899. Mitosis in Noctiluca miliaris and its bearing on the nuclear relations

of the Protozoa and Metazoa. Jour. Morph., 15, 715-772, pls. 40-42.
CHAMBERS, R.

1917. Microdissections studies. II, The cell aster; a reversible gelation

phenomenon. Jour. Exp. Zool., 23, 483-504, pl. 1.
DoFLeEIn, F.

1911. Lehrbuch der Protozoenkunde. Eine Darstellung der Naturgeschichte
der Protozoen mit besonderer Bericksichtigung der parasitischen
und pathogenen Formen. (Ed. 3, Jena, Fischer), xii+1043 pp.,
951 figs. in text.

FoA, A.

1904. Richerchi sulla riproduzione dei Flagellati. II, Processo di divisione
delle Triconymphi. Rend. R. Accad. dei Lincei, Cl. Sci. Fis. Mat. e
Nat., Rome, (5), 13, 618-620, 5 figs. in text.

Grassi, B.
1911. Intorno ai Protozoi dei Termitidi. Jbid., (5), 20, 725-741.
Grassi, B., and Sanpras, A.

1893. Constituzione e sviluppo della societa dei Termitidi. (Catania, Gala-

tola), pp. 1-151, pls. 1-5.
HARTMANN, M.

1910. Bau und Entwicklung der Trichonymphiden (Trichonympha hertwigi).

Festschr. z. Hertwigs, pp. 349-392, pls. 27-30, 3 figs. in text.
Hickson, S. J.

1903. The Infusoria or corticate Heterokaryota, in Lankester, Treatise on

Zoology, 1, sec. 2, pp. 361-426, 97 figs. in text.
Kent, S.

1880-82. A manual of Infusoria. (London, Bogue), 1, 2, 1-913, pls. 1-50.

1884. Notes on the infusorial parasites of the Tasmanian white ant. Papers
and Proc. Roy. Soc., Tasmania, 1884, 270-273.

Koro, C. A., and CHRISTIANSEN, E. B.

1915. On binary and multiple fission in Giardia muris (Grassi). Univ. Calif.

Publ. Zool., 16, 30-34, pls. 5-8, 1 fig. in text.
Korom, C. A., and Swezy, O.

1915. Mitosis and multiple fission in trichomonad flagellates. Proce. Am.
Acad. Arts Sci., 51, 289-378, pls. 1-8, 7 figs. in text.

1919a. Parasites of the termites, I. On Streblomastix striz, a polymastigote
flagellate with a linear plasmodial phase. Univ. Calif. Publ. Zool.,
20, 1-20, pls. 1, 2, 1 fig. in text.

1919b. Studies on the parasites of the termites, II. On Trichomitus termitidis,
a polymastigote flagellate with a highly developed neuromotor sys-
tem. Ibid., 20, 21-40, pls. 3, 4, 2 figs. in text.

1919¢. Flagellate affinities of Trichonympha. Proc. Nat. Acad. Sci., 5, 9-16,
figs. 1-10.

1919d. A monograph of the unarmored Dinoflagellata. Mem. Univ. Calif. In
press.

1919] Kofoid-Swezy: Trichonympha campanula 83

Lewy, J.
1877. On intestinal parasites of Termes flavipes. Proce. Acad. Nat. Sci.

Philadelphia, 1877, 146-149.
1881. The parasites of the termites. Jour. Acad. Nat. Sei. Philadelphia, (2),
8, 425-450, pls. 51, 52.
Pocue, F.
1913. Das System der Protozoa. Arch. Prot., 30, 125-321, 1 fig. in text.
Porter, J. F.

1897. Trichonympha and other parasites of Termes flavipes. Bull. Mus. Comp.

Zool., Cambridge, 31, 48-68, pls. 1-6.
SENN, G.

1900. Flagellata, in Engler and Prantl, Die natiirlichen Pflanzenfamilien.
(Leipzig, Englemann), 1, part 1, a, pp. 93-192, text figs. 63-140.

1911. Oxyrrhis, Nephroselmis und einige Euflagellaten. Zeitschr. wiss. Zool.,
97, 605-672, pls. 30, 31, 8 figs. in text.

SHarp, R. G.

1914. Diplodinium ecaudatum with an account of its neuromotor apparatus.

Univ. Calif. Publ. Zool., 18, 48-122, pls. 3-7, 4 figs. in text.
Stein, F.

1878. Die Organismus der Flagellaten nach eigenen Forschungen in syste-
matischer Reihenfolge bearbeitet. Der Organismus der Infusions-
thiere. 3. Abth., Die Naturgeschichte der Flagellaten oder Geissel-
infusorien. 1. Hilfte, Den noch nicht abgeschlossenen allgemeinen
Thiel nebst Erklarung der simmtlichen Abbildungen enthaltend,
(Englemann, Leipzig), pp. 1-154, pls. 1-24.

WItson, E. B.

1911. The sex chromosomes. Arch. mikr. Anat., 77, 249-271, 5 figs. in text.

1913. A chromatoid body simulating an accessory chromosome in Pentatoma.
Biol. Bull., 24, 392-410, pls. 1-3.

Yocom, H. B.

1918. The neuromotor apparatus of Euplotes patella Ehrbg. Univ. Calif.

Publ. Zool., 18, 337-396, pls. 14-16, 1 fig. in text.

EXPLANATION OF PLATES

All figures of Trichonympha campanula sp. nov. from Termopsis angusticollis
Walker, stained with iron haematoxylin and drawn with camera lucida. Mag-
nification 800, unless otherwise stated.

PLATE 5

Fig. 1. Normal trophozoite showing the three zones of flagella, surface ridges
of the body, and centroblepharoplast. X 300.

Fig. 2. Optical section of anterior part of the body, showing the differen-
tiations of ectoplasm and endoplasm with the longitudinal myonemes in the latter,
and part of the centroblepharoplast. 300.

Fig. 3. Surface view of anterior end showing surface ridges, the cuplike
depression with end of centroblepharoplast at its base and its covering operculum.
Internally the ectoplasm and endoplasm may be seen with the vacuoles near the
base of the centroblepharoplast.

Fig. 4. Semidiagrammatic cross-section of body in the region of the nucleus,
showing origin of flagella from crests of surface ridges, ectoplasm, endoplasm,
and the longitudinal myonemes in the latter. X 300.

Fig. 5. Optical section of anterior portion of the body showing striate appear-
ance of ectoplasm, with its different layers, and the circular vacuole at the base
of the centroblepharoplast. X 300.

Fig. 6. Optical section of entire body showing extent of differentiated ecto-
plasm and the two regions of endoplasm. Dark granules are probably remnants
of ingested bacteria. X 300.

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+*PLATE 6

Fig. 7. Vertical view of centroblepharoplast complex in rounded-up form.
Cuplike depression and operculum have disappeared. Oblique fibers radiating out
from lobes of centroblepharoplast.

Fig. 8. Cross-section of distorted individual showing relative extent of endo-
plasm and ectoplasm. X 300.

Fig. 9. Lateral view of anterior end showing centroblepharoplast complex,
oblique fibers and alveolar layer.

Fig. 10. Centroblepharoplast complex of individual in early prophase. Oper-
culum and depression have disappeared, end of centroblepharoplast drawn out to
a point.

Fig. 11. Vegetative nucleus showing the central region of chromatin granules,
the heterochromosome and its vesicle, the alveolar zone and the outer, granular
region.

Fig. 12. Cross-section of anterior end of body showing the centroblepharo-
plast, myonemes and surface ridges.

Fig. 13. Vertical view of centroblepharoplast complex of the early prophase
stage.

Fig. 14. Vegetative nucleus showing the chromatin-encrusted network. Other
parts as in figure 11.

Figs. 15-22. Nuclei of the early prophase stages in individuals which present
no other sign of division.

Fig. 15. Early prophase; alveolar zone has disappeared, chromatin moving
out from the granules along the threads of the network.

Fig. 16. Later stage of the same. Note size of heterochromosome and its
vesicle.

Fig. 17. Formation of chromosomes; vesicle surrounding heterochromosome
has disappeared.

Fig. 18. Appearance of distinct, paired chromosomes.
Figs. 19-22. Various figures of fully formed chromosomes.

[86]

. |

PLATE 7

Fig. 23. Chromosomes appearing in the nucleus before other signs of division
can be detected in the body. Figures 25-29 were drawn from similar individuals.
x 300. ;

Fig. 24. Rounding up of the body preparatory to division; part of the centro-
blepharoplast complex and alveolar layer shown. X 300.

Fig. 25.—Longitudinal division of the chromosomes.

Fig. 26.—Breaking up of the chromatin-encrusted network beginning at one
side of the nucleus.

Fig. 27. Network thickly encrusted with chromatin showing free ends.

Fig. 28. Later stage of the same with the threads beginning to split.

Fig. 29. Chromosome formation nearly completed.

Fig. 30. Beginning of division of the centroblepharoplast complex. Note the
spindle-shaped areas of endoplasm appearing between split.

Fig. 31. Optical section of rounded-up individual of the prophase period.
x 300.

[88]

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UNIV. CALIF. PUBL. ZOOL. VOL

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PLATE 8

Fig. 32. Prophase with division of the centroblepharoplast completed in the
tubular part but no split yet appearing in the ectoplasmie structures. An unusual
figure. X 300.

Fig. 33. Splitting of the centroblepharoplast and the formation of the
paradesmose between the bases as they separate.

Fig. 34. A later stage of the same with the halves forming new tubes con-
nected at the tip. Note development of spines at tip.

Fig. 35. An earlier stage of the same showing the split beginning at the base,
with the ectoplasmic structures drawing apart.

Fig. 36. Splitting completed; paradesmose elongates as the new centro-
blepharoplasts move apart.

Fig. 37. Vertical view of an early stage of splitting of centroblepharoplast.

Fig. 38. Vertical view of individual in prophase with the divided centro-
blepharoplasts connected by paradesmose. Part of oblique fibers and alveolar
layer shown. Note aster arrangement of alveoli around centroblepharoplasts.
x 300. (

Fig. 39. Prophase nucleus showing the breaking up of the chromatin-
encrusted network.

Fig. 40. Paradesmose, centroblepharoplasts and their related fibers in the
prophase, with the nucleus elongating preparatory to formation of mitotic spindle.

[90]

PLATE 9

Figs. 41, 42. Two views of the same figure showing different structures.
41. The paradesmose, centroblepharoplast and oblique fibers. Immediately below
and intermingled with these are the structures shown in 42: prophase nucleus and
alveolar layer.

Fig. 43. Nucleus becoming closely attached to the paradesmose.

Fig. 44. Nucleus showing fifty-two chromosomes with marked chromomere
structure. X 1250.

Fig. 45. Prophase nucleus in the tangled-skein stage.

Fig. 46. Prophase nucleus with looped chromosomes.

Fig. 47. Same stage. In both figures heterochromosome remains isolated.

Fig. 48. Beginning of the tangled-skein stage (?); chromosomes at one side
of nucleus stain deeper than those on the other.

Fig. 49. Marked tendency of chromosomes to assume paired arrangement.
Fig. 50. Tangled-skein stage.

Fig. 51. Individual in prophase showing completion of division of ectoplasmic
structures. Only a few of the surface ridges are indicated. > 300.

Fig. 5la. Enlarged view of nucleus of last figure. Chromosome number
reduced to 26.

[92]

a

PLATE 10

Nuclei on this and the following plates have the reduced number of chromo-
somes. In some figures the paradesmose is shown in position, i.e., above the
nucleus. In others it is omitted or only indicated by faint lines to secure
clearness.

Fig. 52. Late prophase; nucleus elongated and attached to the parades-
mose.

Fig. 53. Same stage; tubular part of paradesmose omitted.

Fig. 54. Chromosome arrangement previous to spindle formation.

Fig. 55. Spindle fibers extending from centroblepharoplasts through
nuclear membrane and attached to chromosomes.

Fig. 56. Showing process of unfolding of chromosomes in the metaphase.
Heterochromosome distinct. Vertical view, with paradesmose uppermost and
outside nuclear membrane.

Fig. 57. Lateral view of same stage with paradesmose above the nucleus.

Fig. 58. Same stage viewed from below, i.e., the interior of the body.
Paradesmose is on opposite side of nucleus.

Fig. 59. Metaphase; paradesmose partly imbedded within the nucleus.

Fig. 60. Final division of the chromosomes; heterochromosome distinet and
still undivided. Entire nucleus elongating.

Fig. 61. Early anaphase; heterochromosome divided.

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PLATE 11

Fig. 62. Anaphase; heterochromosome divided without apparent attach-
ment to spindle fibers.

Fig. 63. Unusual appearance of chromatin granules strung along the
spindle fibers. X 1250.

Fig. 64. Anaphase; heterochromosome dividing.

Fig. 65. Telophase of nucleus; constriction of the nuclear membrane.
X 1250.

Fig. 66. Daughter nucleus with division completed.

Fig. 67. Telophase with nuclear constriction advancing. Spindle fibers
disappearing at one pole, still intact at the other. X 1250.

Fig. 68. Telophase showing surface ridges of the body. Centroblepharo-
plasts still connected by the paradesmose. X 300.

Fig. 69. Telophase of nucleus.
Fig. 70. Daughter nucleus after constriction and prior to rounding up.
Fig. 71. Greatly elongated nuclear band still connecting daughter nuclei.
x 300.
g. 72. Completion of nuclear division with paradesmose still persisting.
Figs. 73, 74. Sister nuclei recently divided.
5. Completion of nuclear division. Paradesmose beginning to fade

[96]

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yy “* it -

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PLATE 12
Fig. 76. Late telophase; nuclei have become reorganized; alveolar layer
shown. X 300.
Fig. 77. Nucleus at the beginning of reorganization.
Fig. 78. Later stage showing chromosomes forming a coarse network.

Fig. 79. Plasmotomy; all ectoplasmic structure except oblique fibers are
omitted. X 300.

Fig. 80. Optical section of individual shown in figure 76 at the point
marked by the arrow. Note basal granules and basal portion of flagella.

Fig. 81. Reorganization process of nucleus.
Fig. 82. Early stage of the same process.

Fig. 83. Later stage showing the vesicle forming around the heterochromo
some.

Fig. 84. After completion of plasmotomy. Nuclear reorganization not yet
begun. X 300.

Fig. 85. Somatella prior to plasmotomy. Note lack of synchronism in
development of ectoplasmic structures. X 300.

[98]

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IN

ZOOLOGY
Vol. 20, No. 4, pp. 99-116, plates 13-14, 1 figure in text July 14, 1919

STUDIES ON THE PARASITES OF THE TERM-
ITES IV. ON LEIDYOPSIS SPHAERICA
GEN. NOV., SP. NOV.

/ Corary of

BY

CHARLES ATWOOD KOFOID anp OLIVE SwEZy Lagi ‘P
% J ] 4
Lip }
: b>
CONTENTS
PIPER ITS rn NEXIS Hj] OY sarasota odes bts hates ds ddse so 4058 isd devas nc snass osc_Jeedexcasdescksaea vera flosn~csdsesacvsccuisesonsced
SSRN STS TEN) gs ca a Be ea aN ee ag eg nha BCs na non a.nd dps nce cc wadaes SS aaodezon a zanetw ances
SURES) DEVEL VAD CC SE ee PSE
Neuromotor system ..... Se
Ectoplasmie structures ..
Surface ridges ..............
ILA ROTO Organs Icky ee 103
CO) Ube pare lho SI See re ee
Centroblepharoplast
LAUR (SO) ENO) Ey oi aca a ee
Endoplasmic structures
ROHL MeN YON OINOS tsar Seon cece a cen seh = eect oct encdonaetucer tenses toceesesastacucs 105
BUA NIS es a sla RESUS oe a 8a ccd awdss nse bsbc ded as idvgeatacdhedtsseennsacaia-asee 106
BEA Rts CARER ES 1 Lhe oes 895- G9 So a tae sak oY MSAD a9 ain a a sdb I059 ae seSbeESS Ss insds sae daavediaesao—dd 107
MUVIBIOMUNOL CCLOPlASMIC, StU CUTS sass aco c coca cence cate cea center sneeteacseecenseedosnces 107
Mitosis a
Taf sCif 3) 00 ase ce Si era a a ee aR 108
IWC TAD RASC sc cenecseweesen. 3 asetabd attuas! osSa Masia dis eead tin: susdacedsSaied sasadacdassdcaascnasdoacsecades 109
PNT ASO) esses oss. Maatoced sds oSts da asc oasas ss ieee i asses saddel sadacn asa sedasaisadss sdshddsas casa 109
LD CSAP ES STE a a en eed 110
Relationships ......

RSSREARI TT EAA ae aS eae oa Sa faaasst aba saad adn dacs locn secs tssdelacsdiveed
Literature cited

100 University of California Publications in Zoology [ Vou. 20

INTRODUCTION

In our previous studies on the parasites of the termite, Termopsis
angusticollis Walker, we have described three of the curious and
highly interesting protozoans in this faunal complex. These are
the flagellates Streblomastiz strix (1919a), Trichomitus termitidis
(1919b), and Trichonympha campanula (1919c). A fourth member
of this group remains to be noticed. This flagellate is closely akin
to Trichonympha campanula yet presents some striking differences
which, in our opinion, give it a position generically distinet from that
species. We therefore propose for it the name Leidyopsis sphaerica
gen. noy., sp. nov., naming it in honor of the pioneer investigator of
this group, the American naturalist Dr. Joseph Leidy.

It is less abundant than are the other members of this association
of termite parasites. It is found in the lumen of the intestinal tract
and is in no ease attached to the walls. In this location, as also in
its activities, it resembles Trichonympha campanula. The mass of
flagella attached to the anterior portion of the body stream backward,
partly clothing its rotundity. In the living animal the action of the
flagella is similar to that of its larger relative. Waves of contraction
pass from the proximal to the distal ends of the flagella without
cessation. Owing to the differences in the shape of the body its
anterior end is slightly less mobile than is the case in Trichonympha
campanula. Its rate of progression is also somewhat slower, as though
impeded by the rotundity of the body in moving through the seething
mass of organisms found in the intestinal tract.

MORPHOLOGY -

In its morphology Leidyopsis presents a type of structure which
is as highly differentiated as that found in Trichonympha. It differs
from it mainly in the extent and distribution of the specialized struc-
tures as compared with the remainder of the body. These differences
will be pointed out more specifically below.

The outstanding feature of its morphology, next to its abundant
supply of flagella, is its neuromotor system, which is of the tricho-
nymphid type (Kofoid and Swezy, 1918c). Correlated with this
are the ectoplasmie differentiations, while the remainder of the body
presents no structural features which mark an advance over that of
the simpler flagellates. The evolutionary development of organelles

1919] Kofoid-Swezy: Leidyopsis sphaerica 101

in Leidyopsis is thus marked only in its neuromotor system, while
the remainder of the body still shows a low degree of development.
Its mode of nutrition is holozoic, as shown by the food particles in
the endoplasm, yet no eytostome or other organelles for food taking
are present.

SHAPE AND SIZE

In size Leidyopsis sphaerica presents less variation than has been
found in the three other flagellates previously described from the
same habitat. This is probably due, however, to the paucity of our

Ax

jaar ees ienete caw ewan tO

Fig. A. Diagrammatic figure of Leidyopsis sphaerica gen. novy., sp. nov. Eeto-
plasm drawn in sections to show structure of its different parts.

Abbreviations: alv. l., alveolar layer; bas. gr., basal granules; centrobleph.,
eentroblepharoplast; ect., ectoplasm; end., endoplasm; f. b., food bodies; j.,
flagella; long. my., longitudinal myonemes; n., nucleus; obl. f., oblique fibers;
oper., operculum; per., periplast; sur. rdg., surface ridges. X 400.

material, as a larger number of individuals would present room for
greater variations. It is nearly spherical in shape, its length only
a few microns greater than its width, due to the cone-shaped pro-
jection of the anterior end of the body. The length varies from
165 to 190» and the width 160 to 185y. Figure 1 of plate 13 is that
of an individual 182 in length and 176u in breadth. These pro-
portions vary considerably, particularly in stained material where the
body becomes flattened and relative proportions almost entirely lost.
Measurements are of value only when made from the living organism.

The body is perfectly symmetrical in outline, a condition found
in only a few protozoans. The shape is that of a sphere with the

102 University of California Publications in Zoology [ Von. 20

anterior end drawn out into a short cone-shaped projection. This
is terminated by a small, transparent, caplike structure, the oper-
culum (fig. A, oper.), which covers a cup-shaped depression (pl. 13,
fig. 10). This is similar to the structure of the anterior end of
Trichonympha campanula. No evidences have been found which
would indicate the function of this peculiar formation. It is the
only part of the surface of the body bearing any resemblance to a
eytostomal opening into the endoplasm, yet we have no evidence that
it is used for that purpose.

The surface of the anterior and sometimes even of the middle
portions of the body is marked by longitudinal ridges which extend
from the operculum posteriorly (fig. A, surf. rdg.). In the posterior
regions these are entirely lacking, the surface having a smooth,
unbroken outline.

NEUROMOTOR SYSTEM

The neuromotor system of Leidyopsis resembles that of T'richo-
nympha. It consists of a highly developed, anteriorly located
eentroblepharoplast, numerous flagella, and two sets of fibers, the
oblique fibers in the ectoplasm and the longitudinal myonemes in the
endoplasm. The transverse myonemes of Trichonynvpha are not
found in this form and the others are not so well developed as are
those of that genus.

EcTopuaAsMIC STRUCTURES

The division of the body into ectoplasm and endoplasm is clearly
marked only in a narrow zone surrounding the cone-shaped anterior
end of the body. Here it presents the three distinet layers so striking
in the ectoplasm of T'richonympha, but these soon fade out, though
the myonemes and surface ridges can be traced farther posteriorly,
the latter usually having a length of one-third to one-half that of
the body. Anteriorly the ectoplasm is thick around the base of the
cone, becoming thin posteriorly until it merges into the thin periplast
of the middle and posterior regions of the body (fig. A). It is com-
posed of an outer layer of surface ridges ( fig. A, surf. rdg.), an
alveolar layer (alv. l.), and a narrow, inner ectoplasmie layer. These
are traversed by the oblique fibers and also contain the basal granules
of the flagella. These separate structures will now be discussed in
detail.

1919] Kofoid-Swezy: Leidyopsis sphaerica 103

SurFrace Riwces: The outer layer of ectoplasm is raised in narrow
longitudinal ridges (fig. A, swf. rdg.) from the crests of which spring
the flagella. The ridges extend from the base of the operculum at
the anterior end posteriorly for about one-third to one-half the length
of the body. Anteriorly they are very narrow and placed close
together, the ends forming an irregular, wavy line around the base
of the operculum (pl. 13, fig. 10). Posteriorly they spread out in
fan-shaped or radiating lines, imereasing in number by the inter-
position of new ridges. Their course is longitudinal without spiral
twisting, and when the limits of the differentiated eectoplasm are
reached they disappear in the thin periplast of that region.

Locomotor ORGANELLES: These are confined to the anterior third
of the body, and consist of long flagella, approximately equal in length
and arising in longitudinal rows from the crests of the surface ridges.
They are most numerous anteriorly on the cone-shaped portion of the
body, where they form a dense mass which retains the dark color of
iron haematoxylin. A single flagellum alone does not show the stain
nor do the ends which stream out from this mass. Collectively, how-
ever, they are usually stained so darkly as to obscure the nucleus and
other structures beneath them. The spreading apart of the surface
ridges distally results in a distinet thinning of the coating of flagella
behind the narrowed anterior end of the body.

Each flagellum arises from a minute basal granule below the
surface ridges, passes up through the ridge and leaves the crests as
a single thread. Each basal granule is connected with a minute fibril
from the oblique fibers, apparently in the same manner as in T'rico-
nympha campanula. These fibrils and basal portions of the flagella
give a finely striate appearance to the layers of ectoplasm through
which they pass.

The flagella stream outward and backward over the surface of the
body and in the living flagellate are in constant motion.

OsLIQuE Freers: These are not so prominent in Leidyopsis as
in Trichonympha, owing partly to their smaller extent, and partly
to the difficulty of differentiating them from the mass of dark flagella.
When the flagella have been sufficiently destained to allow observa-
tion of the structures beneath them, the latter have also completely
lost their color, and only the most careful focusing enables the observer
to distinguish between the flagella and the equally slender oblique
fibers immediately beneath.

The oblique fibers form an integral part of the centroblepharoplast

104 University of California Publications in Zoology [ VoL. 20

complex. They arise from its basal lobes as branches which break
up into the minute, threadlike oblique fibers. These follow an oblique
course posteriorly, continually giving off branches, part of which go
to the basal granules of the flagella and the remainder cross and
intercross in an irregular anastomosing network through the eectoplasm
of the body (fig. A, ob. f.). Near the distal limits of the ectoplasmie
zone these fibers fade out and disappear.

In their structure they are homogenous and not granular, and
their width is about equal to that of the flagella. The smaller branches
leading out to the basal granules are more slender. At the anterior
end, near the basal lobes of the centroblepharoplast, they take a darker
stain than is the case farther posteriorly, though this may be largely -
due to their slightly greater thicknéss in the posterior region.

CENTROBLEPHAROPLAST: The oblique fibers are intimately related
to the centroblepharoplast. This organelle in Leidyopsis differs but
little if any from that of Trichonympha. It forms a tubular or rod-
shaped structure in the pointed anterior end of the body (fig. A,
centrobleph.; pl. 13, figs. 1, 3), from the ‘base of which stream out the
oblique fibers.

The tubular part of the centroblepharoplast is composed of a
number of rods or strands which extend to the base of the cuplike
depression at the anterior end of the cone-shaped portion of the
body. Here the ends are joined together by a circular band of darkly
staining material, the center of which is occupied by the core of endo-
plasm (pl. 13, figs. 2, 10, 13). The tube extends posteriorly to near
the base of the cone where it expands into a collar-like structure com-
posed of several large, irregular lobes, which may sometimes be united
into a solid band encircling the base of the tube (pl. 13, fig. 3).
Distally these lobes fray out into the oblique fibers.

The center of the tube is occupied by a slender core of endoplasm
continuous with the endoplasm of the body. This extends up to the
base of the small, cup-shaped depression, where it may be seen as a
light area within a dark ring (pl. 13, fig. 10).

The tubular part of the centroblepharoplast complex may present
some modifications, such as the formation of short, spinelike processes
extending out from the tip (fig. 3) or along its sides (fig. 1). At the
time of division the cuplike depression and operculum disappear,
leaving the tip of the centroblepharoplast complex exposed at the
surface. The entire structure takes a deep stain with iron haema-
toxylin, making it a conspicuous part of the body in stained prepara-
tions.

1919] Kofoid-—Swezy: Leidyopsis sphaerica 105

Anvreouar Layer: This is closely associated with the oblique fibers,
occupying the same region of ectoplasm. It is difficult to demonstrate
and sometimes appears to be entirely lacking. This, however, seems
to be due to the small extent of the ectoplasmic layer which it
covers in those eases. In only a few specimens does it extend as far
posteriorly as do the other ectoplasmic structures. It seems to be
the first part to disappear in the thinning of the ectoplasm, which
takes place a short distance behind the base of the cone.

The alveoli are most prominent at the time of division and appear
in surface view as small, colorless spheres, varying considerably in
size, and not closely packed together (pl. 14, fig. 24). In the ordinary
trophozoite the arrangement is more compact, with the individual
alveoli of smaller size.

ENDOPLASMIC STRUCTURES

The endoplasm may be divided into two regions, anterior and
posterior, but with no definite boundary line separating them. The
distinction between the two portions is rather less pronounced in this
form than in the different species of Trichonympha.

The anterior part is relatively small, extending but a short dis-
tance behind the nucleus (pl. 13, fig. 1). It is granular, without
alveoli or food inclusions. The part immediately surrounding the
nucleus is usually more dense than the remaining portion, evidently
the result of greater metabolic activity.

The posterior region of endoplasm is coarsely vacuolate in structure
and is often abundantly filled with food bodies (fig. A, f.b.). These
are sometimes found enclosed in food vacuoles but more frequently
are found lying free in the plasma. They consist of particles of
wood, small flagellates, bacteria, or other small bodies that may be
present in the intestinal contents. The method of ingestion of these
is entirely unknown. No eytostome is present unless it is possible for
the cup-shaped depression at the anterior tip of the body to assume
that function. Neither the feeding reactions nor defecation of Leidy-
opsis have been observed.

LonerrupinaL Myonemes: These myonemes are found in the
outer zone of endoplasm, immediately beneath the ectoplasm of the
anterior part of the body, and coextensive with it. They are granular
in structure and form straight, longitudinal lines radiating out from
the region of the centroblepharoplast (fig. A, long. my.). Their
connection with that structure, if any exists, could not be clearly
detected.

106 University of California Publications in Zoology [ Vor. 20

Near the distal limits of the ectoplasmic differentiation the longi-
tudinal myonemes fade out in the endoplasm, without showing distinet
attachment areas. Their function here, as in Trichonympha, seems
to be concerned with the mobility of the anterior tip of the body.
The globular form of Leidyopsis as compared with the elongate one
of Trichonympha allows for much less activity, yet the same type of
sidewise movements, though greatly restricted, may be observed in
the living, active flagellate.

Nucueus: The nucleus is found in the anterior part of the body,
a short distance posterior to the centroblepharoplast (fig. A, n.). It
is a rotund ellipsoid, with its longer axis, which lies in the transverse
plane of the body, exceeding its shorter axis by nearly a third of its
own length. No rhizoplast could be detected between it and the centro-
blepharoplast.

The structure of the nucleus is like that of Trichonympha cam-
panula. The membrane is thin and overlies a narrow granular zone,
beneath which is an alveolar zone. These alveoli are rounded out-
wardly where they abut upon the granular zone, with the remaining
facets closely pressed together. The alveoli are fairly uniform in
size, yet in stages which show distinct chromosome changes prior to
the prophase of division, great modifications may often be noticed.
The alveoli may be fewer in number and larger in size. This may
be the result of a breaking down of the walls between several adjacent
alveoh, since in the early prophase their disappearance is usually
complete.

The remainder of the nucleus inside the alveolar zone is occupied
by an irregular linin network in which loose ends may often be
detected. The network is usually encrusted with chromatin, with
large granules of the same substance at the nodes. The disposition
of the chromatin, however, varies considerably in different individuals
and is probably conditioned by the chromosome cycle, which here
apparently follows a course similar to that previously outlined for
Trichonympha campanula (Kofoid and Swezy, 1919c). The nuclei
shown in figures 5 to 8 on plate 13, give different phases of nuclear
structures in individuals which do not yet give other indications of
the approach of division. In these nuclei the network progressively
breaks up, the chromatin moving out from the granules along the
threads which become thicker and split lengthwise (fig. 6). From
these threads the definitive chromosomes are produced.

1919] Kofoid-Swezy: Leidyopsis sphaerica 107

The nucleus of Leidyopsis is further distinguished by the presence
of a small, coiled chromatin rod similar to the heterochromosome of
Trichonympha campanula. This is isolated in a small, clear vesicle
at one side of the central chromatin mass and partially imbedded
within it.

BINARY FISSION

The process of binary fission in Leidyopsis sphaerica shows a
close similarity to the various phases of binary fission in T'richo-
nympha campanula. It is characterized by the longitudinal division
of the centroblepharoplast complex and of the ectoplasmiec structures,
the formation of a paradesmose, precocious, longitudinal splitting
of the chromosomes and pseudosynapsis. Owing to the scanty num-
bers of these flagellates which have been present in any one host, we
cannot present the full details of the different stages. The close
similarity between the phases that we have secured and those of
the other species, however, would seem to suggest that the remaining
stages of Leidyopsis are also similar. In the following discussion,
therefore, the different phases are interpreted in the hight of our fuller
knowledge of the mitotic phenomena of Trichonympha.

Division oF EcropuasMICc STRUCTURES

The first evidences of division in the extranuclear structures of
the body are found in the centroblepharoplast. This structure divides,
beginning at the base and splitting longitudinally to the tip. The
basal masses become separated into two equal parts (pl. 13, figs. 12,
13), taking with them their attached fibers and motor organelles.
This divides the entire ectoplasmie layer, leaving a constantly increas-
ing strip of endoplasm between them as they move apart. As the two
halves of the centroblepharoplast separate, a broad band, the parades-
mose, is formed between them (pl. 13, figs. 15, 16). This is attached
directly to the bases, leaving the tubular portions of the centroble-
pharoplasts standing out from it at right angles, or nearly so. In
figures 15 and 16 these parts are omitted, as the structures are viewed
from the posterior end of the organism and show the relations of the
paradesmose and the basal lobes of the centroblepharoplasts. Here,
as in Trichonympha, with the final separation of the tips of the centro-
blepharoplast, each half develops into a tube similar to the original
structure. This seems to take place immediately after separation of
the halves.

108 University of California Publications in Zoology [ Vor. 20

The further course of division of the ectoplasmie structures shows
a continued separation of the two portions until they come to lie on
opposite ends of the organism, connected by the spindle and para-
desmose (pl. 14, fig. 17). The relatively large portion of the body
which is not covered with ectoplasmic structures and flagella, renders
this separation very conspicuous in these stages. The completion of
these structures for each daughter cell is partly the result of new
outgrowths and partly the readjustments of those derived from the
parent cell. At the time plasmotomy occurs these may be completed
or may still be in the process of formation (pl. 14, figs. 24, 25).

Mirosis

The first evidences of the approach of mitosis may be looked for
in the nucleus. How early these appear cannot be stated, but, as in
the case of Trichonympha, the relative abundance of individuals show-
ing nuclear organization leading to the appearance of distinet chromo-
somes, would suggest that it begins soon after the completion of a
previous division period.

Propuase: The chromatin of the nucleus may be disposed in
large granules (pl. 13, fig. 2) or in smaller granules with the network
connecting them thickly encrusted with chromatin (fig. 4). In the
change from one condition to the other there seems to be a reduction
in the amount of chromatin. The nucleus, however, becomes enlarged
hence the reduction may be more apparent than real. The chromatin
of the granules continues to move out along the network until the
threads of the latter have become greatly thickened and the granules
have disappeared (pl. 13, fig. 5). This produces a coarse, heavy
network in which many free ends may be detected (fig. 12). These
are most frequently found at one side of the nucleus, with the opposite
side staining more densely and presenting an unbroken outline. <A
further disintegration of the network is shown in figure 9 of plate 13.
Here its component parts are forming threads which begin to take on
the appearance of chromosomes on one side of the nucleus, while the
other still retains its network formation.

The threads thus formed split longitudinally (pl. 13, fig. 6), while
at the same time becoming thicker, with the chromatin arranged in
distinct chromosomes. The outer alveolar zone of the nucleus dis-
appears, though this may frequently occur at an earlier stage. The
chromosomes, which at first are straight or irregularly twisted or bent

1919] Kofoid-Swezy: Leidyopsis sphaerica 109

(pl. 18, figs. 7, 8, 11), gradually assume a V-shaped form, and the
cloudy appearance surrounding them disappears, leaving them dis-
tinet against a clear background (fig. 16).

These different phases of nuclear organization are almost identical
with those previously described for Trichonympha campanula. There
is some suggestion in figure 16 of plate 13 that the chromosomes are
arranged in pairs, as in Trichonympha. This point, with an exact
count of the number of chromosomes, could not be as clearly made
out here as in the other species, owing to the small number of individ-
uals under observation. The number of chromosomes seems to be
slightly less than that of Trichonympha, though this cannot be stated
with certainty. In the prophase this seems to be forty-eight (pl. 13,
fig. 15), with a reduction to twenty-four in the later stages (pl. 14,
figs. 18-22). The process of pseudosynapsis by means of which this
reduction takes place, cannot be figured from our material but is
evidently similar to the same process in Trichonympha.

MerapHase: This stage also has been lacking in our material.
The appearance of the following anaphase (pl. 14, fig. 17) would
suggest in part the probable mode of procedure. In the late prophase
the nucleus had taken its place close against the paradesmose (pl. 13,
fies. 15, 16), and elongated until its length is equal to that of the
paradesmose. Spindle fibers are formed from the ends of the parades-
mose or bases of the eentroblepharoplasts, and to these the chromo-
somes become attached, with a single fiber from each pole attached to
the end of the chromosome lying nearest it. The nuclear membrane
remains intact throughout the entire process of mitosis.

AnapHase: A slight shortening of the spindle fibers assists in the
separation of the chromosomes, but apparently this is more dependent
upon a lengthening of the entire nucleus in the equatorial plane than
upon any other factor. The spindle fibers show but little contraction
up to the time of their disappearance in the late telophase, and the
chromosomes are not drawn to the poles (pl. 14, fig. 23). The nucleus
becomes greatly elongated and constricted in the middle until the
two halves are connected by a slender line of nuclear material. The
paradesmose also increases in length.

As the chromosomes separate the heterochromosome may usually
be found near the ends of the two groups (pl. 14, fig. 19). As in
Trichonympha it is the lagging chromosome and is apparently the last
one to divide. In a later stage this assumes a coiled shape preparatory
to the formation of the vesicle by which it is later enclosed (fig. 20).

110 University of California Publications in Zoology [ Vor. 20

Its course in the prophase has not been followed but it apparently
retains its isolated position throughout.

TeLopHase: As the two centroblepharoplasts with their related
structures move towards opposite ends of the cell, the slender band
connecting the two halves of the nucleus becomes ruptured, while the
paradesmose fades out and begins to disappear at its middle portion
(pl. 14, fig. 21). The daughter nuclei at this time are spindle-shaped
both ends drawn out to a slender point, with the chromosome lying
in a roughly subparallel band near the center. The point of the
nucleus opposite the poles is withdrawn (fig. 20), the spindle fibers
disappear, and the nucleus loses its connection with the centro-
blepharoplast (fig. 18) and begins to round up. ‘This part of the
process is usually completed before the chromosomes begin to undergo
reorganization (pl. 14, figs. 22, 23, 25). Plasmotomy may occur
before this takes place (fig. 25) or it may*be delayed until reorganiza-
tion of the nucleus and the reformation of the neuromotor system
of each daughter cell has been completed (fig. 24).

RELATIONSHIPS

The close similarity of the various phases of division, combined
with the striking resemblances in their morphological characters,
indicate at once a close relationship between Leidyopsis and Tricho-
nympha. These resemblances are found in the differentiations of the
ectoplasm, the neuromotor system, and the interrelations of its various
parts, the lack of a distinct eytostome, and the division of the endo-
plasm into a uniformly granular anterior portion and a posterior part
filled with coarse alveoli and food particles, indicative of holozoic
nutrition.

The differences between these two genera are few, but are im-
portant from a taxonomic standpoint. The most striking one is found
in the number and arrangement of the flagella. In Trichonympha
these cover two-thirds or more of the surface of the body and are
divided into three distinct groups, an anterior, middle and posterior
eroup. The anterior group is composed of long flagella, the middle or
lateral group, which is the largest in extent, of short, cilia-like flagella,
and the posterior group of flagella, which are twice or even three
times the length of those in the anterior group. In Leidyopsis the
middle and posterior groups of flagella are lacking, leaving only the
group of long flagella at the anterior end of the body.

1919] Kofoid-Swezy: Leidyopsis sphaerica 111

In view of the taxonomic importance attached to the number of
flagella in other orders of the Flagellata, where these organelles serve
as the chief features of classificatory value, these differences in their
flagellar coating seem to create a generic distinction between the two
forms and not a specific one only. We have therefore proposed a new
genus in the order of Trichonymphidae for this form with the follow-
ing characters:

Leidyopsis gen. nov.: Trichonymphidae with ectoplasmie differen-
tiations found only on the anterior third of the body, the remainder
of which is covered with a thin pellicle; neuromotor system consisting
of one anterior group of long flagella, oblique fibers, longitudinal
myonemes, basal granules and centroblepharoplast. One species only,
the type, Leidyopsis sphacrica sp. nov. from intestine of Termopsis
angusticollis Walker from Berkeley, California.

SUMMARY

1. Leidyopsis sphaerica is a flagellate of the Trichonympha type,
with the same structural characteristics but with a lower type of
evolutionary development.

2. It is characterized by the presence of a neuromotor system
consisting of a highly developed centroblepharoplast, oblique fibers,
basal granules and related flagella, restricted to a single anterior
zone, a differentiated ectoplasm on the anterior third of the body.
surface ridges from the erests of which spring the flagella, and an
alveolar layer. Longitudinal myonemes are found in the endoplasm.

3. The nucleus is anterior in position and is distinguished by the
presence of a heterochromosome contained within a small vesicle.

4. Nutrition is holozoic but its methods of feeding are unknown.

5. Division is of the trichonymphid type. The centroblepharo-
plast, with its connecting motor organelles and ectoplasmic structures,
divides longitudinally with the formation of an extra-nuclear para-
desmose between them as the two parts separate.

6. Division of the chromosomes is longitudinal, and takes place
prior to the appearance of division in other structures of the body.
About forty-eight chromosomes are formed. This number is reduced
in pseudosynapsis to twenty-four.

7. The nuclear membrane remains intact throughout mitosis, with
the spindle fibers arising from the ends of the paradesmose and centro-
blepharoplast and passing through it.

112 University of Califorma Publications in Zoology [ VoL. 20

8. Leidyopsis is a member of the family Trichonymphidae Kent, in
the order Hypermastigina Grassi. It stands close to the genus T'richo-
nympha, to which it is related both morphologically and in its division
processes.

Transmitted October 25, 1918.
Zoological Laboratory, University of California,
Berkeley, California.

1919] Kofoid-Swezy: Leidyopsis sphaerica 113

LITERATURE CITED

Korom, C. A., and Swezy, O.

1919a. Parasites of the termites. I, On Streblomastiax striz, a polymastigote
flagellate, with a linear plasmodial phase. Univ. Calif. Publ. Zool.,
20, 1-20, pls. 1, 2, 1 fig. in text.

1919b. Studies on the parasites of the termites. II, On Trichomitus termitidis,
a polymastigote flagellate with a highly developed neuromotor sys-
tem. Ibid., 20, 21-40, pls. 3, 4, 2 figs. in text.

1919c. Studies on the parasites of the termites. III, On Trichonympha campa-
nula sp. nov. Ibid., 20, 41-98, pls. 5-12, 4 figs. in text.

EXPLANATION OF PLATES

All figures of Leidyopsis sphaerica gen. noy., sp. nov., from Termopsis angusti-
collis Walker, stained with iron haematoxylin and drawn with camera lucida.
Magnification 800 unless otherwise stated.

PLATE 13

Fig. 1. Optical section of trophozoite showing thickness and extent of ecto-
plasm, centroblepharoplast and nucleus with the two portions of endoplasm.
x 300.

Fig. 2. Vertical view of anterior end showing centroblepharoplast with central
endoplasmic core, oblique fibers and nucleus slightly misplaced.

Fig. 3. Centroblepharoplast with a few of the radiating oblique fibers.

Fig. 4. Nucleus of the vegetative trophozoite; note the inner region filled
with a coarse network with granules interspersed along its nodes, the surrounding
alveolar zone and the outer granular region beneath the nuclear membrane.

Fig. 5. Nucleus showing the heterochromosome and its vesicle. Alveolar
region has disappeared.

Fig. 6. Early prophase nucleus showing formation and splitting of the
chromosomes.

Fig. 7. Early prophase with the alveolar region breaking up.
Fig. 8. Same stage as in figure 7.
Fig. 9. Another phase of the breaking up of the chromatin network.

Fig. 10. Anterior portion of the body, showing the cuplike depression with
its covering operculum, the tip of the centroblepharoplast, and the surface ridges.

Fig. 11. Nucleus showing the full number of chromosomes.

Fig. 12. Beginning of the splitting of the centroblepharoplast and ectoplasmie
structures.

Fig. 13. <A slightly later stage of same process as shown in figure 12.

Fig. 14. Separation of the centroblepharoplast with the completion of the
paradesmose connecting them.

Fig. 15. Same stage as in figure 14.

Fig. 16. Prophase of nucleus and the first appearance of the spindle fibers.
Dark areas at the end of fibers are parts of the centroblepharoplasts.

[114]

PLATE 14

Fig. 17. Anaphase; note flagella and other ectoplasmic structures attached
to the centroblepharoplasts. X 300.

Fig. 18. Telophase; paradesmose has been absorbed, the spindle fibers and
connection of nucleus and centroblepharoplast are lost, but nuclear reorganization
has not yet begun.

Fig. 19. Enlarged nucleus of figure 17. One lagging heterochromosome may
be seen.

Fig. 20. Telophase of nucleus; spindle fibers still present as well as attach-
ment to centroblepharoplast. Heterochromosome may be found near ends of the
other chromosomes. X 1250.

Fig. 21. Telophase; remains of paradesmose are seen above nuclei; flagella
are omitted and the oblique fibers shown with their attachment to the centro-
blepharoplasts.

Fig. 22. Telophase of nucleus. It has rounded up and the chromosomes are
losing their parallel position preparatory to forming a network.

Fig. 23. Telophase: beginning of constriction of the body. Note different
relative positions of nuclei.

Fig. 24. Telophase; nuclear reorganization nearly complete as well as the
formation of the new ectoplasmic structures. The alveolar zone, oblique fibers
and flagella are shown.

Fig. 25. <A daughter flagellate, after plastmotomy, which here has taken place
before the reorganization of the nucleus.

[116]

UNIV. CALIF PUBL. ZOOL. VOL 20 | KOFOID AN SWEZ

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ZOOLOGY

Vol. 20, No. 5, pp. 117-144, plates 15-17, 2 figures in text April 23, 1920

ON THE MORPHOLOGY AND MITOSIS OF
CHILOMASTIX MESNILI (WENYON), A
COMMON FLAGELLATE OF THE
HUMAN INTESTINE

BY

CHARLES A. KOFOID
AND
OLIVE SWEZY

CONTENTS
PAGE
TIER ERR BUN a ee ee Maly
NGI TAI RNS Se ee cS 119
Morphology: :.----.csns--- = Alilt)
The active flagellate ...... 25 dally)

-- 123

The neuromotor system -... =
--. 129

The encysted flagellate .... :
AVIS ORIS tees e cos ccs, eat se esa Soot = 133)
IDS GIDUSSMIOS cet Bee ao ie ee 135
SIPURA: ee eeteee hee ee ere ee a eo ee 137
SLTIREIADARIURG) OYE ee ea yc 138

INTRODUCTION

The present paper records certain observations on the free and
encysted stages of Chilomastix mesnili (Wenyon), a common flagellate
of the human digestive tract, made, in part during the course of
examinations of stools of soldiers, primarily for Endamoeba dysen-
teriae, and in part at Berkeley in conjunction with the work of the
Division of Parasitology of the Bureau of Communicable Diseases
of the California State Board of Health. The army examinations
were made at the United States Debarkation Hospital No. 3, New

118 University of California Publications in Zoology [Vou. 20

York, N. Y., from December 28, 1918, to May 30, 1919, on 3301 stools
from 2300 men who had been overseas, mainly in France, and from
576 men, chiefly of the medical detachments and food handlers, who
had seen only home service. In the former group there were 97
persons infected or 4.2 per cent, and in the latter 20, or 3.5 per cent.
At Berkeley 1836 stools of 534 persons have been examined, with
28 cases or 5.3 per cent of infection.

Although a common and widely distributed parasite of man,
this organism has often been regarded as a tropical form. It has
undoubtedly been confused, in much of the clinical literature, with
other intestinal flagellates, especially with Trichomonas, and _ its
structure, relationships, and encysted stages have been imperfectly
described and figured and sometimes even misinterpreted as those of
another flagellate. Conclusions as to its pathogenicity are also at
variance.

The data here presented add to our knowledge of its wide dis-
tribution, and give, for the first time, a full analysis of its finer
structure, especially of its neuromotor apparatus and of its mitosis
in the eneysted stage. This affords a satisfactory basis for conclusions
as to its systematic relationships, a suggestion as to the holdfast fune-
tion of the eytostome, and a critical basis for the accurate detection
of this genus and its differentiation from the other parasites with
which it is associated in the intestine of man and other mammals.

We present a full analysis of the structure of Chilomastiz,
especially of the neuromotor system, and by the homology of its
elements find that Chilomastix is closely related to Giardia, and is a
transition form from the monozoie to the diplozoic polymastigote
flagellates.

Our analysis has added the following organs to those hitherto
detected: the centrosome, nuclear rhizoplast, peristomal rhizoplast,
peristomal fiber, paradesmose, and the spiral groove (as a permanent
organ). We have also detected and analyzed intracystie mitosis.
Our interpretations of the precise relations of the flagella to the
blepharoplasts and of the organs of the eytostomal rim, which we
designate as parastyle and parabasal, differ from those of previous
writers and make possible a fuller comparison of Chilomastix and
Giardia, and establish intimate relationships between the two genera.

1920] Kofoid—Swezy: Morphology and Mitosis of Chilomastiz 119

ACKNOWLEDGMENTS

Acknowledgments are made to Lt. Col. E. H. Schorer, M.C.,
U.S.A., in charge of the Army Laboratory, for his efforts to provide
facilities and staff for the work, and to 2nd Lt. S. I. Kornhauser for
his technical aid in making preparations upon which this work is
based.

A grant from the Division of Medicine and Related Sciences of
the National Council of Research made possible the codperation of the
junior author.

MORPHOLOGY
Tue Active FLAGELLATE

The shape of the body of Chilomastix is normally elongate pyriform,
with the broad end anterior and the sides more or less convex, accord-
ing to the degree of extension or contraction. Its length is generally
two to four times its greatest width which is 0.2 to 0.3 of the total
length from the anterior end. The anterior end is rather broadly
rounded, sometimes flattened dorso-ventrally, and the curvature of
the two sides is dissimilar. This dissimilarity is masked in the other
irregularities due to the spiral groove. This asymetry is fundamental ;
it is caused by the relative positions of nucleus and ecytostome, but is
variously modified by the movements of the latter and by the torsion
of the body. In ventral view (fig. A) the eytostome lies to the right
side of the body (left of the figure) and the nucleus to the left. These
relations persist in the cyst and at mitosis.

Posteriorly the body contracts rather abruptly into a short taper-
ing tail, whose length is 0.2 to 0.3 of the total length. This tail may
stretch out in a long thread, sometimes nearly as long as the body,
when autotomy of the cytoplasm is in process, showing its great
plasticity. It is apparent that the eytostome of Chilomastix is mor-
phologically more highly differentiated than that of Trichomonas,
Eutrichomastiz, or Embadomonas. Specialization in function may
well attend this structural development. Adhesion of the parasite
to the intestinal wall of the host by Giardia, and possibly also by
Chilomastix, is certainly one factor of no small clinical significance

120 University of California Publications in Zoology [Vou. 20

in providing a structural and functional basis for the persistence of
infections by these two flagellates during and after palliative treat-
ment.

The dimensions of the active flagellate are quite variable, both in
size and in extension. Twenty active individuals averaged 19.6» in
length, with a range of 13 to 24. Stained flagellates range from 9.6

SS ee a ae SSeS Ss Sones ece prim. bleph.
“Deeet sesh os ess esasscs se sec. bleph.

tr. rhiz.
Sas prim. bleph.

——.nuc. rhiz.

Fig. A. Chilomastix mesnili (Wenyon). Normal flagellate viewed from the
ventral or oral side and showing all the structures of the body. X 6370.

Fig. B. Cyst of Chilomastiz mesnili, viewed from the ventral or oral side.
X 6370.

Abbreviations: cent., centrosome; cent. k., central karyosome; cyst, cyst wall;
cyt., eytostome; cyt. fl., eytostomal flagellum or undulating membrane; int. rhiz.,
intranuclear rhizoplast; J. a. jl., left anterior flagella; nue., nucleus; nue. rhiz.,
nuclear rhizoplast; par. b., parabasal body; parast., parastyle; perist. f., peri-
stomal fiber; prim. bleph., primary blepharoplast; r. a. fl., right anterior flagellum;
sec. bleph., secondary blepharoplast; spir. gr., spiral groove; tert. bleph., tertiary
blepharoplast; tr. rhiz., transverse rhizoplast.

1920] Kofoid—Swezy: Morphology and Mitosis of Chilomasticx 121

to 15u, having contracted in the process of fixation. The small forms,
3p in length, noted by Prowazek and Werner (1914), Wenyon (1915),
and Chalmers and Pekkola (1918), may be Chilomastix. We find
such small forms with the larger ones but have not been able to prove
eritically that they are Chilomastiz.

The cytoplasm of Chilomastix is enclosed in a very resistant, elastic
pellicle, which permits rapid changes in contour and deformation.
It is crowded in the active stage with vacuoles of varying size, some
of which contain bacteria (pl. 15, fig. 4), though generally they show
only a fluid material. We find no evidence of a specialized region
of ingestion in the eytostomal pouch, though the locality where the
parastyle and parabasal are in juxtaposition is apparently adapted to
this end. There is no specialized structure for the elimination of
dejecta. This appears to be accomplished, in some instances, by the
autotomy of cytoplasm and its contents at the posterior tip. In one
instance a long rod-shaped bacillus was seen lying transversely in
such a terminal mass.

The spiral groove (spur. gr., figs. A, B) is a peripheral modification
of the cytoplasm, seemingly peculiar to the genus Chilomastix, which
is intimately connected with the very evident torsion of the body in
the active flagellate stage. It is a special trough or channel impressed
in the cytoplasm of the body, running from the dorsal face of the
anterior end posteriorly in a spiral course from the mid-dorsal to the
right side and obliquely across the ventral face to the left, making
approximately a full turn in its sinistral course. This trough in
contracted, staimed individuals is a narrow, shallow channel about
0.25 of the diameter of the nucleus in width (pl. 15, fig. 1). In active
flagellates 1t may be a deep, wide trough, which may be as wide as
the nucleus anteriorly and cleave the body for nearly a third of its
diameter, or it may be a narrow depression tapering posteriorly and
terminating near the very tip. The floor of this groove is somewhat
less coarsely granular than the rest of the peripheral cytoplasm. It
is evidently a contractile area varying in the depth of its indentation.
In the eyst (spir. gr., fig. B; pl. 16, fig. 10) it is a meridional, faintly
marked, clear streak on the right side of the nucleus and eytostome,
running nearly from pole to pole. It appears to have lost entirely
all trace of the torsion of the active flagellate stage after the encysted
phase is established.

The nucleus (nue., fig. A) 1s a spheroidal, rarely ellipsoidal, strue-
ture located close to the anterior border of the body in the active

122 University of California Publications in Zoology [Vou. 20

flagellate, where it usually is appressed against the blepharoplasts
(pl. 15, fig. 5). In other cases it is shghtly withdrawn, spinning out
the rhizoplast between the centrosome on its anterior face and the
primary blepharoplast. It lies to the left of the cytostome, asym-
metrically and excentrically in the cytoplasm as a whole, both in the
free and encysted stages, thus adding an important factor in the
fundamental asymmetry of the organism.

As eneystment approaches, and even before the cyst wall is formed,
the nucleus withdraws from the anterior end posteriorly to a position
slightly in advance of the middle of the cyst.

The close physical connection between the blepharoplasts at the
base of the flagella and the nucleus, the metabolic center of the cell,
throughout the period of flagellar activity, is indicative of the intimate
relation which the nucleus bears to these energy-expending structures.
It can not be merely the physical tug of the moving flagella which
pulls the nucleus to its anterior position, for the blepharoplasts retain
this anterior location when the nucleus moves posteriorly, the con-
nection between them being retained merely by the slender nuclear
rhizoplast. The rounding-up process in the cytoplasm of the eneysting
individual doubtless exerts some pressure leading to spatial readjust-
ments, but the translation of the nucleus appears to be out of propor-
tion to this single factor. In the active phase of the organism the
nucleus is nearest to the center of metabolic activity, and in the passive
phase of eneystment it tends to assume a place as near as possible to
the center of the cytoplasmic mass, the eytostomal pouch appearing to
hold it off from the fully central location.

The presence of the centrosome on its anterior border gives evi-
dence of a permanent polarization of the nucleus. The distribution
of the chromatin within the nucleus lends support to this interpreta-
tion. There is a tendency for the chromatin, in the encysted stage,
to accumulate at or near the anterior and the posterior poles of the
nucleus, adacent to and opposite to the centrosome (pl. 16, figs. 8, 9),
in plaques flattened against the membrane. In numbers of instances
a radial fiber can be traced from the centrosome (fig. B) to the central
karyosome, as in Giardia, where the intranuclear rhizoplast occupies
a similar position. Several radial fibers within the nucleus are often
found in the cysts. In other instances (pl. 16, figs. 11, 14) the chro-
matin is broken into several smaller blobs or plaques, which are
distributed on the membrane irregularly and are sometimes connected
by linin threads to the central karyosome. The intranuclear rhizoplast

1920] Kofoid—Swezy: Morphology and Mitosis of Chilomastix 123

is found less frequently in Chilomastix than in Giardia and is not so
readily demonstrated. In some nuclei (fig. A; pl. 15, fig. 5) the
chromatin is confined to a thin uniform sheet on the inner face of
the nuclear membrane and to the central karyosome.

The central karyosome (fig. A, cent. k.) is a small, spheroidal
or angular body at or near the center of the nucleus, joined to the
centrosome by the intranuclear rhizoplast (int. rhiz.) and elsewhere
to the peripheral chromatin masses by radial linin fibers.

Tue NeuroMoToR SYSTEM

In Trichomonas the centroblepharoplast is the common point of
origin of all fibrillar parts of the neuromotor system and generally
lies close to the nuclear membrane. From it diverge the flagella,
marginal fibril, paradesmose, rhizoplast, and, at mitosis, the parades-
mose. In Chilomastix a more complex condition exists in that the
centroblepharoplast complex is broken up into no less than four gran-
ules, each giving rise to two or more outgrowths, and all connected
in sequence on a fiber which has its origin in the central karyosome
of the nucleus.

We use the term neuromotor system here, as elsewhere (Kofoid,
1916; Kofoid and Swezy, 1919), to designate the integrated fibrillar
system uniting the karyosome, centrosome, blepharoplasts, flagella,
and other motor organs, and the fibers of the oral region into one
continuous, structural unit. In Chilomastix this consists (fig. A)
of the centrosome (cent.), three blepharoplasts (prim. bleph., sec.
bleph., tert. bleph.), nuclear rhizoplast (nue. rhiz.), transverse rhizo-
plast (tr. rhiz.), three anterior flagella, one posterior cytostomal
flagellum (cyt. fl.), one parastyle (parast.), the parabasal body (par.
b.), and the peristomal fiber (perist. f.). Taken together these consti-
tute the locomotor and feeding organs of Chilomastic. It is not
improbable that the eytostomal region has also a holdfast function.

This system is most clearly demonstrated in the encysted stage,
in which the cytoplasm is free from confusing and obscuring food
particles, and, what is far more important, the migration of the
nucleus from the anterior end to its median-lateral position exposes
the blepharoplasts and their connections. The approach of mitosis
permits a wider separation of the elements and a clearer inspection
of their structure and relations, especially of the blepharoplast com-
plex, the parabasal, and parastyle. It is essential to have thoroughly

124 University of California Publications in Zoology [Vou. 20

decolorized cysts and to study lateral as well as ventral views to obtain
a clear conception of this organ system.

The centrosome (figs. A, B, cent.) in Chilomastix is a discrete and
separate structure, independent, in both free and encysted stages,
of the blepharoplasts. There is no centroblepharoplast here, as in
Trichomonas, where the two organs are combined in one granule, but
the relations of the two are similar to those found in Giardia. It is
a small granule flattened against the anterior end of the nucleus
where it forms a small hemisphere on the nuclear membrane. As
mitosis ensues in the eyst, it divides and one daughter centrosome
migrates posteriorly on the membrane of the nucleus (pl. 16, figs.
13, 15), spinning out the paradesmose between the two. In early
stages the paradesmose is applied to the nuclear membrane (pl. 17,
figs. 17, 19), but, in later stages, as the daughter nuclei part, it
stretches out between them across the intervening cytoplasm (pl. 17,
fig. 23). At no stage have we seen the centrosome detached from the
nuclear membrane.

The blepharoplast in Chilomastix is a complex of three granules.
In the free stages it is lodged on or near the anterior end of the
nucleus and its structure is generally obscured by the crowding of
organs anteriorly in a restricted area (pl. 15, figs. 2-6). It consists
of three granules or individual blepharoplasts, which may be desig-
nated, according to their positions in relation to the nucleus, as the
primary (figs. A, B, prim. bleph.), secondary (sec. bleph.), and ter-
tiary (tert. bleph.) blepharoplasts respectively. In the free stage
they are appressed against one another so that there may appear
only two blepharoplasts, and the free flagella seem to emerge rather
close together. Closer inspection, however, reveals the fact that the
right flagellum is slightly removed from the other two. In stained
flagellates (pl. 15, figs. 2-6) the nucleus erowds upon the blepharo-
plasts, obscuring the centrosome and rhizoplasts, while the two
blepharoplasts of the right side generally appear as a single granule,
often somewhat larger than the tertiary blepharoplast. The two (in
reality three) granules usually lie at the anterior end of the nucleus
and eytostome, but only one of them is actually attached to the peri-
stomal fiber.

It is in the eneysted stage that the structure, individuality, and
relationships of the three blepharoplasts can be clearly distinguished
(pl. 16, figs. 7, 8) as follows. The left or primary blepharoplast (fig.
B, prim. bleph.) gives rise to two anterior flagella (pl. 15, fig. 1), is

1920] Kofoid—Swezy: Morphology and Mitosis of Chilomastix 125

connected posteriorly to the centrosome on the anterior face of the
nuclear membrane by the nuclear rhizoplast (fig. B, nwe. rhiz.) and
transversely to the secondary blepharoplast (fig. B, sec. bleph) by the
transverse rhizoplast (fig. B, tr. rhiz.). It is somewhat widely sep-
arated from the other two blepharoplasts at the anterior end of the
eytostome in this stage, and also from the nucleus which has migrated
postero-laterally. Because of its connection with and more intimate
relationship to the nucleus, this blepharoplast should be regarded as
primary and the other two as of secondary and tertiary status.

The right anterior or secondary blepharoplast (fig. A, sec. bleph.)
in the free flagellate (pl. 15, fig. 4) les close to or appears to be fused
with the right posterior or tertiary one in a more or less elongated
body. In some eysts not heavily decolorized the two granules can not
be differentiated. In some favorably oriented and strongly decolor-
ized eysts it is possible to distinguish the secondary (sec. bleph.) from
the tertiary blepharoplast (tert. bleph.) and to see the short peri-
stomal rhizoplast connecting the two (pl. 16, fig. 10). The secondary
blepharoplast gives rise anteriorly to the single right flagellum which
is slightly detached from the other two (pl. 15, fig. 3), and posteriorly
to the straight parastyle which underhes the left rim of the eytostome,
and also to the short peristomal rhizoplast which joins this secondary
blepharoplast to the tertiary blepharoplast.

The tertiary blepharoplast (fig. C, ter. bleph.) is at the anterior
end of the rim of the eytostome. It gives rise anteriorly to the peri-
stomal rhizoplast connecting it with the secondary blepharoplast and
posteriorly to the undulating cytostomal flagellum, to the looped peri-
stomal fiber (perist. f.) and to the curved parabasal (par. b.) which
is attached to and posteriorly underlies the peristomal fiber. It is
often obscured by the secondary blepharoplast and by the thickened
anterior ends of the peristomal fiber.

The peristomal fiber is a fine thread encircling the so-called
cytostome in its immediate rim and originating in the tertiary
blepharoplast. Its proximal ends, as they emerge from this granule,
are thickened and continue so for 0.25 to 0.33 of the length of the
cytostome, while beyond this section it is a delicate fibril which
encireles the remainder of the opening. Owing to its location in
the immediate surface of the body, and to its overlying the parastyle
on the left side and the parabasal on the right, it is easily confused
with these structures or overlooked, especially if the stain is not
sufficiently decolorized. Furthermore, the varying degrees of contrac-

126 University of California Publications in Zoology (Vou. 20

tion and expansion of the cytostome give differing pictures of the
relations of the peristomal fiber to the underlying organs.

The deeply stained periphery of the cytostome of Chilomastix owes
its appearance to two structures whose analysis in preparations is most
difficult and whose morphological interpretation offers an interesting
problem. These organs are the parastyle and the parabasal body.
They so much exceed the peristomal fiber in volume and stainability
that they appear largely to have been the structures interpreted as
the cytostome and drawn as its margin by previous investigators
(Brumpt, 1913; Prowazek and Werner, 1914; Kuezynski, 1914; and
De Fonseca, 1916), except Chalmers and Pekkola (1918). The last
named note the distinction between the peristomal fiber and the
underlying stained structures.

The parabasal (fig. A, par. b.) is a deeply staining, curved rod
which runs posteriorly from the tertiary blepharoplast. Its course
follows approximately that of the right margin of the ceytostome but
posteriorly it extends beyond this opening and lies deep in the ecyto-
plasm, curving to the left around the posterior end of the oral pouch,
extending anteriorly for a short distance on its left side, and ending
in a fine point near the posterior end of the parastyle.

In some eases (pl. 16, fig. 8) it appears to adhere to the peristomal
fiber for a short distance, in others it 1s free from that fiber throughout
its whole length. The parabasal is imbedded in the right wall of the
oral pouch. In imperfectly decolorized individuals this whole wall
may retain the stain or the fiber be buried in the lateral sheet of
stained material. The curvature of the parabasal varies according
to the state of its contraction. When the cytostome is wide open the
parabasal may be exposed (pl. 16, fig. 10) imside of the cytostomal
opening, and when the lips are contracted and the cytostome reduced
to a constricted, slipper-shaped opening (pl. 16, fig. 11), the parabasal
is outside of the opening on the right side of the body. It usually
entends posteriorly beyond the end of the cytostomal opening (pl. 16,
fig. 13) but may be ineluded within it (pl. 17, fig. 16). These varying
pictures of its relations to the cytostome are records of the mobility
and exhibit stages of the contraction of the overlying peristomal fiber.
As Chalmers and Pekkola (1918) state, the inner and outer lips may
contract so as almost to touch each other, forming overhanging lobes
(pl. 16, fig. 8) in the anterior half of the opening. Such a lobe may
form on one side only. These movements of the cytostome are sug-
gestive of the food-gripping function observed by the authors cited

1920] Kofoid-—Swezy: Morphology and Mitosis of Chilomastix 127

above and also of a sucker or holdfast action of this region which we
have oceasionaly seen in the free flagellates. The structure, lateral
position, and relation to the cytostome of this parabasal suggest its
analogy to the antero-laterally located, deeply stained, curved struc-
ture near the rim of the cytostome of Giardia, wihch we have called
the anterior peristome (Kofoid and Christiansen, 1915), but we
believe it to be the homologue of the parabasal, and that the peri-
stomal fibers of the two genera are homologues. It appears to bear
the same morphological relation to the cytostomal flagellum that the
parabasal bears to the undulating membrane in Trichomonas. Chal-
mers and Pekkola (1918) also have called this structure the parabasal.

We have found no evidence that the parabasal of flagellates is
a motor or contractile organ and have elsewhere (Kofoid, 1916)
advaneed the interpretation that it is a reserve body connected with
metabolism consequent upon the motor functions of the neuromotor
system. While its changes in shape in Chilomastix are far more
noticeable than those of the parastyle, this might well be due to the
intense activity of the adjacent cytostomie flagellum and of the peri-
stomal fiber and parastyle, rather than to its own intrinsic movement.

In one point our findings are at variance with those of Chalmers
and Pekkola (1918), namely, the origin of the parabasal. They state
(p. 225) that it arises from the second or anterior chromatic particle,
our secondary blepharoplast. We find that in every case where it is
possible to separate the anterior and posterior right blepharoplasts
the parabasal is joined to the posterior one only, that is to the right
posterior or tertiary blepharoplast.

The parastyle (fig. A, parast.) is a slender, straight, or slightly
eurved rod, much smaller and usually more decolorized than the
parabasal. It originates from the secondary blepharoplast, and runs
posteriorly in the left wall of the oral pouch, almost to the posterior
end of that cavity, sometimes overlapping the recurved end of the
parabasal and more or less obscured by the peristomal fiber above it.

The relative shapes of parastyle and parabasal, as seen in a series
of free flagellates and cysts, are instructive as to their relative fune-
tions. While the parabasal exhibits a great variety of shapes showing
the contractility and mobility of the region in which it lies, the para-
style is straighter, has much slighter curvature, and its shapes likewise
appear to be correlated with the state of contraction of the cytostome,
as though it were passively moved by the activity of that region.
There is lack of evidence that it is an active motor organ. It appears

128 University of California Publications in Zoology (Vou. 20

to have the same relation to the neuromotor system that the right
axostyle has in the neuromotor system of the right side of Giardia.
We have called it a parastyle because it is alongside the ecytostome.
It differs from the axostyle of Giardia in not ending in a free flagellum.
The parallelism in the persistence in the cysts of Giardia of axostyles
and parabasals and of the parastyle and parabasal in Chilomastix
is significant of the homology of these structures in the two genera.

The flagella of Chilomastix are four in number and fall into three
categories, the two left anterior (fig. A, l. a. fl.), the right anterior
(nr. a. fl.) and the eytostomal (cyt. fl.). The three anterior ones are
free throughout their whole course, while the fourth is attached to
the cytoplasm and proceeds posteriorly within the cytostome as an
undulating membrane. The two left anterior flagella arise from the
primary blepharoplast. In locomotion these two flagella act in
unison, beating together in the backward stroke, the wave of con-
traction in the inner one slightly preceding that in the outer one.
These two flagella are thrown down against the left side of the body
while the right anterior one beats in the opposite direction and is
thrown down against the right side of the body with a tendeney to
lie for a time in a trailing position. It arises from the secondary
blepharoplast. The length of these three anterior flagella is about
equal to the length of the body.

The cytostomal flagellum (fig. B, cyt. fl.), or undulating membrane,
originates from the tertiary blepharoplast and runs posteriorly in the
floor of the oral pouch within the cytoplasm to or near to the end
of the cytostome. In action it moves in rapid waves of contraction
running posteriorly and almost equalling the width of the oral pouch
in amplitude. The rate, in moribund individuals whose quiescence
permits observation, is two to four beats per second. The amplitude
of the lateral movement dies out distally. We have never been able
to see any evidence of a free end of this flagellum. The fiber which
constitutes it persists in the eneysted stage and can be traced by
careful focussing.

This flagellum corresponds to the filament in the margin of the
undulating membrane of Trichomonas, Trichomitus, and Eutricho-
mastix, the eytostomal flagellum of Chilomastix being, in reality, a
diminutive undulating membrane enclosed in the eytostomal pouch.
It lies adjacent to the parabasal the location and length of which are
correlated with those of this flagellum, as in the trichomonad flagel-
lates. Its presence has led to confusion of Chilomastix with Tricho-
monas in many clinical accounts of human flagellates.

1920] Kofoid-Swezy: Morphology and Mitosis of Chilomastix 129

Tue ENcYSTEeD FLAGELLATE

The eysts (fig. B; pls. 16, 17) of Chilomastix are small, ovoidal,
pyriform, or lemon-shaped bodies in some cases remarkably similar
in general appearance to the smaller cysts of the species of amoeba
with which they are associated in the intestine of man. In fresh
smears in physiological salt solution the cysts are bluish green bodies
with distinct walls and often a few highly refractive granules strewn
within. In iodine-eosin stain the cysts quickly become tinged with
the iodine to a hght yellow, and, according to the amount and
diffusion of the contained glycogen, gradually assume an olive or
brownish tint. If the glycogen is massed, the brown color is very
evident in the glycogen vacuole, if it is-diffuse, a dark olive or brown-
ish tint spreads through the whole cyst. If glycogen is not present
the eyst remains yellow. As the iodine penetrates, the outlines of
the nuclear membrane and of the fibrillar sling in the walls of the
eytostome become visible. The cysts are not uniformly lemon-shaped.
In not a few cases all trace of the pyriform contraction is lacking and
the cyst is ovoidal, spheroidal, or broadly ellipsoidal (pl. 16, figs.
alae)

In the pyriform cysts the length is from 1.21 to 1.24 times the
greatest diameter, which is slightly below the level of the middle of
the body, assuming that the narrower end is the anterior one. They
are thus broadly pyriform in contour. In other instances the neck
of the cyst is not contracted and the contour becomes broadly ovoidal
with the length 1.24 to 1.31 times the greatest diameter which is located
at 0.6 of the total length from the anterior end. In still other instances
the cyst may be spheroidal (pl. 16, fig. 8), with the longer axis reduced
to 1.08 times the greatest diameter and the anterior projection forming
only a very obscure and slight eminence. It is only in lateral view
that this departure from sphericity is detected.

The cyst varies from 6.5 to 9 in length and averages 7 to 7.5p,
while its greatest diameter is from 5.8 to 7.51 and averages 6.5y. Its
dimensions are thus very close to those of a red blood corpuscle, and
to the small races of Hndolimax buetschlii and Endamoeba dysen-
teriae, an important point in the microscopical diagnosis of these
infections.

The cyst wall is a transparent, homogenous substance, without
structural modification except at the tip of the stout projection, where
it is locally thickened across the end to double the thickness elsewhere,

130 University of California Publications in Zoology [Vou. 20

or to about 0.254. This localized: thickening, together with the pro-
jection, forms convenient diagnostic characters for the identification
of these eysts, and for their distinction from ovoidal and ellipsoidal
eysts of Endolimax buetschlii or from the occasional ellipsoidal or
subspheroidal one of Hndamoeba dysenteriae.

The body of the flagellate may not completely fill the eyst, retract-
ing away from the anterior projection in stained cysts. Elsewhere it
lies in intimate contact with the wall of the cyst.

The orientation of the cyst is determined by the position of the
flagellate within it. Normally or usually the anterior end of the
flagellate, which is distmguished by the location of the compound
blepharoplast, appears to be directed towards the tapering end of the
eyst (pl. 16, figs. 7, 10). There are, however, cysts in which this
relation of cyst and flagellate is reversed (pl. 16, figs. 9, 11). This
condition appears to be brought about by the turning of the flagellate
within the eyst after the wall is completed and the form and orienta-
tion of the cyst determined. The rounded subspheroidal eysts (pl. 16,
fig. 8) may be caused by the rotations of the flagellate within the cyst
during its more plastic condition.

The organs of the free-moving flagellate, with the exception of
the free flagella, are all visible in the stained cysts. In stained cysts
with diffuse glycogen (pl. 16, fig. 10) the cytoplasm was rather
uniformly and finely alveolar. In a few eases the glycogen was
clumped in clouds, rather diffusely margined, scattered in the cyto-
plasm (pl. 16, fig. 8). In stained cysts these glycogen masses are
dissolved out, leaving coarser vacuoles (pl. 16, fig. 9). Very rarely
was the glycogen condensed in one or more dense, deeply staining,
iodophylic masses which became dark brown in iodine-eosin and dis-
solved out, leaving large vacuoles (pl. 16, fig. 7), when treated with
aqueous solution, as in the iron haematoxylin staining method. Dobell
and Jepps (1917) have figured two such glycogen-laden eysts.

The amount of glycogen within the cyst decreases with age, as it
does in the glycogen-bearing cysts of the three species of human
intestinal amoebae. There is great variation in the cysts in different
stools in the amount of glycogen contained therein, but less variation
among the cysts of a given stool. Older binucleate cysts lack the
glycogen vacuoles.

The other cytoplasmic contents of the cyst are the chromatoidal
bodies. These may or may not be present and when found vary greatly
in number and location. They are ellipsoidal, ovoidal, or spheroidal

1920] Kofoid—Swezy: Morphology and Mitosis of Chilomastix 131

masses of deeply staining substance, taking, in iron haematoxylin, the
same stain as the chromatin within the nucleus (pl. 16, fig. 11). These
bodies are from 0.25 to 0.9 in greatest diameter, homogenous in strue-
ture, and lie near the borders of the eytostome and nucleus. In these
particulars they resemble the chromatoidal structures found near
the parabasal in the cytoplasm of Trichomonas and called by us
(1915) ‘‘chromidia.’’ Their variability corresponds to that of the
chromatoidal rods of the cysts of Hndamoeba, with which they are
associated in the faeces, and suggests that they are of the same nature,
and meet similar needs of the organism during encystment. They
contain reserve materials of unknown, but probably nuclear origin,
contingent upon encystment.

The organelles of the flagellate are retained to an unusual degree
in the encysted stage (fig. B). The nucleus (nwe.) is spherical, or
spheroidal with its longer axis parallel to the major axis of the cyst.
It is generally located a little in front of the middle of the body
and to the right side of the cytostome, considerably posterior to its
customary position in the free stage. It is 2 to 2.54 in diameter.
Its membrane may be very delicate with one, or several scattered
chromatin plaques, or it may be rather heavily encrusted with chro-
matin in addition to one or two large plaques which appear to bear
a fairly constant relation to the rhizoplast and axis of the body.
These two plaques are usually unequal in size, the larger often anterior
and at the junction of the rhizoplast with the nucleus, and the smaller
one at or near the opposite pole. These relations are not constant,
however, the posterior mass being the larger in some instances (pl. 16,
fig. 8), while in others there is only a single anterior plaque (pl. 16,
fic. 10). There is in most nuclei a central karyosome, less well-defined
than in the free stage, and often somewhat excentric in location. From
it there pass peripherally fine threads to the chromatin plaques or
other smaller chromatin aggregates on the nuclear membrane. <A
clear, fluid-filled zone lies between the central karyosome and the
peripheral chromatin.

The structure of the nucleus is suggestive of a permanently polar-
ized organization not unlike that found in Giardia by Kofoid and
Christiansen (1915) and by Boeck (1917). This polarization is fur-
ther emphasized by the presence of a small granule on the anterior
end of the nucleus into which the rhizoplast passes. We interpret this
granule as the centrosome (cent., fig. B), since its location and mor-
phological relations to the nucleus, rhizoplast, and blepharoplast are

132 University of California Publications in Zoology (Vow. 20

homologous with those of a like strueture in Giardia, where this
granule plays the rdle of the centrosome at mitosis. This centrosome
is seen with difficulty, if at all, owing to its juxtaposition to the large
chromatin plaque and sometimes to the position of the nucleus which
may be so turned as to obscure it. At mitosis in Chilomastix this
granule divides and the daughter centrosomes form the paradesmose
between them as a fiber on the nuclear membrane (pl. 16, fig. 15).

A comparison of the position, structure, and relations of the
blepharoplasts in the free flagellate with those of the eneysted stage
indicates that the nucleus in its postero-lateral migration to near the
center of the cytoplasmic mass has not only drawn out the long rhizo-
plast from the left blepharoplast but has also parted the blepharoplasts
more or less widely (pl. 16, figs. 8, 11).

In the encysted stage the primary blepharoplast (fig. B, prim.
bleph.), has lost its two anterior flagella, is often quite small, dis-
located laterally to the left and connected by the nuclear rhizoplast
(nue. rhiz.) to the centrosome (cent.) on the anterior face of the
nucleus. The transverse rhizoplast (tr. rhiz.) joins it to the secondary
blepharoplast (sec. bleph.) which has lost its single right flagellum.
This granule and the preceding are often about equal in size to
the primary blepharoplast. The tertiary blepharoplast hes near the
ventral surface at the anterior end of the cytostome, receives the peri-
stomal rhizoplast, and gives rise posteriorly to three structures: (1)
to the eytostomal flagellum which, unlike the free flagella, persists as
a deeply staining fiber during the early phases of the cyst, (2) to the
eurved parabasal, a deeply staining rod in the right wall of the oral
pouch, and (3) to the peristomal fiber in the margin of the opening
into the cytostome. The latter is so delicate and so superficial in
location as to require great care in differentiating it from the under-
lying parastyle and parabasal.

The unity, fibrillar continuity, and elasticity of the neuromotor
system is again demonstrated in the structure and behavior of the
centroblepharoplast complex of Chilomastix during encystment.
Though divided into centrosome and three blepharoplasts the fibrillar
continuity of the complex is maintained unbroken in encystment and
mitosis.

The cytostomal complex is clearly recognizable in the eyst, both
in the iodine-eosin stain and in the iron haematoxylin, as an elongated
or slipper-shaped loop with a contracted region anteriorly. A dark
thread, the peristomal fiber, outlines the opening into the shallow

1920] Kofoid—Swezy: Morphology and Mitosis of Chilomastix 133

cavity of the oral pouch. The same structural peculiarities which exist
in the neuromotor apparatus in the free flagellate are present in the
eyst except for the free flagella. There is little if any change in the
size of its constituent elements in the eyst.

The spiral groove of the body (fig. B, spir. gr.) loses its spiral
course and becomes a faint meridional shadow at the right of the
eytostome, distinguishable in the iron haematoxylin preparation as a
narrow, clear region extending from the anterior end of the encysted
flagellate nearly to its posterior end (pl. 16, figs. 10-14).

MITOSIS

Binary fission in the free flagellates has not been found in our
stained faecal smears, though constant watch has been maintained
for it. In the eneysted stages, however, we have been more fortunate
in that we have been able to secure a fairly representative series show-
ing the main steps in the mitosis of this form in the first two divisions
in the eyst. Many of the minute details of this process have thus far
eluded us, as well as the plasmotomy of the two or more daughter
flagellates thus formed and their liberation from the eyst.

The onset of mitosis is apparently not foreshadowed in the behavior
of the chromatin contents of the nucleus, since this retains its usual
structure until the new neuromotor organelles are at least partially
developed (pl. 16, figs. 10, 11, 18, 14). The process is begun by the
division of the centrosome and blepharoplasts. The earliest stages
we have been able to find (pl. 16, fig. 13) show these granules dupli-
eated and each set of blepharoplasts connected by the normal com-
plement of rhizoplasts to the as yet undivided nucleus. The origin
of the rhizoplasts appears to be by division of the original rhizoplasts.
The centrosomes spin out between them as they separate a darkly
staining thread, the paradesmose (pl. 16, figs. 13, 14), which les
on the nuclear membrane.

The duplication of other parts of the neuromotor apparatus occurs
by outgrowth and not by division, as seems to be the case with the
blepharoplasts and rhizoplasts. The new cytostome, and its peri-
stomal fiber and associated parabasal, parastyle, and cytostomal
flagellum, arise as outgrowths of the new secondary and tertiary
blepharoplasts, appearing at first as minute loops and threads (pl. 16,
fig. 13) which gradually enlarge until, in the final stages, they equal

134 University of California Publications in Zoology (Vou. 20

the old organelles in size (pl. 17, fig. 24). The old cytostome, with
its peristomal fiber, parabasal, parastyle, and cytostomal flagellum,
remains intact throughout the process of mitosis, though later stages
would seem to indicate that the entire complex is somewhat reduced
in size and its eytostomal flagellum can no longer be found (pl. 17,
figs. 22, 23). It retains its original position while the new cytostome,
with its related organelles, follows the migrating centrosome and
eventually comes to lie at the opposite end of the body (pl. 17, figs.
18-22). With the completion of mitosis and the disappearance of
the paradesmose, the activity of the encysted flagellate somatella may
bring the second neuromotor complex and nucleus back to their orig-
inal anterior position, so that the two groups of organelles come to
he side by side (pl. 17, fig. 24). The mobility which we have observed
in the dividing schizonts in binary and multiple fission in free
trichomonad flagellates (see Kofoid and Swezy, 1915) appears to
oceur, to some extent, in the schizonts of Chilomastix while still within
the eyst.

During the development of the new neuromotor organelles the
daughter centrosomes move farther apart on the nuclear membrane
(pl. 16, figs. 13-15; pl. 17, figs. 16-17) until they come to lie at
opposite poles of the nucleus (pl. 17, fig. 18), drawing out between
them the slender thread which forms the paradesmose. This may be
seen in stained preparations as a dark line which follows the contour
of the nucleus but may not be attached to it (pl. 17, fig. 20). In later
stages, it extends through the cytoplasm between the centrosomes on
the nuclear membrane of the two daughter nuclei (pl. 17, fig. 23).

The nuclear membrane is retained intact throughout the entire
process of mitosis. It loses its encrusted chromatin before the forma-
tion of the spindle, becomes considerably enlarged and is drawn out
into a spindle shape as mitosis progresses, and is finally parted by
equatorial constriction into the two daughter nuclei.

The behavior of the chromatin and its formation into chromosomes
we have not been able to follow in all details. The amount of chro-
matin apparently increases considerably with the onset of mitotic
phenomena and decreases in the final phases, as may be noted by a
comparison of figures 17 to 20 on plate 17 with the other figures given
on the same and preceding plates. A spireme is formed as a rather
thick, continuous thread (pl. 17, fig. 17) coiled in the axis of the
nucleus. This spireme later shows transverse constriction into five
unequal masses prior to its movement into the equatorial plate (pl. 17,

1920] Kofoid—Swezy: Morphology and Mitosis of Chilomastix 135

fig. 19). In the equatorial plate stage (pl. 17, fig. 18) the chromosomes
appear as large, dense rounded masses, connected with the centrosomes
at the poles by numerous spindle fibers. The number of these granules
or chromosomes is rather difficult to determine at this stage, but in
the anaphase (fig. 20) five were clearly distinguishable. They vary
considerably in size, and stain intensely with iron haematoxylin.
The spindle fibers disappear as the chromosomes move to the poles
(pl. 17, fig. 22) and each granule becomes elongated in a narrow cone
at its point of attachment to the pole of the nucleus where the centro-
some lies.

In the reorganization of the nuclei the chromatin is found massed
in groups of rather large granules at the center of the nucleus (pl. 17,
fig. 24). Later the chromatin takes on the form of a small compact
karyosome connected with the periphery by narrow fibers and the
remainder of the chromatin collects on the nuclear membrane (pl. 17,
fie. 23).

The plane of division of Chilomastix is morphologically longitud-
inal. This is shown in the prophase in the splitting of the blepharo-
plasts and their separation (pl. 16, figs. 13-15; pl. 17, figs. 16, 17).
With the formation of the relatively large spindle in the rigid confines
of the cyst some adaptation is necessary for the accommodation of the
two schizonts. This adaptation appears in the initial posterior migra-
tion of one of the centrosomes carrying with it the newly formed
neuromotor apparatus, resulting in an apparently transverse division
of the nucleus (pl. 17, figs. 19-22).

It is probable that one or two other nuclear divisions follow in the
eysts, though we have not been able as yet to find them in infected
stools in which we have secured the first division by keeping the
stools for as much as four days at room temperatures.

DISCUSSION

Chilomastix is asymmetrical in the distribution and function of
the anterior flagella, in the location and organization of the neuro-
motor apparatus, and in the course of the spiral groove. This groove
has the same torsion as the transverse flagellum and girdle of the
dinoflagellates and the undulating membrane of Trichomonas. This
torsion is sinistral. It has the same twist as the thread of a left-handed
screw.

136 University of California Publications in Zoology [Vou. 20

When binary fission occurs in the free and eneysted stages the
newly formed neuromotor apparatus, which is attached to the centro-
some at one of the poles of the dividing nucleus and goes into one
of the daughter individuals, has the same asymmetry as that of the
parent individual which goes into the other daughter. The structures
at the two poles are not mirror images each of the other. Bilateral
symmetry of the two as yet unseparated daughter cells is not estab-
lished by mitosis.

The structures which we have discovered in the neuromotor organ-
ization of Chilomastix are, in detail, similar to and homologous with
those of the right cell in the binucleate Hexamitidae and most closely
with those of the cell on the right side of Guardia. The centrosome,
nuclear rhizoplast, blepharoplast complex, peristomal fiber, parabasal,
and parastyle are homologous with the like organs in Giardia, save
that the parastyle is represented by the axostyle with its free terminal
flagellum in Grardia, but has no flagellum in Chilomastix. The three
anterior flagella of the former are represented by the lateral, postero-
lateral and ventral flagella of the latter, while the undulating flagellum
of the eytostome seems to have disappeared in Giardia. The asym-
metry of the cytostomal region of the two genera is of like pattern
and the spatial relations of this region to nucleus, centrosome and
blepharoplast are strikingly similar in the two organisms.

It is obvious from the comparative morphology of these two genera
that they are rather closely related and that the Hexamitidae, inelud-
ing Giardia, must be regarded as an offshoot from Chilomastix or its
near relations. Giardia differs from Chilomastix in two very impor-
tant particulars: (1) it is binucleate and (2) it is bilaterally sym-
metrical with the left cell the mirror image of the right, which is the
morphological equivalent of Chilomastix.

This binucleate bilateral organism could be derived from sinistral
Chilomastiz only by a complete morphological reversal of the funda-
mental asymmetry of the left cell, with its nucleus and neuromotor
apparatus, in the binucleate body and the coincident suspension of
plasmotomy. Such a process, which is not unlike molecular reversal
in principle, might establish the bilateral binucleate Hexamitidae.

The morphological relations of Chilomastix and Giardia are pro-
foundly significant and instructive as to the method by which bilater-
ality has appeared among the highly specialized, asymmetrical, uni-
cellular organisms.

1920] Kofoid—Swezy: Morphology and Mitosis of Chilomastix 137

SUMMARY

1. Chilomastix mesnili has a deep spiral groove running posteriorly
from right over to left as a permanent cell organ distinet from but
adjacent to the cytostome. It persists in the cyst as a meridional
structure.

2. The neuromotor apparatus consists of centrosome, nuclear rhizo-
plast, three blepharoplasts and connecting rhizoplasts, the primary
giving rise to two flagella, the secondary to one and to the parastyle,
the tertiary to the parabasal, the peristomal fibril, and the eytostomal
flagellum or undulating membrane.

3. The centroblepharoplast complex is thus subdivided into four
granules, the centrosome and three blepharoplasts having continuous
rhizoplast connections with the central karyosome of the nucleus.

4. The nucleus is polarized with the centrosome anterior and the
spireme forms in its longitudinal axis. Binary fission in the eyst is
morphologically longitudinal.

5. The blepharoplast-rhizoplast chain splits lengthwise at mitosis

.and the remainder of the neuromotor complex appears to be produced
de novo by outgrowths from the blepharoplasts prior to the spireme
stage. The daughter centrosomes are connected by a paradesmose.

6. In mitosis the nuclear membrane remains intact and its con-
struction is spatially transverse. The daughter nuclei are for a time
connected by:the paradesmose but lie at opposite poles of the eyst, but
may later change their position.

7. The neuromotor system of Chilomastix is strikingly similar to
that of the right half of Giardia in symmetry and its constituent
elements.

8. The two daughter individuals have the same symmetry; they
are each equivalent to the right half of Giardia.

9. The bilateral symmetry of the two-celled Giardia could arise
only by a complete morphological reversal from the sinistral to the
dextral type of one of the two daughter schizonts at mitosis.

10. The genus Chilomastix is closely related in structure to and
may be the source of the bilateral binucleate Hexamitidae.

Transmitted March 30, 1920.
Zoological Laboratory,
University of California,
Berkeley, California.

138 University of California Publications in Zoology [Vou. 20

LITERATURE CITED
Boeck, W. C.
1917. Mitosis in Giardia microti. Univ. Calif. Publ. Zool., 18, 1-26, pl. 1,
1 fig. in text.
Brumpt, E.
1913. Précis de parasitologie. Ed. 3 (Paris, Masson), xxviii+ 1011+ 32 pp.,
4 pls., 698 figs. in text.
OHALMERS, A. J., and PEKKOLA, W.
1918. Chilomastix mesnili (Wenyon, 1910). Ann. Trop. Med. and Parasit.,
11, 213-264, pls. 6-7.
DosE.L, C., and Jepps, M. W.
1917. On the three common intestinal entamoebae of man, and their differ-
ential diagnosis. Brit. Med. Journ., 607-612, 14 figs in text.

Fonseca, O. O. R. Da
1916. Estudos sobre os flagelados parasitos dos mamiferos do Brazil. Mem.
Inst. Oswaldo Cruz, 8, 5-40, pls. 1-2, 4 figs. in text.
Koro, C. A. ;
1916. Biological and medical significance of the intestinal flagellates. Proce.
Sec. Pan Amer. Sci. Cong., 1915-1916, 10, 546-565.

Korom, C. A., and CHRISTIANSEN, E. B.
1915. On binary and multiple fission in Giardia muris (Grassi). Univ. Calif.
Publ. Zool., 16, 30-54, pls. 5-8, 1 fig. in text.
Koror, C. A., and Swexzy, O.
1915. Mitosis and multiple fission in trichomonad flagellates. Proc. Amer.
Acad. Arts and Sci., Boston, 51, 289-378, pls. 1-8, 7 figs. in text.
1919. Studies on the parasites of the termites. I-IV. Univ. Calif. Publ.
Zool., 20, 1-116, pls. 1-12, 9 figs. in text.

KuczynskI, M.
1914. Untersuchungen an Trichomonaden. Arch. Prot., 33, 119-204, pls.
11-16, 4 figs. in text.
PROWAZEK, S., and WERNER, H.
1914. Zur Kentniss der sog. Flagellaten. Arch. Schiffs-u. Tropenhyg., 18,
155-167, pl. 10, 1 fig. in text.
SWELLENGREBEL, N. H.
1917. Ueber die Cystenbildung des Chilomastix mesnili Wenyon. Arch. Prot.,
38, 89-93, pls. 1, 2, 1 fig. in text.
WEnyon, C. M.
1910. <A new flagellate (Macrostoma mesnili n. sp.) from the human intestine,
with some remarks on the supposed cysts of Trichomonas. Parasit.,
3, 210-216, pl. 16, 2 figs. in text.

EXPLANATION OF PLATES

All figures of Chilomastix mesnili were drawn with camera from Schaudinn-iron
haematoxylin preparations at a uniform magnification of 4800. Finer structures
have been worked out with high-power monobjective binocular microscope with
strong illumination, fluorite and apochromatic objectives, wide-angle condenser and
immersion oil between slide and condenser.

PLATE 15

Fig. 1. Normal trophozoite showing the spiral groove, the cytostome with
its peristomal fiber, eytostomal flagellum, parabasal and parastyle, the nucleus
anteriorly located, and the three blepharoplasts and centrosome.

Fig. 2. Small flagellate which is preparing to encyst or may have just escaped
from a cyst.

Fig. 3. Free flagellate showing the deep notch of the spiral groove. The
blepharoplast complex appears as a single granule.

Fig. 4. The large vacuoles show the position of glycogen masses in the living
flagellates. The black rods represent ingested bacteria. Spiral groove obliterated
by contraction.

Fig. 5. Chilomastix viewed from the ‘‘dorsal’’ or aboral side. The cytostome
is partly engulfed by the deep spiral groove.

Fig. 6. Chilomastix of the more slender type.

| 140 |

UNIV. CALIF. PUBL. ZOOL. VOL. 20 [ KOFOID-SWEZY] PLATE 15

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PLATE 16

Fig. 7. Eneysted Chilomastix showing the typical pyriform cyst with its wall
at the anterior end thickened,

Fig. 8. Spheroidal cyst with large chromatoidal bodies near the parabasal
and parastyle.

Fig. 9. Cyst viewed from the aboral side. The body within the cyst has
become inverted with the anterior end at the large end of the cyst.

Fig. 10. Cyst showing normal appearance of encysted flagellate. Spiral
groove shown as clear line on the right side. Parabasal exposed within the cyto-
stome.

Fig. 11. Ovoidal cyst containing numerous chromatoidal bodies in cytoplasm.
Body inverted within the cyst.

Fig. 12. Subspheroidal cyst.

Fig. 18. Prophase of mitosis. Centrosome, blepharoplasts and rhizoplasts
have divided, and the new cytostome with parabasal and parastyle has appeared.

Fig. 14. Prophase of mitosis. A slightly later stage with the cytostome en-
larging.

Fig. 15. A still later stage of the prophase, with the new cytostome fully

developed and the chromatin of the nucleus beginning its reorganization for
mitosis.

| 142 |

UNIV, CALIF. PUBL. ZOOL. VOL. 20 [KOFOID-SWEZY] PLATE 16

PLATE 17

Fig. 16. Prophase of mitosis. The backward migration of one centrosome
and the new cytostome has begun.

Fig. 17. Prophase of mitosis. The chromatin has become arranged in a
coiled spireme in the axis of the nucleus.

Fig. 18. Equatorial plate stage. Note the posterior position of the new cyto-
stome and the paradesmose connecting the two centrosomes. Body inverted within
the eyst.

Fig. 19. Abnormal type of anaphase.

Fig. 20. Anaphase. Five chromosomes of unequal size may be distinguished.
Note the separation of the paradesmose and the nuclear membrane.

Fig. 21. Anaphase. Note the spindle fibers still connecting the two groups
of chromosomes. Body inverted in eyst.

Fig. 22. Late anaphase. Note the conical shape of the chromosomes. The
old cytostome appears reduced in size.

Fig. 23. Telophase. Reorganization of the nuclei completed but paradesmose
still persisting. Note shortening and thickening of the parabasals and decrease
in the paradesmose.

Fig. 24. Reorganization of the neuromotor apparatus of each set completed
but that of the nuclei still in progress. Note absence of paradesmose, anterior
location of the blepharoplasts, and parallel position of the schizonts.

[144]

[KOFOID-SWEZY] PLATE 17

UNIV. CALIF, PUBL. ZOOL. VOL. 20

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Blo, a texte Apures, way, POLS: ox 5 Se ee a Se fa nec bose Sbbcsasuanee

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

2 CR Fo eS 7 ba Tepe 62 fi ii a A a ie a ah Ie SUNN Sen UN Sl as SNR PIL hn ep Co
Index, pp, 537-545.

0 18, 1. Mitosis in Giardia microti, by William C, Boeck. Pp. 1-26, plate 1, Octo-
WTS) ead | wy fete Ree RAAT Ri I AN SEO Oe Sl SEN wre COGS ARUN OE Tap SR ale Se Oda Wet ea

2. An Unusual Extension of the Distribution of the Shipworm in San Fran-
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8. Description of Some New Species of Polynoidae from the Coast of Cali-
fornia, by Christine Essenberg. Pp. 45-60, plates 2-3. October, 1917 ....

4, New Species of Amphinomidae from the Pacific Coast, by Christine Essen-
berg. Pp. 61-74, plates 4-5. October, 1907 anc onic eeteecenenenee
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convivus Stal, by Irene McCulloch. Pp. 75-88, 35 text figures. Decem-

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Swezy. Pp. 89-102, 12 figures in text. December, 1917 ......22.20..2-..--s-0- es

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SL aa Se rere EE INS gee 2S Se a SN Be oi ee
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be = 435-144. 1 fipure in texts January; 1918.25 ec sn A
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me) Faeces, by William C. Boeck... Pp. 145-149. December, 1917-.......0.2....2.....
~ 10. The Musculature of Heptanchus maculatus, by Pirle Davidson... Pp. 151-170,
d2itipures: in text. March,-1918 soe er Ser Be ei tea cdepccctecaceaateas

11, The Factors controlling the Distribution of the Polynoidae of the Pacific
SA pie Coast of North America, by Christine Essenberg. Pp. 171-238, plates 6-8,
ees mfilpnres-in text. Maren 2O1825 50 c8 eves Pe ee es
a aes 12. Differentials in Behavior of the Two Generations of Salpa democratica
4 Relative to the Temperature of the Sea, by Ellis L. Michael. Pp. 239-298,
ihn plates 9-11, 1 figure im text. March, 1918. 2.2.2... ce cceee elec eeccceanee nce ee enero

SI -:18. A Quantitative Analysis of the Molluscan Fauna of San Francisco Bay, by
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15

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UNIVERSITY OF CALIFORNIA PUBLICATIONS—(Continued)

14. The Neuromotor Apparatus of Euplotes patella, by Harry B. Yocom, Pp
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. The Significance of Skeletal Variations in the Genus Peridinium, by AL i
Barrows. Pp. 397-478, plates 17-20, 19 figures in text. June, 1918. ee =

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17. The Cercaria of the Japanese Blood Fluke, Schistosoma japonicum Kat-—
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Atwood Kofoid and Olive Swezy. Pp. 1-20, plates 1-2, 1 figure in text.
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IN
ZOOLOGY
Vol. 20, No. 6, pp. 145-168, 9 figures in text June 28, 1920

A CRITICAL REVIEW OF THE NOMENCLATURE
' OF HUMAN INTESTINAL FLAGELLATES,
CERCOMONAS, CHILOMASTIX, TRICHO-
MONAS, TETRATRICHOMONAS,
AND GIARDIA

BY

CHARLES ATWOOD KOFOID

Eten” yan is trey tip

JUL 19, 1920 j

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UNIVERSITY OF CALIFORNIA PUBLICATIONS
IN

ZOOLOGY
Vol. 20, No. 6, pp. 145-168, 9 figures in text June 28, 1920

A CRITICAL REVIEW OF THE NOMENCLATURE
OF HUMAN INTESTINAL FLAGELLATES,
CERCOMONAS, CHILOMASTIX, TRICHO-
MONAS, TETRATRICHOMONAS,

AND GIARDIA

BY f

|

CHARLES ATWOOD KOFOID :

CONTENTS

PAGE
TED TEAYG REO LEG) ese ee 145
TO SEYS OTIS ISRO SS Ee See 147
Cercomonas not as yet proved to be a human parasite -........-.....0..---.0-..----- 147
C@hilomastiscd avai et Mo quaim=W a Cony) ees cence ae sce reas ceecaee techn nent eine eeceee cecncens 151
Trichomonas hominis (Davaine)...-...............2...------ sees 1hGY5)
Trichomonas of the mouth and respiratory tract ... 156
Tetratrichomonas, a synonym of Trichomonas ....................2-.--2::0---0--000000-0--* 159
Tritrichomonas gen. nov. for trichomonads with three flagella .................. 160
Giardia enterica (Grassi) 160
SSRIS ee ea ... 164

TLRS DTM UME: COLAO Ls era ei oe Ret eo ee 2 a sere tee 165

INTRODUCTION

The conquest and control of the intestinal protozoan infections of
man, which investigation is showing to be both frequent and varied,
involves among many other important factors the accurate determina-
tion and differentiation of the various flagellate and other protistan
species of the intestinal fauna. This is fundamental for the detection
of etiological factors of disease, especially in view of the prevalence
of varied and multiple infections.

For purposes of comparative protozoological investigation and of
clinical and therapeutic progress, it is eminently desirable that the

146 University of California Publications in Zoology | Vou. 20

nomenclature of these organisms be established upon as sound a basis
as may be feasible with our present knowledge of the organisms and
in the light of the accumulated literature, much of which is confusing,
incomplete, and even contradictory.

The early microscopists who first explored the invisible living world
turned their crude instruments to the dejecta of man and found therein,
as elsewhere in organic material in the early stages of decay, a horde
of bacteria and of flagellates feeding thereon. They were hampered
in their investigations by the rapid movements of the monads, by
the fact that the technique of modern staining was undeveloped, by
the poor definition of their lenses which failed to reveal the number
and relation of the flagella, by the lack of organization of scientific
bibliography, and by the absence of monographic works. The science
of protozoology was, as yet, undeveloped.

These observers saw the commoner flagellates of man, sketched and
described them briefly and imperfectly, especially as to the flagella
which now constitute the bases of classification. They often mis-
interpreted the undulating membrane as a row of cilia, and either
failed entirely to see the other flagella, or did not correctly resolve
their numbers. Added to these difficulties is the fact that clinicians
and zoologists have each alike been uninformed, in many eases, as to
the literature in the other’s field when they have ventured to christen
some newly discovered flagellate from the intestine of man.

As a result of these conditions the nomenclature of these flagellates
has been in a continual state of confusion from the early discoveries
of Miiller (1773), Donné (1837), Davaine (1854, 1860, Lambl (1859,
1875), and Grassi (1879, 1881) to the present time.

After some experience at the U. S. Army Laboratory, New York
City, and in the Division of Parasitology, Bureau of Communicable
Diseases, California State Board of Health, of the practical difficulties
in the identification of intestinal flagellates with existing facilities, and
a persistent effort to establish on sound bases the names at present
widely but not universally used for these organisms, and after a
careful perusal of the pertinent literature, the following conclusions
are offered as a solution of the correct nomenclature, under the
Zoological Code of Nomenclature, of the common flagellates of man.
We offer our conclusions for criticism, with the hope that they may
be recognized as conforming to the Code of Nomenclature, and that
they may conduce ultimately to stability of nomenclature in this
common ground of medicine and zoology.

1920] Kofoid: Nomenclature of Human Intestinal Flagellates 147

DISCUSSION
Crercomonas Nor As YET PROVED TO BE A HUMAN PARASITE

This genus was originally described by Dujardin (1841) for free-
living flagellates from infusions. It was characterized by the posses-
sion of one anterior flagellum and a posterior prolongation. It was
therefore natural that human parasitic flagellates in which there was
seemingly one anterior flagellum and a caudal process should be
referred to this genus. In the clinical literature to the present day,
this genus is a convenient catchall for most inadequately determined
flagellates of man. This reference results in part from the fact that
the first flagellate to be described from the intestine of man was called
Cercomonas by Davaine (1854).

The genus Cercomonas, however, has not been critically established
as a parasite of man by any of the many extensive surveys of the
protozoan intestinal fauna which have been made in recent years in
connection with the World War. No competent protozoologist has
described, figured, and critically discussed any Cercomonas as an
undoubted parasite of man in connection with these tens of thousands
of stool examinations. There are, however, many reports of occurrences
of Cercomonas and of Cercomonas hominis, and some of C. parva and
C. crassicauda, but they all fall, in my opinion, into some one of the
following categories.

1. Uneritical allocations to Cercomonas of some flagellate not
assignable to Trichomonas or Giardia. In most eases, if not all, the
organism present and thus reported was Chilomastix davainer
Moquin-Tandon, a name which we adopt in leu of C. mesnilt
(Wenyon). This conclusion rests upon statistical evidence such
as that of Matthews and Smith (1919) who compile the results of
23,024 stool examinations of 4068 persons. mainly dysenterics, with
669 infections of Giardia intestinalis, or 16.4 per cent, 148 of Chilo-
mastic mesnili, or 3.6 per cent, and 29 of Trichomonas intestinalis, or
0.7 per cent; and that of Kofoid (1920), who compiles the results of
3218 examinations of 2876 soldiers, with 168 Giardia intestinalis, or
5.9 per cent, 117 Chilomastix mesnili or 4.1 per cent, 6 Trichomonas
intestinalis or 0.2 per cent, and 4 occurrences of Embadomonas intes-
tinalis, and 8 of Tricercomonas intestinalis. It thus appears that,
according to statistical probabilities. any flagellate in addition to

148 University of California Publications in Zoology [ Vox. 20

Giardia and Trichomonas present in any numbers in any group of
human eases, is Chilomastix.

Additional support of this conjecture is derived from the fact that
nearly all of the clinical writers reporting Cercomonas, such for ex-
ample as Guastalla (1909), Mello Leitao (1913), Prentiss (1915), Chace
and Tasker (1917), and Simon (1918), have taken no note of Chilo-
mastix. The same is true of certain reports of larger numbers of cases
surveyed, as for example those of Christie (1915), 3 per cent in 100
natives of Sarawak, and Smithies (1918), who reports 36 cases in
1000 examinations in Augustana Hospital, Chicago. It is very prob-
able that some of the earlier clinical literature reviewed by Janowski
(1897) reporting Cercomonas deals in reality with Chilomastiz.

2. In the second category belong certain cases of critical determina-
tion of flagellates in stool cultures or in stools as species of Cercomonas,
such as that of Wenyon (1910a) who found Cercomonas longicauda in
a culture from a human stool, and that of Hartmann and Chagas
(1910), who found C. parva n.sp. in a lke culture. The first of
these species occurs in infusions, but neither was regarded by the
authors as of parasitic origin in the cultures. These investigators give
critical figures of the organisms and there is no doubt that they were
correctly refered to the genus Cercomonas.

Less certainty as to status attends the reports of the occurrence of
Cercomonas hominis in the stools of dysenteric patients by Fantham
and Porter (1916a@) and of Fantham (1916), who also later (19166)
reported two infections of C. parva in 1305 dysenterie patients in
England, and 3 in 14 special cases in hospitals in Liverpool. No
figures or accounts of the organisms thus identified are given by these
authors and the two reports appear to deal with the same group of
examinations. Porter (1918) found 10 infections by C. parva among
375 South African natives, but none in 104 autopsies, but in her account
details are lacking which would establish the parasitism of the
organisms. Woodcock (1916) reports one case of Cercomonas among
26 dysenteric patients at the Dardenelles. Castellani (1917) lists it
from the Balkans, and Sangiorgi (1919) from Italian soldiers at
Venice. Cutler and Williamson (1919) list C. crassicauda among the
intestinal flagellates of man without citation of its occurrence as such.
Since Chilomastix was well known to these investigators and they are
familar with intestinal flagellates and the literature thereof, the
organisms they have referred to Cercomonas must belong to one of
two genera, either to Cercomonas or to Craigia. If to the former, they

1920] Kofoid: Nomenclature of Human Intestinal Flagellates 149

may have been dealing with the coprophilic infections of stale stools;
if the latter, with active flagellates of parasitic origin belonging to
Craigia rather than Cercomonas.

3. Judging from the occurrence of Cercomonas in culture from
human stools, as shown by Wenyon (1910) and Hartmann and Chagas
(1910), it is probable that this genus contains coprophilic species which

Figs 1-4. Cercomonas longicauda Dujardin. After Wenyon, 1910, figs. 9, 10,
19. Magnification about 2000. Fig. 1. Lateral view of free stage showing
flattened side along which the posteriorly directed intracytoplasmic flagellum
runs. Figs. 2 and 3. Encysted stages, cyst wall not shown.

Figs. 5-7. Cercomonas parva Hartmann and Chagas (1910, pl. 4, figs. 4, 2,
and 12). X 2500. From culture of human faeces. Fig. 5. Free stage, showing
axostyle with terminal posterior flagellum. Fig. 6. Free stage from life. Fig. 7.
Encysted stage with cyst wall.

150 University of California Publications in Zoology | Vou. 20

multiply in the human stool after its evacuation from the bowel, and
that the stool may be inoculated either after evacuation by cysts from
dust or water, or by living flagellates in hospital receptacles, or by
cysts which have safely passed through the intestine but have not
developed at body temperatures. It may well be that the reports of
C. hominis by Fantham and Porter (1916) and of C. parva by the
former, and by Porter (1918), and by other competent protozoologists,
rest upon the coprophilic basis, rather than upon a parasitic one.
There has been as yet, however, no adequate evidence presented to
establish the presence of either the living Cercomonas or of their cysts
in fresh stools in numbers characteristic of parasitic infections. While
it may be that Cercomonas is represented by a parasitic species in the
human intestine, a survey of the literature shows that the evidence
thereof is quite unconvincing.

4. It is possible that the uniflagellate phase of Craigia hominis
(Craig) which does oceur in fresh human stools (Craig, 1911, and
Barlow, 1915) might be mistaken in fresh preparations for Cercomonas.
Craigia usually lacks the tapering posterior end of the species of
Cercomonas, but may sometimes be attenuate posteriorly. In stained
preparations it shows an unusually large central karyosome which is
not characteristic of Cercomonas. It is likewise accompanied by the
amoeboid stage and sometimes by the multiple encysted phase in the
human stool. The possibility of the confusion of Craigia with Cerco-
monas in much of the literature should be borne in mind.

From a consideration of the points above enumerated we conclude
that there is no proof that Cercomonas is represented among the
human intestinal flagellates, though it may occur in the human
coprozoic fauna. The species thus found are Cercomonas longicauda
Dujardin and C. parva Hartmann and Chagas. The species Cerco-
monas hominis Davaine is not today recognizable as a member of the
genus Cercomonas in human stools. Davaine’s (1860) forms belong
to other genera, as we shall show later.

It is highly desirable that all flagellates reported as Cercomonas
by any investigator of human flagellates or disease associated there-
with, be carefully worked out from stained preparations and accur-
ately figured, that cultures be attempted, and that necessary precau-
tions be taken to determine whether the flagellate has a coprozoie or
a parasitic source.

1920] Kofoid: Nomenclature of Human Intestinal Flagellates ilbyl

CHILOMASTIX DAVAINEL (Moqurn-TANDON )

The question now arises as to what flagellates Davaine (1854, 1860)
really had as the basis of his description of Cercomonas hominis vars.
A and B. After considerable experience in the examination of living
human intestinal flagellates from fresh stools and cultures thereof
(except Giardia), and a critical reading of Davaine’s earlier (1854)
and later (1860) accounts of the organisms discovered by him, we
conclude that (1) Davaine had two different flagellates, his vars. A
and B. This rests on his own decision, the accumulated statistical
evidence of recent years, and the contrasts in his descriptions and
figures; (2) the larger form, his var. A, is Chilomastix, and the smaller
one, Trichomonas. This decision is supported by the relative sizes of
Chilomastix, 19.6 (13-24) and Trichomonas, 5-lip. Chilomastixr
sometimes has the dimensions as small as those given by Davaine. A
second and most conclusive evidence is Davaine’s (1860, p. vi, not
in 1854) statement ‘“‘trait longitudinal vers |’extrémité anterieure,
donnant l’apparence d’un orifice buceal?;’’. Chilomastix is the only
human flagellate with well-defined. elongated c¢ytostome except
Embadomonas, which is much smaller and apparently very rare. This
eytostome serves today as the best available criterion for the certain
identification of Chilomastix in both free and encysted stages. The
cytostome of Trichomonas is not elongated nor so bordered by a
cytostomal rim as to be easily seen as is that of Chilomastiz. The
tapering posterior end (extrémité amincie) is also a distinguishing
characteristic. Davaine’s (1860) figures of his form may be har-
monized without difficulty, save in the matter of the number of
flagella, with our present knowledge of Chilomastic.

We therefore conclude that Davaine’s (1860) Cercomonas hominis
var. A is the organism later deseribed by Wenyon (1910) as Macro-
stoma mesnili and now generally called Chilomastix mesnili. A survey
of the literature indicates that Brumpt (1912) and Chalmers and
Pekkola (1917), investigators of Chilomastix, all agree that Cerco-
monas hominis var. A Davaine is the same organism as Chilomastixc
mesmili, though Brumpt later (1913) places an interrogation mark
after the synonym and Chalmers and Pekkola quote Cercomonas
hominis as a synonym pro parte without noting his varieties.

Since Davaine’s Cercomonas hominis is a complex of two species,
the withdrawal of either one of these under a new name leaves hominis
as the residual name for the species not withdrawn. This withdrawal

152 University of California Publications in Zoology [ Vou. 20

was first made by Moquin-Tandon (1860, p. 398) who called Davaine’s
larger form (his var. A) C. davainet, and the smaller one (his var. B)
C. obliqua (p. 399). Accepting page sequence in Moquin-Tandon
(1860) as establishing the priority of C. davainei as the first withdrawn
species, var. B becomes residual and retains the name hominis, while
C. obliqua Moquin-Tandon becomes a synonym of C. hominis. Davaine’s
sequence (of vars. A and B) cannot be used to establish the priority of
the subsequent reviser’s (Moquin-Tandon) names. It follows from
the evidence here presented that the correct name for the human
intestinal flagellate which Davaine called Cercomonas hominis var. A
is Chilomastix davainet (Moquin-Tandon).

There remains one obscure feature of this problem, namely the
question of priority between Davaine’s (1860) ‘‘Traité des Ento-
zoaires’’ and Moquin-Tandon’s (1860) ‘‘Eléments de Zoologie Médi-
cale.’’ Both bear the same date, were printed at the same press and
were issued by the same prominent publishing house, J. B. Bailliére
et Fils, publishers for the Imperial Academy of Medicine in Paris.
The English translation of Moquin-Tandon’s work by R. T. Hulme
apeared in London in the following year. The date of the preface in
Moquin-Tandon’s smaller book is September 1, 1859, and the flagellates
are treated in the closing pages. The date of the preface of Davaine’s
larger book is September 30, 1859, but the flagellates are treated in
the first pages of the book in a ‘‘Synopsis’’ paginated separately in
Roman numbers.

The Bibliotheque Nationale of Paris in reply to an inquiry writes
that ‘‘L’ouvrage de Moquin-Tandon Elements de zoologie medicale
est daté de 1859 et figure au journal de la libraire (Dépot légal) du
24 Septembre, 1859, sous le No. 8501. Le Traite des Entozoaires de
Davaine figure du méme journal du 3 Décembre, 1859, sous le No.
10774. Sauf retard l’editeur a effectuer le depot, il est dont un peu
posterieur.”’

These dates evidently refer to registration merely, rather than to
actual deposit of the volumes following publication, since the publisher
dated them both 1860.

There is fortunately decisive internal textual evidence that Moquin-
Tandon had before his eyes Davaine’s figures when he wrote his
descriptions of Cercomonas davainei and C. obliqua. We reproduce
both Davaine’s and Moquin-Tandon’s descriptions herewith:

From Davaine’s (1854) original article in which no specific names

9?

and no figures are given we quote:

1920] Kofoid: Nomenclature of Hwman Intestinal Flagellates 153

‘<Les cercomonas des selles, au contraire, ont des organes qui ne laissent
aucun doute sur leur nature animale; ils ont 8 milliémes de millimétre de
longuer; leur forme est ovoide, un peu variable; le tegument est mou, blanchatre;
un corpuscule ou deux, trés-petit, nucléaire, peut quelquefois étre percu a
l’intérieur. L’extrémité antérieure est pourvue d’un filament vibratile d’une
minceur extréme, trés-long, que 1’on devine d’abord par |’agitation qu’il exerce
dans le liquide ambiant, et que 1’on n’apergoit qu’a de courts intervalles et avec
une attention soutenue; a4 1’extrémité opposée existe un autre filament plus
épais, aussi long ou plus long que le corps, qui s’agglutine quelquefois aux
corpuscules environnants et autour duquel, dans ce cas, la cercomonas oscille
comme la lentille du pendula autour de sa tige. Ces cercomonas sont extréme-
ment agiles, ce qui rend l’observation de leurs caractéres trés-difficile. La
cercomonas des selles du malade atteint de fiévre typhoide m’a paru différer
de celle des cholériques, en ce que le filament caudal s’insére un peu sur le cété
du corps, et en ce qu’il offrait, en outre, un mouvement d’ondulation dans tout
de contour.’’

From Davaine, ‘‘Traité des Entozoaires,’’ 1860, p. vi:

CERCOMONAS DE L’HOMME (CERCOM. HOMINIS, DAVAINE)
Variété ou espéce A
Corps pyriforme, variable, long de 0 mm, 01 4 0 mm, 012; extrémité amincie
se terminant par un filament caudal épais aussi long que le corps; filament
flagelliforme antérieur situé A l’extrémité obtuse, opposé au précédent, trés long
(deux fois aussi long que le corps?) et mince, toujours agité, trés difficile 4 voir;
trait longitudinal vers 1’extrémité antérieure, donnant l’apparence d’un orifice
bueeal?; point de nucléus bien appréciable. Locomotion assex rapide, quelque-
fois suspendue par l’agglutination du filament caudal aux corps environnants;
l’animal oscille alors, comme un pendule, autour du filament.
Ces protozoaires existent en nombre quelquefois considérable dans les
garderobes récentes des malades atteints du choléra.
Variété ou espéce B
Plus petite que la précédente: corps moins pyriforme, 4 contours moins
arrondis, long de 0 mm, 008; deux filaments, 1’un antérieur, l’autre caudal,
situés un peu latéralement; longueur des filaments non déterminée; locomotion
trés rapide. Cette variété se rapproche des Amphimonas.
Protozoaires observés en grand nombre dans les déjections d’un individu
atteint de fiévre typhoide.

From Moquin-Tandon, ‘‘Eléments de Zoologie Médicale,’’ pp. 398,
399:

*¢9° CERCOMONADE (Cercomonas).—M. Davaine a découvert, en 1853 dans
les déjections encore chaudes des cholériques, une espéce de Cercomonade qui
s’y trouvait en nombre trés considérable.

Plus tard le méme savant a observé deux fois le méme animaleule dans les
selles de quelques malades atteints de diarrhée simple. L’épidémie du choléra
régnait encore.

J’ai désigné cet Infusoire, dans mes cours, sous le nom de Cereomonade de
Davaine (Cercomonas Davainei). [Name in footnote. ]

Ces animaleules présentent 8 millimétres de longueur. Leur corps est obové
ou pyriforme, un peu variable, trés obtus en avant et fortement atténué. Leur

154 University of California Publications in Zoology — [Vou. 20

tégument est mou et blanchatre. Un ou deux corpuscules, trés petits, nucléaires,
peuvent quelquefois étre apercus 4 l’intérieur. En avant, on remarque un
filament vibratile d’une minceur extréme, trés long et trés flexueux, naissant
brusquement de bord antérieur, que 1’on devine d’abord par 1’agitation qu’il
exerce dans le liquide ambiant, et que 1’on n’apercoit qu’aé de courts intervalles
et aves une attention soutenue; a 1’extrémité opposée existe un autre filament
plus épais, du moins 4 son origine, se confondant avee le partie postérieure, de
la longueur du corps ou un peu plus long, presque droit, roide, s’agglutinant
quelquetois aux corpuscules environnants, et autour duquel, dans ce cas, la
Cercomonade oscille comme la lentille du pendule autour de sa tige....

Une autre espéce de Cercomonade a été rencontrée une fois chez un jeune
homme atteint d’une fiévre typhoide bien caractérisée, sans aucun phénoméne
cholérique. C’est encore 4 M. Davaine que nous devons sa connaissance.

Cette seconde espéce différe un peu de la premiére; elle est plus petite et
plus ovoide; son filament antérieur parait de méme long et flexueux; il se
détache moins brusquement. Le filament caudal nait un peu sur le céte; il est
proportionnellement plus petit et ne se confond pas 4 son origine avee
l’extrémité du corps. Cette Cercomonade offre un mouvement d’ondulation
dans tout le contour. qui semble quelquefois légerément sinueux.

On pourrait appeler cette espéce Cercomonade oblique (Cercomonas obliqua).’’
[Name in footnote. ]

aoe

Fig. 8. Cercomonas hominis var, A. After Davaine (1860, fig. 1 [1]). xX 467.
Note the undulating membrane characteristic of Trichomonas hominis.

Fig. 9. Cercomonas hominis var. B. After Davaine (1860, fig. 1 [2]). X 467.
Note the cytostome characteristie of Chilomastix davainei.

Tn his earlier account Davaine (1854) makes no mention whatsoever
of the smaller size and more ovoid form of his var. B which Moquin-
Tandon notes. The latter must therefore have had Davaine’s later
figures and possibly also the text before him when he wrote his
description. Davaine’s figures appear only in his later (1860) work.

There is some question as to Moquin-Tandon’s access to Davaine’s
text for he omits reference to the ‘‘orifice buecal’’ in C. davainei (C.
hominis var. A) and states of C. obliqua (C. hominis var. B) “‘son
filament antérieur parait de méme long et flexueux’’ while Davaine
says of it “‘longeur des filaments non déterminée.’’

Moquin-Tandon was professor of medical natural history in the
Faculté de Médecin at Paris and his book appeared in French and in
English editions, but in the face of this publicity his treatment of
Davaine’s flagellates appears to have escaped the notice of all con-
temporary and subsequent parasitologists, including that of his com-

1920] Kofoid: Nomenclature of Human Intestinal Flagellates 155

patriots Railliet (1889, 1895), Verdun (1907, 1913), Neveu-Lemaire
(1908), and Brumpt (1910, 1913), as well as that of Leuckart and his
followers. His names fulfill all requirements under the Code of
Nomenclature and have a just claim for consideration.

In view of the internal evidence that Moquin-Tandon had before
him Davaine’s figures and his acknowledgment, ‘‘C’est encore a M.
Davaine que nous devons sa connaissance’? we award priority to
Davaine’s Cercomonas hominis over C. obliqua Moquin-Tandon, but
establish his Cercomonas davainei on the reviser’s privilege of naming
one (but not both) of the two species included under Davaine’s C.
hominis.

TRICHOMONAS HOMINIS (DAVAINE)

As stated above, the human intestinal Trichomonas, the smaller
species described by Davaine as Cercomonas hominis var. B, is the
Trichomonas of the human intestine. The grounds upon which this
conclusion rests are as follows.

(1) The statistical frequeney in which Giardia, Chilomastix, and
Trichomonas are revealed in any series of examinations of diarrheic
stools, makes it highly probable that Davaine found Trichomonas in
the course of his examinations of choleraic stools.

(2) There is no evidence that he had found Giardia except the
phrase (1854, 1860) ‘‘un corpuscle, ou deux, trés petit, nucléaire’’ in
his account of his var. A (Chilomastix davainet). The two nuclei
might have been due either to mitosis or to a confusion with Giardia.
There is no additional evidence of Giardia whatever. There is nothing
in his account of his var. B to suggest Giardia. We therefore eliminate
this genus and have only Trichomonas left as a probability.

(3) Conclusive evidence that Davaine had found Trichomonas
appears in his first paper (1854) where two characteristics are noted in
the phrases ‘‘le filament caudal s’insere un peu sur le cote du corps et
en ce qu’il offrait, en outre, un mouvement d’ondulation dans tout le
eontour.’’ These are the undulating membrane and the postero-lateral
departures of the free end of the lateral marginal flagellum. He omits
reference to the undulations in his later (1860) treatise. They are
the main features upon which identification is made today of the living
Trichonomas. They are not together characteristic of any other
human intestinal flagellate. Chilomastix has a eytostomal undulating
membrane found only with greatest difficulty, and the posterior end

+

156 University of California Publications in Zoology [Vou. 20

of the body is fairly symmetrical. Tricercomonas has a laterally dis-
placed caudal prolongation but no undulating membrane. Davaine’s
Cercomonas hominis var. B, which has both characters, was therefore
the human intestinal Trichomonas and its correct specific name is
hominis on the ground that Davaine had two species in his original
C. hominis and one of these, his var. A, was detached by Moquin-
Tandon (1860) as Cercomonas davainei, leaving Davaine’s var. B as
the residual species to retain the specific name hominis on the prin-
ciple of elimination. This name therefore replaces T. intestinalis
Leuckart (1879) and 7. confusa Stiles (1902) proposed later as names
for the human intestinal Trichomonas.

TRICHOMONAS OF THE HuMAN MoutH AND RESPIRATORY TRACT

There is much uncertainty as to whether or not there is more
than one species of Trichomonas in man. Species have been described
from the intestine (hominis, intestinalis), from the urogenital tract
(vaginalis), from the mouth (Tetratrichomonas buccalis, hominis), and
from the lungs (pulmonalis). Two points of view and two possibilities
are open. (1) There is but one species in man which is modified by
the tissues and secretions which surround it, thus giving rise to the
morphological differences that have been detected among Trichomonas
from the different organs. This is the view of Jollos (1913). (2) There
are at least two species, the intestinal one and the one in the vagina.
There is also the possibility that there is a third species in the mouth
and that this may be the same as the one in the lung, though infection
in the lung by the intestinal form carried there in the blood stream,
or by invasion from hepatic abscess accompanying Endamoeba dysen-
teriae, is also possible. Culture and inoculation of Trichomonas from
these different organs and much more morphological work accompany-
ing these experimental investigations will be necessary to determine
the number of species. For the present it seems advisable to treat the
Trichomonas from the intestine (7. hominis (Davaine)), from the
vagina (7. vaginalis Donné), from the mouth (7. buccalis (Goodey
and Wellings) ) and questionably that from the lung (T. pulmonalis),
as separate species pending investigation.

The recent work of Bensen (1909), Brumpt (1913) and Lynch
(1915a, b) is indicative that there are morphological distinctions
between 7. hominis and T. vaginalis, and the investigations of Goodey
and Wellings (1917) upon the flagellate of the mouth is suggestive

1920] (ofoid: Nomenclature of Human Intestinal Flagellates 157

that this may be a distinet species also; but more data are desirable
on this matter.

The discoverer of the flagellate of the mouth was O. F. Miiller,
the early Danish explorer of the microfauna of ponds and ditches, who
ransacked a culture made from the tartar between dirty human teeth
and found therein (1773, 1776) and figured (1786) tiny organisms
which he included in his genus Cercaria as C. tenax, together with
dinoflagellates and larval stages of trematodes. These parasites from
the mouth have not the slightest relation to larval trematodes, though
their possession of a short tail afforded a justification for this alloca-
tion in the days of medieval morphology and systematics. His
diminutive sketches show no flagella but the pyriform shape and the
short asymmetrically curved, pointed, posterior projection are sugges-
tive of the projecting axostyle and flagellum of Trichomonas.

Bory de Saint Vincent (1824) in one of the last volumes of the
““Eneyclopédie Méthodique’’ (fide Moquin-Tandon, 1860, p. 400)
established the genus Virgulina for tailed animaleules and included
therein Miiller’s species V. pirenula.

In 1838-1845 Mandl (fide Moquin-Tandon, 1860) rediscovered
Miiller’s ‘animaleules in the tartar about human teeth subjected
to microscopical examination in distilled and boiled water. He
records their dimensions as from 0.05 to several hundredths of a milli-
meter, possibly a typographical error for 0.005, and notes their lively
movements. They were said to contain in their bodies calcareous
elements which contribute to the deposition of tartar of the teeth.

Moquin-Tandon (1860) in his ‘‘Eléments de Zoologie Médiecale’’
revives Miiller’s name tenaz, follows Mandl in referring it to Bory’s
genus Virgulina and rejects Bory’s specific name pirenula. He notes
the prevalence of this organism in the tartar of patients on low diet
for several months and states that it forms the greater part of the
mucus coat on the tongue of persons with disturbed digestion. Neither
Mandl (1838-1845), nor Moquin-Tandon (1860), nor any subsequent
investigator of the flagellates of the human mouth, seems to have noted
the resemblance of Miiller’s species to Trichomonas; and Davaine
(1854, 1860), Leuckart (1863), and all subsequent investigators of
the flagellates of the human digestive tract seem to have overlooked
Miiller’s discovery and its possible relation to the organisms with
which they were dealing.

Since only one flagellate from the human mouth has thus far been
definitely identified in the course of modern investigation of the

158 University of California Publications in Zoology | Vou. 20

microfauna of that habitat, it is obvious that Miller (1773), Mandl
(1838-1845), and Moquin-Tandon (1860) may have been dealing with
that flagellate.

However, Miiller’s figures are so imperfect and his description so
meagre that there is no certainty whatever that he had a Trichomonas
and there is still less a clue to the specific identity of the moving
organism he discovered. It may well be that with additional investi-
gation more than one kind of flagellate will be found in the mouth.

In view of the lack of critical evidence we reject Miiller’s Cercaria
tenax as unidentifiable with Tetratrichomonas buccalis Goodey and
Wellings and as an indeterminable species. The specific name tenaxr
is therefore not available and Trichomonas buccalis (Goodey and
Wellings) becomes the correct name for the trichomonad flagellate of
the mouth.

It is possible that the trichomonad of the lung and that of the mouth
belong to the same species, but this is not as yet proved and Tricho-
monas pulmonalis Schmidt (1895) is so inadequately characterized as
to be unidentifiable. As deseribed it cannot be distinguished from
T. intestinalis and T. vaginalis, and the specific name pulmonalis
should be allowed to lapse as indeterminable.

One factor complicating the status of these several species is the
question as to the number of flagella which in the earlier accounts
(Davaine, 1854, 1860, and others) was reported as one, and in others,
such as Kunstler (1898), as varying (in Trichomonas of the rat)
from 4-8-10 up to 15, while in recent years Wenyon and O’Connor
(1917) admit that there are forms with three, four, and five flagella.

The uncertainty as to the number of flagella in both 7. vaginalis
and T. hominis is shown by Doflein’s (1916) cautious treatment of
this matter. He regards three flagella as typical and four as possibly
resulting from the detachment of the marginal filament of the undulat-
? condition which has
four free flagella, one of them trailing. This conclusion appears to
me incorrect, after a review of the literature, and careful examination
of my own preparations and cultures and of other preparations secured
from Drs. Lynch, E. L. Walker, and F. G. Haughwout. In every
instance where the number of flagella can be accurately determined
we have unquestionably found four anterior flagella and an undulating
membrane with marginal filament present in both 7. hominis and
T. vaginalis.

ing membrane, and a resulting ‘‘Trichomastix’

There is a tendency for the flagella of Trichomonas to adhere to
one another in a single strand, parting only distally. Often only three

1920] Kofoid: Nomenclature of Human Intestinal Flagellates 159

flagella adhere in this bundle and the fourth is thrown down at one
side. This makes determination difficult and leads to the acceptance
of three as the total number of anterior flagella present. Lynch (1915)
found four flagella on 7. hominis in culture. Wenyon and O’Connor
(1917) state that four flagella were found by them in all eases of
T. intestinalis except on two “‘ocecasions,’’ when three and five respec-
tively were found. Kunstler (1884) and Lynch (1915) found four
flagella in 7. vaginalis.

There is so much discrepancy in the results of different investi-
gators as to the number of flagella that one can not be dogmatic and
deny the existence of species in man with only three flagella. We
affirm that the most critical evidence indicates that 7. hominis and
T. vaginalis have four flagella, and that no report of a Trichomonas
with three flagella from man can be accepted as critical until verified
on a number of living flagellates restrained for observation or fixed
by fumes of osmiec acid, 10 per cent formalin, or Gram’s iodine, and
after analysis of a number of carefully stained individuals.

Four anterior flagella have also been found on Tetratrichomonas
buccalis by Goodey and Wellings (1917). The number of flagella on
the Trichomonas of the respiratory tract has not been determined.

Ohira and Nogouchi (1917, February) in a paper published prior
to that of Goodey and Wellings (1917, July) name the Trichomonas
of the mouth Tetratrichomonas hominis, but the specifie name hominis
is preoccupied in Trichomonas by T. hominis Davaine. This necessi-
tates the use of buccalis Goodey and Wellings for this flagellate.

TETRATRICHOMONAS Parist 1910

This was established by Parisi (1910) as a subgenus of Tricho-
monas for a parasitic species (7. prowazeki) from Salamandra, which
had four instead of three free anterior flagella. He assigns to T'richo-
monas sensu strictu these species with only three anterior flagella.
Goodey and Wellings (1917) raise Tetratrichomonas to generic status.

The genus Trichomonas was established by Donné (1837) with
vaginalis as the only, and therefore the type, species. He did not
determine the number of flagella in 7. vaginalis as four, but later
investigators have done this. It follows from this that the genus
Trichonomas is founded on a species with four flagella, that T'etra-
trichomonas is a synonym of Trichomonas, and that its type species,
T. prowazeki, belongs in Trichomonas.

160 University of California Publications in Zoology {| Vou. 20

In view of the fact that the number, disposition, and specialization
of the flagella have been the basis of differentiation in the Poly-
mastigina and that the genera of the order are distinguished by their
flagellar complexes, it seems to be necessary to establish a new generic
name for those species of Trichomonas sensu latu which have only
three anterior free flagella. It is necessary to reserve Trichomonas
for species with four flagella.

We therefore propose the new generic name Tritrichomonas for
all trichomonad flagellates with the following diagnostic characters:

Tritrichomonas nom. gen. nov.

Polymastigote flagellates with three free anterior flagella arising
from an anterior centroblepharoplast. This centroblepharoplast gives
rise to a posteriorly directed, laterally attached flagellum forming the
marginal filament in the sinistral slightly spiral undulating membrane
terminating distally in a short free flagellum; it also gives rise to the
parabasal body along the base of the undulating membrane, and to the
axostyle; eytostome not heavily margined with a deeply staining
fibril. We designate the well-known Trichomonas augusta Alexeieft
as the type species of T'ritrichomonas.

THE GENUS GIARDIA

Giardia Kunstler (1882), pp. 347-349.

Giardia Kunstler (1883), pp. 52-54, pl. 2, figs. 1-4.

Dimorphus Grassi (1879), pp. 445-448. Nee Dimorphus Haller, 1878
(Arachnida); nee Dimorpha Jurine 1807 (Hymenoptera); nee Dimor-
phus Gray 1844 (Mollusea): Hodgson 1841 (Aves).

Megastoma Grassi (1881), pp: 575-580. Nee Megerle von Mihlfeld (1818)
(Mollusea); Blainville 1825? (Mollusca); Swainson 1837 (Aves);
Costa 1850 (Pisces).

Lamblia Blanchard (1888), pp. 18-19.

Giardia Kunstler, Alexeieff (1914), pp. 210-212, figs. 4, 5.

The first description of a species now included in this genus
was that of Lambl (1859 nec 1875) of Cercomonas intestinalis from —
mucus stools of a child. His species is plainly not to be included in
the genus Cercomonas. Davaine (1875) deseribed a species belonging
to Giardia from the rabbit as Hexamita duodenalis, evidently in
ignorance of Lambl’s account, but his species can not be allocated in
Hexamitus.

Grassi (1879) described as Dimorphus muris a species belonging to
Giardia from Mus and Arvicola, and later (1881) from man and the
eat. Dimorphus is, however, a homonym with Dimorphus Haller

1920] Kofoid: Nomenclature of Human Intestinal Flagellates 161

(1878) arachnoid. In his later paper Grassi changes the name from
Dimorphus muris to Megastoma entericum, since ‘‘la denominazione
Dimorphus potrebbe dar luogo ad un equivoco e l’altra muris ora e
diventata insufficiente.’? Megastoma, as Blanchard (1888) indicated
when he proposed the new generic name Lamblia, had already been
preoccupied in no less than four instances cited above.

Prior to Blanchard’s (1888) erection of Lamblia the genus Giardia
had been established by Kunstler (1882, 1883a,b) for a species from
the intestine of tadpoles. The human species is a member of this genus
Giardia as Alexeieff (1914) has shown. Doflein’s (1916, p. 628)
objection to Giardia is in reality an objection to the Code of Nomen-
clature and to the just ethical claims of priority on the part of
Kunstler and Alexeieff, and to sound morphology as appled to
classification.

The generic term Lamblia is, therefore, as Alexeieff (1914) has
recently shown, not tenable, and Giardia Kunstler must be substituted
for it on the grounds of priority if due regard is had to the rules of
nomenclature. This was regrettable from the fact that the term
Lamblia had become entrenched by the custom of many compilers in
both biological and medical literature, especially in textbooks and
general treatises.

Some of the grounds for the inclusion of Lamblia (Megastoma) in
Giardia were noted by Grassi and Schewiakoff (1888) when, in their
discussion of the evident close relationships which they clearly recog- _
nized of the two genera, Megastoma Grassi and Giardia Kunstler, they
based their rejection of Giardia upon the apparent absence of the
eytosome, which was not demonstrated in Kunstler’s (1883) figures or
mentioned in his earlier discussion (1882, 1883).

However, in Kunstler and Gineste’s (1907) later paper this struc-
ture is definitely described and figured in Giardia alata, sp. nov., also
from the tadpole and probably the same species as G. agilis. In any
event Giardia is recognized by its author as possessing the very organ
whose apparent absence led to the generic recognition of Megastoma
and of its successor Lamblia.

In 1907 Kunstler and Gineste discuss the synonymy of the three
genera, Megastoma, Giardia, and Lamblia, and although they recog-
nize the equivalence of the organelles of Giardia and Lamblia, they
conclude, erroneously we believe, that both genera should be main-
tained “‘a la fois’’ although the only differences they can find are in
proportions and size, distinctions which in the general experience of
protozoologists are valid only for the separation of species.

162 University of California Publications in Zoology [ Vou. 20

The recent investigation by Alexeieff (1914) of the finer structure
of Giardia and our own more extended investigation (Kofoid and
Christiansen, 1915 a and b) bring convincing proof of morphological
similarity of the species referred to Giardia to those heretofore included
in Lamblia. They have the same organelles in similar relationships.
Their differences consist solely im contour, shape, proportion, size,
hosts, and shape of the parabasal bodies, and are valid only to separate
species. The species of Giardia and Lamblia should therefore all be
placed in one genus, as in the case of the various species of T'vricho-
monas and Trypanosoma. By the accepted laws of nomenclature
Giardia has priority.

The several species which have been described, especially by Bensen
(1908), are based on shght differences in form and proportions, and
Noe (1908) raises the question as to the validity of these distinctions.
After our experience with the material which we have examined,
which includes five species, Giardia alata, G. muris, G. microti, G.
duodenalis, and G. enterica, and a wide experience with the genus
Trichomonas, we are inclined to regard Noe’s skepticism as not well
grounded. One learns to detect those differences in size and propor-
tions, due to movement and growth, and to recognize others, usually
a group of characters, which constitute the specific distinctions, such
for example as those between G. muris and G@. microti. We believe
therefore that Bensen (1908) was correet in making distinctions
though it may be that not all his species will stand the test of fuller
examination, and that not all his distinctive characters will prove valid.

This matter is one of more than restricted interest for it impinges,
on the one hand, on the general biological question as to how far
specificity of parasites to particular hosts obtains, and on the other
hand on the very practical question as to whether or not rodents,
especially mice, rats, and Belgian hares, are sources of infection to
man, for these flagellate parasites, in mice and men at least, are often
associated with chronic intestinal disturbances (Noe, 1909; Roden-
waldt, 1912; Jollos, 1913; Kofoid and Christiansen, 1915). If the
species are distinct, it is less probable that rodents serve as sources
for human infection. If these so-called specific differences are illusory,
on the other hand, the dangers from rodents as carriers and sources
are greatly enhanced.

There remains the further problem which experiment only can
solve as to whether or not the same species of flagellate may pass
through a series of host species and assume in each, or in certain

1920] Kofoid: Nomenclature of Human Intestinal Flagellates 163

eroups of hosts, a given set of so-called species characters, and another
set in another species or group. The fact that G. muwris occurs in both
Mus and Peromyscus, and possibly also in the rat, without noticeable
modification is an indication of the fixity of at least this species under
different conditions as to hosts. Noe (1908) states that he finds in
man Lamblia which exhibit the whole range in modifications described
by Bensen for his species. It is important to learn in how far Noc’s
conclusions are critical, in how far the resemblances are superficial,
and in how far certain combinations of size, proportion, and structure
distinguish the species thus far described, and in how far these are
correlated with predominant relations to certain species as hosts.

The determination of the correct specific name for the Giardia from
man is entangled not only with the problem of the specific distinctness
of the species of Giardia in other hosts than man, especially in rodents,
but also with matters of synonymy and priority under the Code of
Nomenclature.

In our present opinion the morphological evidence indicates that
the several species normal to rodent hosts differ from that in man, and
that the Giardia of man should receive a separate specific designation.
Fantham and Porter’s (1916b) experimental inoculation of mice by
the Giardia from man does not necessarily invalidate the specific dis-
tinctness of the species of Giardia normally present in mice from that
found in man.

The first Giardia to be deseribed was that from man by Lambl
(1859) as Cercomonas intestinalis, but this specifie name is pre-
occupied in Cercomonas by Diesing’s (1850) transfer of Bodo intes-
tinalis Ehrbg. to Cercomonas, which this reviser treated as a sub-
genus of Bodo.

Davaine (1875) described Hexamita duodenalis from the rabbit.
This also is a Giardia but the species in the rabbit is distinct: from
that in man and duodenalis is therefore not available for the species
from man. Grassi (1881) proposed the name Megastoma entericum
for the flagellates belonging to Giardia which he had found in Mus,
Arvicola, the cat, and man. In 1879 he had given the name Dimorphus
muris to the species from Mus and Arvicola and in 1881 proposed
M. entericum as a substitute with wider application. Since the species
in the mouse is distinct from that in man the name Giardia muris is
applicable only to the former. No data are available as to the status
of the form in Arvicola and infection in the cat is presumably derived
from that in mice. By elimination the specific name Giardia enterica

164 University of California Publications in Zoology [| Vou. 20

(Grassi) becomes available for the Giardia from man in place of
intestinalis (Lambl).

The name Giardia lamblia Stiles (in litt.) proposed in Kofoid and
Christiansen (1915), p. 28) as a substitute for the preoccupied intes-
tinalis becomes therefore a synonym of G. enterica.

SUMMARY

1. Cercomonas is not as yet proved to be a human intestinal para-
site, but coprophilic species of this genus occur in stale stools or in
stool cultures. Cercomonas of clinical literature is probably Chilo-
mastix. Flagellate stages of Craigia may have been reported as
Cercomonas.

2. Cercomonas hominis Davaine (1860) was a complex of two
species. His var. A, named Cercomonas davainei by Moquin-Tandon
(1860), is Chilomastix. The correct name for Chilomastix is not C.
mesnilt (Wenyon) but C. davainet (Moquin-Tandon).

3. Davaine’s Cercomonas hominis var. B is the human intestinal
Trichomonas, the correct name of which is 7. hominis (Davaine).
Cercomonas obliqua Moquin-Tandon (1860), Trichomonas intestinalis
Leuckart (1879), and 7. confusa Stiles (1902) are synonyms of 7.
hominis.

4. The type species of Trichomonas Donné (1837) has four free
anterior flagella, hence Tetratrichomonas Parisi 1910 is a synonym
of Trichomonas. Trichomonas hominis from the intestine and 7.
buccalis from the mouth also have four flagella.

5. A flagellate of the mouth was first described by O. F. Miiller
(1773) as Cercaria tenaz, but the description and figure are too meagre
to permit re-identification. Tetratrichomonas buccalis Goodey and
Wellings (1917) should be transferred to Trichomonas.

6. A new genus Tritrichomonas is established for trichomonad
flagellates with three anterior free flagella with 7. augusta Alexeieft
as type species.

7. Giardia enterica (Grassi) 1881 has priority over Lamblia intes-
tinalis (Lambl) Blanchard 1888, and over Giardia lamblia Stiles 1915.

Transmitted April 19, 1920.
ZOOLOGICAL LABORATORY, UNIVERSITY OF CALIFORNIA,
BERKELEY, CALIFORNIA,

1920] Kofoid: Nomenclature of Human Intestinal Flagellates 165

LITERATURE CITED
ALEXEIEFF, A.
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Bartow, N.
1915. Craigiasis. Am. Jour. Trop. Med., 2, 680-702, pl. 26.
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1908. Bau und Arten der Gattung Lamblia. Zeitschr. f. Hyg. u. Infekt.,

61, 109-114, 6 figs. in text.
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1888. Remarques sur le megastome intestinal. Bull. Soe. Zool. France, 13,

18-19.
Brumpt, E.

1912. Célite a Tetramitus Mesnili (Wenyon 1910) et Célite Trichomonas
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1913. Précis de Parasitologie. Preface de M. le professeur R. Blanchard;
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Bory DE SAINT VINCENT, J. B. G. N.
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1918. Alcune osservazione sulla etiologia, diagnose e cura della dysenteria.

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1917. Chilomastix mesnili (Wenyon, 1910). Ann. Trop. Med. Paras., 11,

213-264, pls. 6, 7, 1 fig. in text.
CuHacr, A. F., AnD TasKer, A. N.

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Assoc., 68, 1528-1531, 1 fig. in text.
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1915. Further investigations into latent dysentery and intestinal parasitism

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1911. The parasitic amoebae of man. (Lippincott, Philadelphia), pp. x +

258, 30 figs. in text.
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33, 262-266.
DaVAINE, C.

1854. Sur les animaleules infusoires trouvés dans les selles de malades
atteints du cholera et d’autres affections. Compt. Rend. Soe. Biol.,
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1860. Traité des Entozoaires et des maladies vermineuses de l’homme et des

' animaux domestiques; ed. 1 (Paris, Bailliére et Fils), pp. xix + xcii
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1875. Monadiens. Dict. encyel. d. sei. méd. sér. 2, 9, 115-130.

Diesine, K. M.,
1850-1851. Systema helminthum (Vindibonae, Braumiiller), 1, pp. xiii + 679.

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DortetIn, F.
1916. Lehrbuch der Protozoenkunde; ed. 4 (Leipzig, Fischer), pp. xv +
1190, 1198 figs. in text.
Donné, A.

1837. Recherches microscopiques sur la nature des mucus et la matiére des
divers écoulemens des organes genito-urinaires chez l’homme et
chez la femme (Paris, Chevalier), pp. iv + 71, pl. 1.

DuJARDIN, F.

1841. Histoire naturelle des Zoophytes. Infusoires, comprenant la physi-
ologie et la classification de ces animaux, et la maniére de les
étudier 4 1’aide du microscope (Suites 4 Buffon, Roret, Paris), pp.
xii + 684, 22 pls.

FantHam, H. B.

1916. Remarks on the nature and distribution of the parasites observed in

the stools of 1305 dysenteric patients. Lancet, 1916, i, 1165-1166.
FantTHAM, H. B., AND PorTER, A.

1916a, The pathogenicity of Giardia (Lamblia) intestinalis to men and to
experimental animals. Brit. Med. Jour., 1920, ii, 139-141.

1916b. Notes on certain Protozoa which may be found in eases of dysentery
from the Mediterranean war zone. Proce. Camb. Phil. Soe., 18, 184—
188.

GoopEy, T., AND WELLINGS, A. W.

1917. Observations on Entamoeba gingivalis from the human mouth with a
note on the trichomonad flagellate Tetratrichomonas buccalis, n. sp.
Parasitology, 9, 5387-559, pls. 20-22.

GRASSI, B.

1879. Die protozoi parassiti e specialmente di quelli che sono nell’uomo.
Gazz. Med. Ital. Lomb., 39, 445-448.

1881. Di un nuovo parasite dell’uomo Megastoma entericwm (mihi). Gazz.
degli Ospitali, Milano, 2, 575-580.

GRASSI, B., AND SCHEWIAKOFF, W.

1888. Beitrag zur Kenntnis des Megastoma entericum. Zeitschr. f. wiss.

Zool., 46, 143-154, pl. 15.
GUASTALLA, N.

1919. Flagellaten im menschlichen Darm. Beitrag zur Kenntniss der In-

fusorienenteritis. Wien. klin. Woch., 22, 1560-1563.
HARTMANN, M., AND CHAGAS, C.

1910. Flagellaten-Studien. Mem. Inst. Oswaldo Cruz., 2, 64-126, pls. 4-9,

3 figs. in text.
JANOWSKEI, W.

1897. Ueber Flagellaten in den menschlichen Faces und tiber ihre Bedeut-
ung fiir die Pathologie des Darmkanals. Zeitschr. f. klin. Med.,
31, 442-492, pls. 4-5.

KUNSTLER, G.

1882. Sur cinque protozoaires nouveaux. Compt. Rend. Acad. Sci. Paris,
95, 347-349.

1883a. Recherchés seu les infusoires parasites. Sur quinze Protozoaires
nouveaux. Ibid., 97, 755-757.

1883b. Histoire naturelle des infusoires parasites. Ann. Sci. Nat. Bordeaux
et du Sud-Ouest, 3, 45-54, pls. 1, 2.

1884. Trichomonas vaginalis Donné. Jour. de micrographie, 1, 317-331, pls.
5, 6.

1920] Kofoid: Nomenclature of Human Intestinal Flagellates 167

KUNSTLER, J., AND GINESTE, C.
1907a. Giardia alata, n.sp. Compt. Rend. Acad. Sci. Paris, 144, 441-443.
1907b. Megastoma, Lamblia ou Giardia? Bull. Soe. Zool. France, 32, 28-32.
Koro, C. A.

1920. Intestinal protozoan infections in United States army troops. Trans.

Amer. Gastroenterol. Assoc. In press.
Kororp, C. A., AND CHRISTIANSEN, E. B.

1915a. On Giardia microti, sp. nov., from the meadow mouse. Univ. Calif.
Publ. Zool., 16, 23-29, 1 fig. in text.

1915b. On binary and multiple fission in Giardia muris Grassi. Ibid., 30-54,
pls. 5-8, 1 fig. in text.

LAMBL, W.

1859. Mikroskopische Untersuchungen der Darmexcrete. Beitrag zur Path-
ologie des Darms und zur Diagnostik am Krankenbette. Viertel-
jahrsehr. prakt. Heilk., 61, 1-58, pls. 1-4, figs. 1-13 in text.

1875. Cercomonas et Echinococcus in hepate hominis. Med. Vestnk., St. Peters-
burg.

LEDINGHAM, J. ©. G., PENFoLD, W. J., AND Woopcock, H. M.

1915. Recent bacteriological experiences with typhoidal diseases and dysen-
tery. With notes on the protozoan parasites by Woodcock, H. M.
Brit. Med. Jour., 1916, ii, 704-711, 4 figs. in text.

LEucKaRT, R.

1863. Die menschlichen Parasiten und die von ihnen herriihrenden Krank-
heiten. Ein Hand- und Lehrbuch fiir Naturforscher und Aertze
(Leipzig, Winter), pp. viii + 766, 268 figs. in text.

1879. Idem, ed. 2,1, Lief. 1, pp. viii + 336, 130 figs. in text.

Lyncu, K. M.

1915a. Trichomoniasis of the vagina and the mouth. Cultivation of the
causative organism and experimental infection. Am. Jour. Trop.
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1915b. Clinical and experimental trichomoniasis of the intestine with culti-
vation of the causative organism. New York Med. Jour., 101,
886-889.

Manp1, L.
1838-1845. Anatomie microscopique (Paris, Bailliére).
MartrHews, A. J., AND SmitH, A. M.

1919. The intestinal protozoal infections among convalescent dysenteries
examined at the Liverpool School of Tropical Medicine (Third
report). Ann. Trop. Med. Parasit., 13, 83-90.

MELLO, LEITAO.

1913. Flagellate dysentery in children. Brit. Jour. Children’s Dis., 10,

60-66.
Movgutn-Tanpon, A.

1860. ‘léments de zoologie médicale (Paris, Bailliére et Fils), pp. xvi + 428,
122 figs. in text.

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Miter, O. F.

1773. Vermium terrestrium et fluviatilium, seu animalium infusorium,
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(Havniae et Lipsiae, Heineck et Taber), v. 1 (1), pp. 30 + 135;
v. 1 (2), pp. 72 + 8; v. 2, pp. xxxvi + 214 + 10.

168 University of California Publications in Zoology [ Vox. 20

1776. Zoologiae Danicae prodromus, seu animalium Daniae et Norvegiae
indigenarum characteres, nomina et synonyma imprimis popularium
(Havniae, Hallageriis), pp. xxii + 274.

1786. Animaleula infusoria fluviatila et marina, quae detexit, systematica
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NEVEU-LEMAIRE, M.

1908. Précis de parasitologie humain. Maladies parasitaires dues a des
végetaux et des animaux (Paris, Rudeval), ed. 4, pp. v + 712,
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Noe, F.
1908. Observations sur le cycle évolutif de Lamblia intestinalis. Bull. Soe.
Path Exot., 2, 93-97.
Parisi, B.
1910. Su aleuni flagellati endoparassiti. Arch. f. Prot., 19, 232-238, pl. 14.
Porter, A.

1918. A survey of the intestinal Entozoa, both protozoal and helminthie
observed among natives in Johannesburg from June to November,
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PRENTISS, C. W.

1914. Infection with the Cercomona hominis. New Mexico Med. Jour.,

December, 1914. Reprint, 5 pp.
RAILuiet, A.

1886. Eléments de zoologie medical et agricole (Paris, Asselin et Houzeau),
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1895, Traité de zoologie médicale et agricole (Paris, Asselin et Houzeau),
ed. 2, pp. xv + 1803, 892 figs. in text.

SANGIORGI, G.
1919. Dissenterie in Albania. Pathologica, 21, 141-144.
Srwon, 8. K.

1918. Cercomonas diarrhea and its management. Southern Med. Jour., 11,

414-417.
SMITHIES, F.

1918. The frequency of protozoic enterocolitis in the Middle West, clinical
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STiLEs, C. W.

1902. The type species of certain genera of parasitic flagellates, particularly

Grassi’s genera of 1879 and 1881. Zool. Anz., 25, 689-695.
VERDUN, P.

1907. Préeis de parasitologie humaine. Parasites animaux et végetaux (les
bactériees exceptées) (Paris, Doin), pp. iii + 727, 4 pls., 310 figs.
in text.

WENYON, C. M.

1910a. A new flagellate (Macrostoma mesnili, n. sp.) from the human intes-
tine, with some remarks on the supposed cysts of Trichomonas.
Parasitology, 3, 210-216, pl. 16.

19106. Some observations on a flagellate of the genus Cercomonas. Quart.

Jour. Mier. Sci., n.s., 55, 241-260, 19 figs. in text.
WENYON, C. M., AND O’ConNor, F. W.

1917. Human intestinal Protozoa in the Near East (London, Bale), pp. 218,

4 pls.

UNIVERSITY OF CALIFORNIA PUBLICATIONS— (Continued)

11. A Study of tke Races of the White-Fronted Goose (Anser albifrons) occur-
ring in California, by H. 8, Swarth and Harold C. Bryant. Pp, 209-222,
2 figures in text, plate 18. October, 1917 22.0... v7
12. A Synopsis of the Bats of California, by Hilda Wood Grinnell, Pp. 223-404,
plates 14-24, 24 text figures. January 31, 1918 oc ens
13. The Pacific Coast Jays of the Genus Aphelocoma, by H. 8. Swarth. Pp.
405-422, 1 figure in text. February 23, 1918 Woo
14. Six New Mammals from the Mohave Desert and Inyo Regions of California,
by Joseph Grinnell. Pp. 423-430.
15. 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 o2......c lec eecceceseeeeeeneenee
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17, The Subspecies of the Mountain Chickadee, by Joseph Grinnell. Pp. 505-
bib,7S- text feuress “Mavi 1 Ob eo SN eo Bed al es a
18. Excavations of Burrows of the Rodent Aplodontia, with Observations on
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eb eRe 9.9 RR Jae ies tl: Pa RSS aN Seal Buy ip pe Fae. tok SP BY RCD POE IRAE OE So ka
Index, pp. 537-545,

Vol. 18. 1, Mitosis in Giardia microti, by William C. Boeck. Pp. 1-26, plate 1. Octo-

1 pn RE AL Sea NIRS oh Jed a ERNE Ie arg Weill Leshan natch SS ae MOc OG tae ale telat a
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,
8. Description of Some New Species of Polynoidae from the Coast of Cali-
fornia, by Christine Essenberg. Pp, 45-60, plates 2:3, October, 1917...
4. New Species of Amphinomidae from the Pacific Coast, by Christine Essen-
berg. Pp. 61-74, plates 4-5. October, 1917 222. eeeenceteneeeeeee
6. Crithidia euryophthalmi, sp, nov., from the Hemipteran Bug, Rurgounthainnd
convivus Stal, by Irene McCulloch. Pp. 75-88, 35 text figures: Decem-
DBT LON Poco ee ae a ol LN oes SR res ree eee abd oA
6, On the Orientation of Hrythropsis, by Charles Atwood Kofoid and Olive
Swezy. Pp. 89-102, 12 figures in text. December, 1917 _..200..
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ARPES bee IS NS a oe ee

. The Function of the Giant Fibers in Earthworms, by John F. Bovard. Pop.
185-144, 1 figure in text. Jamuary, 1918 ooo no. leech ene

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

Faeces, by William ©. Boeck, Pp. 145-149. December, 1917 2...2.0220....... =

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

ie dipures) in text. BEAR Ch 19 ce ree ee oe a a

11. The Factors controlling the Distribution of the Polynoidae of the Pacific
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SUES TT SER G | ARON, A DEB ce Bee eg Bey:

12, Differentials in Behavior of the Two Generations of Salpa democratica
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18. A Quantitative Analysis of the Molluscan Fauna of San Francisco Bay, by
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14, The Neuromotor Apparatus of Huplotes patella, by Harry B. Yocom, Pp,
837-396, plates 14-16. September, 1918 22... .eccecccc cece: eel cc detec ctneeteceenneesee

15. The Significance of Skeletal Variations in the Genus Peridinium, by A. me
Barrows. Pp. 397-478, plates 17-20, 19 figures in text. June, 1918.00...

16. The Subclayian Vein and its Relations in Elasmobranch Fishes, by J.
Frank Daniel. Pp. 479-484, 2 figures in text, August, 1918 -.W

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. Jatuary, 1919.2... .c. cee a eeeeeseeneee ee: re
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15
2.00

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75

15

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Vol. 19. 1. Reaction of Various Plankton Animals with Reference to their Diurnal
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2. The Pteropod Desmopterus pacificus (sp. nov.), by Christine Essenberg.

Pp. 85-88, 2 ‘figures in text..- May, 1919, ub coo

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

gifs Tel tue 8 RE x. 3 aie an eee ey peBloDN, ook ee 1a Pies rst Ara NAR Has AFA De eet Lara Sip Ol YB

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, 8

figures in fext/ October, "1919 oe oe oe saat ig

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6. Binary Fission iu Collodictyon triciliatum Carter, by Robert Clinton Rhodes.

Pp. 201-274, plates 7-14, 4 figures in text.’ December, 1919 20...

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A New Distome from Rana aurora, by William W. Cort. Pp. 283-298,

5 firures, ini text: INovember; 1919.05. tas Be he eu ee ee

9, The Occurrence of a’ Rock-Boring Isopod along. the Shore of San Fran-
cisco Bay, California, by Albert L. Barrows. Pp. 299-316, plates 15-17.

#

DISCem ber LOLS Thiet aby eeepc ann cele adi a tele alr Malpas nc bn decease x

10. A New Morphological Interpretation of the Structure of Noctiluca, and
its Bearing on the Status of the Cystoflagellata (Haeckel), by Charles
A. Kofoid. Pp. 317-3384, plate 18, 2 figures in text. February, 1920...
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Pp, 338-388, plates 19-25, 1 text figure. May, 1920 ieee

Vol. 20. 1. Studies on the Parasites of the Termites. I. On Streblomastix stria, 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
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a Polymastigote Flagellate with a Highly Developed Neuromotor System,
by Charles Atwood Kofoid and Olive Swezy. Pp. 21-40, plates 3-4, 2
firures, intext: Duly, iL O19. 2 sy a eR oe ee met

8. Studies on the Parasites of the Termites. III. On Trichonympha campanula

Sp. Nov., by Charles Atwood Kofoid and Olive Swezy. Pp. 41-98, plates
5-12, 4 figures in texts) Duly, TOL Se eo ES PO MEY

4, Studies on the Parasites of the Termites. IV. On Leidyopsis sphaerica
gen. nov., sp. nov., by Charles Atwood Kofoid and Olive Swezy. Pp.
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5. On the Morphology and Mitosis of Chilomastia mesnili (Wenyon), a Common
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ZOOLOGY
Vol. 20, No. 7, pp. 169-198, plates 18-22, 3 figures in text June 23, 1921

ON THE FREE, ENCYSTED, AND BUDDESGac
STAGES OF COUNCILMANIA LAFLEL&1, SD

A PARASITIC AMOEBA OF THE JUL 14 1993
HUMAN INTESTINE* :

BY
CHARLES ATWOOD KOFOID anp OLIVE SWEZY

’ a. A
fiona} M ysevs~

CONTENTS PAGE
BREE OCU CU OWN oo 2 acne chase cee ce renee Seen oc on cess ances tac eecncescu ins avusstasavacescccescusesccesedssrtacedennssavescoscee 169
pivlist resell smn Guerry Ct HO C'S yeeerccecacsee tesa face! ce Save nce ayece dc Oersna ses Aceee seats yacecacdceseedectenseczteese 171
2S ACOT HINT I Rn eee ee PEE Sees ecusss ote pee eee ee ie acs See eens ee tan eet h a caceeee
The free amoeba
STN) ROO A SLAC TO EIS: ae ee ee ee 175
LDASaGOYE EO) TS Wie ee ee oases ES ee 180
Budding and escape of amoebulae —.............. Ae Aerts ot eat ta Wasshoi 181
GPRD NOE) eee ee oe ae ee as a ee Serle pane edeee eee Soe ee Ee PRES 183
(va PTET], NTN No COE ee eee te eee 185

SS OST CO LC SES a a ee eB i 185
Distinctions between Councilmania lafleuri and Endamoeba coli 86
PMTURYNTAN UI Va cst nears Deck o ceca cha cn vane scceaceounsvatecscnsacicen census Saves Uadocebavcaepincesasescsecssseacbeees 187
LAAN TEEN TEER CTY 6 ee ee a ea enaee eee 188

INTRODUCTION

The perplexing confusion which has long existed regarding the
amoebae of the human digestive tract owes its origin and growth to a
complex of causes, including the difficulties of securing and controlling
fresh normal material, the existence of pathological, moribund, and
abnormal stages in the parasitic organisms themselves, and the presence
in man of multiple infections of molds, yeasts, flagellates, coccidia, and
amoebae, with diverse races and phases in their life histories. The
clinician and protozoologist must learn to distinguish these several
organisms in their various aspects and avoid confusing them if therapy
is to be critically applied, and morphology and life history are to be
correctly interpreted.

* From the Zoological Laboratory, University of California, and the Division
of Parasitology, Bureau of Communicable Diseases, California State Board of
Health.

170 University of California Publications in Zoology [ Von. 20

The intensive study of the protozoan infections of the human diges-
tive tract incident to the prevalence of dysentery during the late war
among troops on the Western, and especially the Near Eastern fronts,
has clearly established the unexpectedly wide distribution of amoebie
infections in man.

In the case of these amoebae the confusion is increased by the
existence of no less than six different valid species of amoebae in
human stools and of size races within some, if not all, of these. These
are the three common species: Endamoeba dysenteriae (Councilman
and Lafleur), Endamoeba colt (Losch), Endolimax nana (Wenyon and
O’Connor), and the rare Dientamoeba fragilis (Dobell and Jepps).
We have recently found a clearly established case of human infection
with a fifth amoeba called Entamoeba muris (Grassi) by Brug (1919),
which Brug and ourselves find to be a normal parasite of wild and
culture rats. Under Endolimax nana we include tentatively the form
known as the ‘‘iodine body’’ which we regarded (see Kofoid, Korn-
hauser, and Swezy, 1919) as the large glycogen-bearing race of Endo-
limax nana.

In connection with Endamoeba coli some differences of opinion
and of observation still persist with regard to its activities, structure,
and life history. Specifically these concern the appearance of the
pseudopodia, the rapidity of formation of pseudopodia and of move-
ment, the ingestion of red blood corpuscles, pathogenicity, the strue-
ture of the karyosome, and the formation of amoebulae by a process
resembling budding from eysts in the stools. These debated points
we hope will be cleared up by the evidence here presented that there
is in man another amoeba hitherto undescribed and heretofore con-
fused with Endamoeba colt, primarily because of its eight-nucleated
cyst.

It is the purpose of this paper to describe this sixth species of
amoeba from the human intestine, which may be present in enormous
numbers, appears to have pathogenic capacities, and has apparently
been passed over as EZ. coli because of its large size, its eight-nucleated
eyst, and its high resistance to stains, all of which tend to obscure its
distinctive characteristics.

We take pleasure in naming this amoeba Cowncilmania lafleurt, in
recognition of the critical discovery in 1891 by these two investigators
of the relation of the amoeba called by them Amoeba dysenteriae to
dysentery and hepatic abscess, a landmark in the medical conquest of
this widespread infection of man.

1921] Kofoid-Swezy: Councilmania lafleuri 171

MATERIAL AND METHODS

The material upon which this study is based is derived from ten
cases under observation for varying periods from July, 1920, to June,
1921. One ease especially has been examined almost daily for over
120 days, the infection appearing, prior to treatment, in nearly every
stool examined from this and some of the other cases under observation.
Hundreds of fresh smears and over nine hundred prepared slides of
both stained smears and sections have been available for study, as have
also several warm stools with an abundance of free amoebae.

Our attention was first drawn to the species by its remarkable
resistance to stain in the usual Schaudinn-iron haematoxylin treatment,
the cysts remaining quite colorless while those of other intestinal
amoebae were stained as usual. It was only after fixing in Schaudinn’s
fluid at 60° C. for several minutes that we were able to stain the cysts
in iron haematoxylin.

We have been able to observe the living organism in the electric
ineubator and to secure preparations of all stages from the free amoeba
to the eight-nucleated cyst and the escaping amoebulae, and are thus
able to determine its distinctive characteristics.

We are deeply indebted to physicians and patients who have made
this investigation possible, especially to Dr. C. L. MeVey, of the staff
of the University Infirmary, and to Dr. R. T. Legge, University
Physician, and to students in their charge who have generously co-
operated to make our observations possible. Acknowledgments are
gratefully made for grants from Mrs. Margaret B. Fowler and an
alumnus of the class of 1900 in support of this work.

ACTIVITIES

In warm liquid stools following a purge of magnesium sulphate,
examined on the warm stage or electric incubator, the free amoebae
predominate and are many of them very active. Some are rounded
up and inactive and among them are probably precystic stages dis-
charged prior to the early phases of encystment.

Amoebae in the active stages creep about with great rapidity. One
elongated amoeba traversed five times its length in one minute and

172 University of California Publications in Zoology | Vou. 20

its whole length in five seconds. There are frequent changes in direc-
tion of locomotion, due either to the swerving of the pseudopodium
as a whole or to the origin of pseudopodia at new areas on the per-
iphery. The nucleus generally lags in the posterior part of the body
when the same general direction is maintained continuously for a
time. The pseudopodia he at or near the level of the substrate.

Fig. A. Free-hand sketches of nine successive phases of formation of pseu-
dopodia in an active amoeboid stage of Councilmania lafleuri during one minute.
X about 600.

There is generally a single pseudopodium in formation at one time.
It is perfectly clear and appears to be composed wholly of ectoplasm.
It is usually broad, from one-fourth to one-half the diameter of the
body in width, though in elongated, traveling individuals it may rarely
equal the width. In rounded-up individuals a slender pseudopodium
one-fifth the diameter in width and as long as the radius may be
thrown out. The tip of the pseudopodium is broadly rounded, rarely
crenated or lobed, never pointed or bifureated. In mobile individuals
the pseudopodium is quickly invaded by the granular, vacuolated endo-
plasm. Pseudopodia may be found at two, rarely three, areas on the

1921] Kofoid-Swezy: Councilmania lafleuri 173

periphery at one time, but usually one of these is advancing and the
others withdrawing.

One of the most noticeable features in connection with the pseudo-
podia of this amoeba is the expulsive suddenness of their formation.
They are shot out almost instantaneously for the greater part of their
length while the remaining extension and peripheral modifications
proceed more leisurely. Invasion by the endoplasm is less rapid than
pseudopodial formation. In the event of change of direction of loco-
motion the withdrawal of the pseudopodium is relatively very slow.
The suddenness and rapidity of formation of the pseudopodia is the
most striking feature of the free stage of this very active amoeba.

THE FREE AMOEBA
Plates 18-20, figures 1-17

The normal free stage of Councilmania laflewri oceurs in diarrheic
and dysenterie stools and in liquid stools after a saline purge and
sometimes in strands of bloody mucus on formed stools. It is rounded
up in cold stools, and when extended becomes sluggish with the drop
in temperature. In rare instances formation of pseudopodia may
continue for as long as thirty minutes in ordinary smears without the
warm stage.

The ectoplasm of Councilmania is most evident in the clear hyaline
pseudopodia. Over the remainder of the body it forms a scarcely
visible peripheral film. In life the ectoplasm of the pseudopodia ap-
pears wholly structureless. In stained preparations it is often some-
what chromophile in the pseudopodia in iron haematoxylin and destains
slowly, exhibiting a dense homogeneous substance of fine texture quite
distinct from the granular and vacuolated endoplasm (pl. 18, fig. 4).
In some amoebae the ectoplasm destains wholly in the pseudopodia
or in their peripheral parts. In rounded-up individuals the ectoplasm
forms only a thin peripheral film, although in certain individuals
which seem to belong to Counctlmania, from their nuclear structure,
there is a wide, vacuolated, clear zone in the periphery.

The pseudopodia of Councilmania differ from those of Endamocba
coli in being free from coarsely granular, vacuolated endoplasm, and
from those of H. dysenteriae in being perhaps rather smaller, restricted
as a rule, in the active state, to a smaller part of the periphery, and
in the expulsive suddenness of their formation. They are also some-
times more chromophile.

174 University of California Publications in Zoology (Vou. 20

The endoplasm, on the other hand, is coarsely vacuolated in the
free stage and may be loaded with food vacuoles containing bacteria,
red blood corpuscles, and occasionally a cyst of Chilomastix (pl. 19,
fig. 6), or other particles from the faeces. This marked vacuolation
and the great abundance of food contents of all sorts distinguish it
at once from Hndamoeba dysenteriae, but lead to confusion with
Endamoeba coli in the absence of pseudopodia.

The food vacuoles and the fluid-filled ones which are seattered
among them may crowd to the periphery of the body locally, or they
may leave a narrow peripheral zone relatively free from such inelu-
sions. They are moved about freely in the mobile endoplasm, as is
the nucleus. The latter often les on the very edge of the endoplasm.

The nucleus in the free stage presents different aspects, according
to the degree of destaining and to phases of the mitotie cycle. The
chromatin is distributed in the peripheral zone on the nuclear mem-
brane and in a karyosome separated from the former by an inter-
mediate clear zone. The peripheral chromatin is broken up into a
relatively thin sheet (as compared with that of Endamoeba coli) of
somewhat uniform particles flattened against the membrane and giving
to it in some instances a crenate Inner margin in optical section.
Occasionally a larger particle is interpolated. This layer is not so
thick as in Endamoeba coli, but is much thicker in the free amoebae
than in the nuclei of the older eysts in Councilmania, and is also thin-
ner in free amoebae when the karyosome is enlarged and mitosis is
approaching.

The karyosome in the resting nucleus is generally somewhat ex-
centric, as it is in EZ. coli. Not rarely it is quite excentric. It may
be spheroidal or angular and has a very narrow, not always concentric,
clear halo about it. This halo can not be found in many nuclei, espec-
ially those in which mitosis is approaching. In such nuclei the karyo-
some takes on the form seen in the cysts and has a much larger volume
of chromatin, which breaks up into particles forming a sphere, ring,
or reniform mass as the spireme stage approaches (pl. 19, figs. 7, 8).

The intermediate zone is free from distinct structural modifications.
There may be a faint reticulum and oceasionally a few minute chrom-
atin granules in it in heavily destained preparations. We are un-
able, with our method of fixation and available eosins, to demonstrate
in it any eosinophile granules such as Dobell (1920, pl. 2, figs. 32-34)
finds in the amoeba which he regards as the free stage of his Jodamoeba
buetschlii. Otherwise there is enough in common between free Cown-

1921] Kofoid-Swezy: Councilmania lafleuri 175

cilmania and these figures of Dobell’s to suggest the desirability of
verification of his interpretation that the free amoeba assigned by him
(his pl. 2, figs. 32-34) to his Zodamoeba buetschlii really belongs to
that species rather than to Councilmania. Stools in which Council-
mania is not found but which carry the eneysted stage of Jodamoeba
should be searched for free amoebae. No ‘‘Zodamoeba’’ has been found
in either free or in encysted stages in the cases from which our free
Councilmania have been derived.

There is usually only a single nucleus in the free amoeba. We
have, however, found a number of amoebae with two nuclei each (pl. 19,
fig. 8) with the karyosomes in the skein stage. They do not appear
to be abnormal. In several instances in stools after prolonged treat-
ment with antimony trioxide amoebae have been found in which no
nucleus whatever can be detected. Many dead and abnormal amoebae
were present in these stools.

THE ENCYSTED PHASES
Plates 20-22, figures 11—28

The precystic and encysted stages of this amoeba occur in non-
diarrheic stools predominantly, free stages being rare and usually
rounded up, moribund, and not reviving into activity on the warm
stage when taken from stools recently cooled. These uneneysted indi-
viduals represent either precystic individuals, those from the lower
parts of the bowel which have had insufficient time to pass into the
eneysted phase, or more often moribund individuals, perhaps from
higher levels in the bowel which, by reason of senescence or other
physiological states, are not proceeding with encystment. The only
activity aroused in a few instances in these individuals on warming is
the slow protrusion or the withdrawal of broad shallow pseudopodia
one-third to one-half of the cireumference in width.

The precystic phases (pl. 19, figs. 6, 7) are uninucleate, rounded
up, with cytoplasm relatively free from food inclusions. The nucleus
does not as a rule differ from that in the free stage, has an enlarged,
often excentric karyosome with less evidence of a clear zone about it,
and considerable peripheral chromatin, and shows no indications of
approaching mitosis.

The encysted stage, on the other hand, has a definite eyst wall,
generally exhibits eight nuclei, each with large, often asymmetrical
karyosome often centrally located, little peripheral chromatin, no

176 University of California Publications in Zoology —_ [ Vou. 20

glycogen, and massed or scattered, somewhat pointed chromatin bodies.
The latter may not be present.

The advanced cysts in the fresh stool examined by light from a
Mazda lamp with blue glass screen have a grayish tone corresponding
to the pale olive, light mineral, or pallid neutral gray of Ridgway’s
“Color Standards and Color Nomenclature.’’ The thick hyaline wall
of the cyst stands off the bacteria and in optical section its innermost

Fig. B Fig. C

Fig. B. Free amoeba of Councilmania lafleuri, with food vacuoles containing
bacteria and red blood corpuscles. Ectoplasm sharply separated from the endo-
plasm. Nucleus with excentrie karyosomes and thin zone of peripheral chromatin.
X 1600.

Fig. C. Budding cyst of Councilmania lafleurt with six nuclei remaining in
the eyst and a seventh in a budding amoebula with chromophile cytoplasm.
Nuclei with somewhat excentric karyosomes of the dispersed type and some
granular peripheral chromatin forming on the nuclear membrane. X 1600.

zone has a dark brownish tinge. The nuclei are indistinct or faintly
outlined. The chromatoidal bodies, when present, are somewhat more
highly refractive than the cytoplasm.

The cytoplasm in the iodine-eosin stain shows more elearly a
coarsely irregular alveolar structure, the chromatoidal bodies are more
distinct, the nuclei and karyosomes become more evident, and the
whole cyst assumes in time a modified chrome yellow tone. Occasion-
ally reninants of food vacuoles and their contents are found in pre-
cystie stages, and rarely in later eneysted phases.

}

1921] Kofoid-Swezy: Councilmania laflewrt 17

The eight-nucleate encysted stage is preceded by the uninucleate,
binucleate, and quadrinucleate stages, which are less abundant than
those with eight nuclei in most stools, but may be found in larger
numbers in liquid stools or in mucus strands on formed stools. Three
successive divisions occur, resulting in two, four, and eight nuclei.
These divisions are approximately synchronous in the one, two, and
four nuclei at the successive mitoses, though slight irregularities in
division rate or even an abnormal or moribund nucleus may occasion-
ally be found. Occasionally a fourth mitosis in the eyst which would
result in sixteen nuclei is indicated by the prophase conditon of the
eight nuclei. In one instance a eyst with eleven nuclei was found.
This last mitosis proceeds sometimes during budding.

In the uninucleate stage (pl. 20, fig. 11) the nucleus is pushed to
the wall by the central vacuole, as are also the nuclei of the binucleate
(pl. 20, fig. 12), and sometimes in the quadrinucleate (pl. 20, fig. 14)
stages, while in the eyst with eight nuclei, and sometimes in the quadri-
nucleate ones, the vacuole has vanished and the nuclei are distributed
through the cytoplasm (pl. 20, fig. 15). The nuclei in these early
stages are much larger than in the later ones! The prevalence of
mitotic phases and the nuclear conditions generally in the stages prior
to the cyst with eight nuclei indicate that the three mitoses follow
rapidly one after the other, though the predominance of the binucleate
phase suggests that this is more prolonged than the quadrinucleate.
Not all stools, even liquid ones, contain these stages, as a consequence,
probably, of recurrent periods of multiplication in the bowel in the
intervals between which such stages are rare or wanting.

There are eight chromosomes in Councilmania lafleurt clearly dem-
onstrable at the metaphase, while Endamocba coli has but six. An
intradesmose connecting distinct chromatic polar masses is prominent
in the prophase-metaphase period of mitosis, in the first and second
mitoses especially, in Councilmania. It lies in contact with the nuclear
membrane.

The term intradesmose is proposed for the chromophile strand be-
tween two polar centrosomes of the spindle, which lies on the inner
face of the nuclear membrane, in contradistinction to the paradesmose
of flagellates on the outer side of the nuclear membrane.

In the precystic stage neither glycogen vacuole nor chromatoidal
bodies are present, but both develop shortly after encystment. The
glycogen vacuole develops first. Many uninucleate cysts have this
vacuole but no chromatoidal bodies, which appear later and usually

178 University of California Publications in Zoology — [Vou. 20

continue from the binucleate stage until even after eight nuclei are
formed and budding begins.

The ‘‘glycogen’’ vacuole (pl. 20, figs. 11-13) is centrally located,
and is relatively very large, in extreme instances filling seven-eighths of
the diameter of the cyst (pl. 20, fig. 11). At first it is spheroidal, but
is indented on one side by the enlarging nucleus. In the binucleate
stage the nuclei at first lie close together, but later are found on op-
posite sides of the vacuole, which they deeply indent. In later stages
the cytoplasm comes more and more to encroach upon the contents of
the vacuole, appearing in optical section as ridges or tapering pro-
cesses (pl. 20, fig. 14).

The structure of the contents of this vacuole in the fresh eysts and
in those in the iodine-eosin stain is that of a homogeneous, undiffer-
entiated substance, though in a few instances a faint reticular mesh
could be detected within it. In preparations passed through aqueous
solutions in staining the contents of the vacuole are entirely removed.
Its substance does not stain brown readily, if at all in some stools, in
iodine-eosin. It remains in such stools wholly unchanged for hours
in this stain. In smears in Best’s carmine and in eelloidin sections in
the same stain it remains unstained or at the most slightly tinged with
pink. In a few stools only were we able to find cysts giving anything
resembling a characteristic glycogen reaction in iodine. In one in-
stance they were in a stool passed after three weeks of treatment with
antimony trioxide and in another in stools after phenolax had been
given. The stool contained a number of moribund and pathological
amoebae, and we found a few advanced eysts in which the central
vacuole stained a deep reddish brown and was somewhat broken up.
This failure to react normally and readily to iodine leaves the nature
of the substance in this vacuole somewhat problematical. It may be
that the heavy cyst wall interferes with the typical glycogen reaction
of the contents as it does with fixation and staining. The occurrence,
period of existence in the life cycle, morphological relations, solubility
in water, and stainability (in a few instances only), all indicate that
it is glycogen, or some substance very similar to it.

The chromatoidal bodies (pl. 20, figs. 11-16; pl. 21, figs. 17, 19)
are quite similar to those of Endamoeba coli, but somewhat stouter
and less acicular. They first appear in the uninucleate, and especially
in the binucleate cysts in the thin film of protoplasm surrounding the
glycogen vacuole as numerous small flecks, blades, granules, or pointed
fusiform bodies staining deeply in iron haematoxylin in the typical

1921] Kofoid-Swezy: Councilmania lafleuri 179

fashion. As the glycogen mass decreases in size and finally vanishes
they come to le nearer the center of the cyst and may appear as a
central bundle visible in fresh smears as a more highly refractive area,
or in stained cysts as a fascicle of pointed splinters, or as scattered
threads, or even in the last stages as small spheroidal masses (pl. 21,
figs. 20, 22).

The chromatoidal substance appears to be involved in the strue-
tural and metabolic processes which result in the formation of the
ridges, the opening of the pore, and the formation of the bud. This
is shown in some instances by the radiation of a tripartite ridge from
the end of the chromatoidal mass, or fascicle, nearest the cyst wall
(pl. 21, fig. 19), or by the formation of the pore and bud at the end
of the fascicle (pl. 22, fig. 23), and by the chromophile nature of the
ridge (pl. 21, figs. 17-21), of the material in the pore (pl. 22, fig. 27),
and of the young bud (pl. 22, figs. 23, 25, 27).

The cysts of Councilmania are quite variable in contour and are
not predominantly spherical as in Endamoeba coli. In one hundred
eysts in a fresh stool examined in iodine-eosin stain, twenty-four were
spherical, forty spheroidal, twenty-two elongated or ellipsoidal, and
twelve asymmetrical.

The cyst wall (pl. 20, figs. 12-16) is 0.8 to 1.5 in thickness and
when fully formed is laminated and triple contoured. It consists in
optical section of a thin, outermost, sharp line, a middle, wide, clear
zone, and an innermost, dark brownish, granular zone nearest to the
cytoplasm. The clear zone is not always evident in the presumably
younger cysts. The cyst wall is generally thicker than in Hndamocba
colt. -

The structure of the nuclei in the cysts is extraordinarily variable
as a result of the prevalence of premitotic and postmitotic phases and
of the fact that the karyosome is relatively large, sometimes asymmet-
rical and centrally located or shghtly or even decidedly excentric, and
somewhat subdivided. It often takes on a crescentie or reniform con-
tour and is rarely seen in the cysts in the form of a single central, or
excentric, spheroidal granule. In fresh stools, especially in those some
of whose cysts are budding, the karyosome may have the form of a
stout, reniform, or semicircular spireme with large chromomeres (pl.
21, figs. 17-22), or the chromomeres may be scattered throughout the
nucleus (pl. 21, fig. 18).

The nuclear membrane is very thin but distinct in iron haema-
toxylin and is not heavily inerusted with peripheral chromatin in

180 University of California Publications in Zoology — [Vou. 20

nuclei in the eysts. There may be no trace of peripheral chromatin
or only a layer of fine granules on its inner surface with an occasional
larger granule or two in this region, but not so heavy an incrustation
as in #. coli and E. dysenteriae. The intermediate zone is filled with
a substance which is often much less deeply stained than the surround-
ing cytoplasm, but in other eases may be slightly stained in iron haema-
toxylin as an indistinct reticulum. We find in the nuclei of cysts no
trace of inner clear halo and outer granular network in the inter-
mediate zone such as Dobell (1920) figures for Endamoeba coli in some
nuclei of the eneysted stage.

The lightly incrusted nuclear membrane and the wide, lightly
stained, intermediate zone make the nuclei of the cysts with eight
nuclei stand out in the darker cytoplasm with a distinctness not equaled
in other amoebie cysts of human stools. This is noticeable in prepar-
ations and is evident in some of the figures of Cowncilmania which
appear in the older literature among the figures of Endamoeba coli,
such, for example, as those of Prowazek (1911, pl. 17, fig. 18). This
feature, the predominant asymmetry or lack of sphericity of the eysts,
and the peculiar karyosome make possible the eritical distinetion be-
tween the cysts of Councilmania and those of Endamoeba colt.

There is no critical, eytclogical evidence from Cowncilmania which
lends the least support to the hypothesis of autogamy in amoebic cysts
proposed and defended by the Schaudinn-Hartmann school of proto-
zoologists.

,

DIMENSIONS

The average size of the free amoebae when rounded up was 28
(20-35) microns in the case of ten measurements of amoebae from one
stool. Smaller amoebae are sometimes seen which probably belong
to this species. The largest free amoeba we have seen in stained prep-
arations measured 35 by 63 microns. The amoebulae on escape from
the cyst have a diameter of 7 to 8 microns. The eysts range from 8 to
34 microns in diameter. In one fresh stool in which 150 of the more
spherical cysts were measured the range in longest diameter was from
11 to 34 microns, with the mode at 16 microns, the average at 16.5, and
114 eysts included between 16 and 20 microns. The curve of distri-
bution of. these measurements showed a marked skew towards the
smaller diameters, and probably represents a small race of the species.

1921] Kofoid-Swezy: Councilmania lafleuri 181

BUDDING AND ESCAPE OF AMOEBULAE
Plates 20-22, figures 15 to 28

The genus Councilmana appears to differ from all other intestinal
amoebae of man in the occurrence in fresh stools, and therefore pre-
sumably also in the lower level, of a reproductive process of repeated
budding resulting in the escape of amoebulae from the eyst. Such a
process has not been observed by us in the eneysted stages of Hnda-
moeba dysenteriae, FE. coli, or in Endolimax nana. We find no record
of such a process in the eysts of any parasitic intestinal amoeba other
than Councilmama, except possibly in Endamoeba muris as deseribed
by Wenyon (1907), but otherwise interpreted by him.

We have designated the process as one of budding since it may be
preceded by the formation of a ridge or intracystiec process which
results in a protrusion of protoplasm through a small opening in the
cyst wall, in the escape of a nucleus into this lobe, and in the subse-
quent detachment of an amoebula, to be followed by a repetition of the
process until the cyst is emptied of all its nuclei.

The evidence upon which this conclusion rests is given in some
detail since Dobell (1920) rejects as ‘‘an incorrect and arbitrary series
of stages’’ the account of Mathis and Mercier (1917), who described
the emergence of amoebulae from the eysts of an amoeba which they
ealled E. coli, but which is undoubtedly Cowncilmania. Dobell is cor-
rect, it seems to us, in denying the existence of these phenomena in
E. coli, but not in discrediting the observations of these investigators.
Their observations were correct as far as they went, but they apply to
Councilmana rather than EF. coli. We agree with Dobell in not ae-
cepting their interpretation of a gamogenic-schizogamie cycle in EL. coli,
and find no evidence for such a double eyele in Cowncilmania.

The intracystie ridge which precedes and attends the early phases
of the formation of the bud first appears as a chromophile, deeply
staining tract which becomes an elevated ridge or keel of varying
width which may run halfway round the cyst, or even farther, within
the wall. It may be a narrow dark thread (pl. 21, fig. 19), or a blunt
process (pl. 21, fig. 17), or a broader ridge (fig. 21). It is generally
direct in its course, but is sometimes bifureated at one end (pl. 21,
fig. 19). In optical section it forms a distinct, elevated ridge on the
contour of the cytoplasm within the cyst wall.

182 University of California Publications in Zoology — [ Vou. 20

In a number of instances this chromophile territory seems to be
definitely related to the chromatoidal mass and to proceed from one
end of this structure (pl. 22, fig. 23). It appears to derive its chromo-
phile material, which may be diffused throughout its extent, from the
deeply stained substance of the chromatoidal body. Not all eysts have
chromatoidal bodies and not all ridges are deeply chromophile, but they
are generally more deeply stained than the cytoplasm.

The point of emergence of the eytoplasmie bud is a minute circular
pore in the wall two or three microns in diameter. We are able to
find this opening only in those cysts in which the bud is present. The
pore is sometimes near the middle of the ridge, but is more often at
one end. We find no evidence of a fixed relation of the pore to a par-
ticular part of the ridge. The diameter of the pore is less than that
of the nucleus, which must therefore be elongated in passing through
it. The eytoplasm in the pore is sometimes more deeply chromophile,
as though condensed by the constriction (pl. 22, fig. 27).

The sequence of events in the budding process is as follows: The
protoplasm protrudes through the pore (pl. 22, fig. 25), the nucleus
slips out into the protoplasmic bud (pl. 21, fig. 22), the.bud detaches
itself as an amoebula (pl. 22, fig. 23), and a new bud forms and another
nucleus creeps out. The process is repeated until the eyst is emptied
of all its nuclei and as many amoebulae have escaped as there were
nuclei in the cyst. We have found a few instances of residual proto-
plasm without nuclei left in the eyst, and no evidence of plasmotomy
of amoebulae within the eyst.

The evidence for this budding process is the fact that the proto-
plasmie bud has been found without a nucleus, that nuclei have been
seen at and near the pore, that more than one nucleus has never been
seen in the bud, that the protoplasmic bud is always relatively small
as compared with the original parent mass which grows smaller as the
nuclei therein become fewer, that a small detached amoebula with a
typical nucleus has been seen near the pore, and that in one instance
a cyst with a bud in a fresh smear after an interval of an hour was
found to have lost the bud. We have not as yet been able to watch
the detachment of active amoebulae in the warm stage. Cytolysis of
the bud and contents of the eyst was observed in one instance.

The number of amoebulae produced is eight as a rule, since there
are normally eight nuclei in the matured cyst. There is, however,
evidence that the number of nuclei and amoebulae may exceed this,
though we have never seen cysts with sixteen nuclei. Cysts with a

1921] Kofoid-Swezy: Councilmania lafleuri 183

bud and nine nuclei, some of them in the premitotie phase, are fre-
quently seen. This condition suggests the occurrence at times of a
fourth division resulting ultimately in sixteen eystic nuclei whose
escape into the bud proceeds during mitosis, which is not synchronous
in all of the eight nuclei. One cyst without a bud but having eleven
nuclei has been found (pl. 20, fig. 16). In the course of our exami-
nation of preparations we have found cysts with buds and seven, six,
four, three, two, and one nucleus respectively within the eyst. Con-
tinued search may be expected to reveal those with a bud and five
nuclei.

The escape of the amoebulae from the cysts within the bowel is
indicated by the fact that budding eysts occur in fresh, formed stools,
and in liquid stools after a saline or other purge. We find no evidence
that the process of budding arises or continues long in cold stools which
have been kept under observation for three weeks after deposition.

Bud formation is a normal process in Councilmania. It appears
in fresh warm stools, both with and without antecedent purge, and in
patients not under drug medication. The protoplasmic ridge is not a
fold of the cyst wall, but rather a protoplasmic structure. It appears
in cysts in untreated smears as well as in stained slides. It can not
be induced by sudden or by slow heat, by cooling, or by the action of
reagents used in the preparation of slides. It is not due to trauma
and can not be produced by pressure. The cysts in which it appears
show no indications in nuclei or cytoplasm of abnormality.

OCCURRENCE

Councilmania lafleuri evidently has a cosmopolitan distribution
similar to that of the other human intestinal protozoa. The eleven
cases which we have observed in Berkeley afford no clue to the precise
source of their infections. They were not contacts, and therefore pre-
sumably acquired their infections independently of one another. All
but one, himself a physician, were patients under physician’s care for
intestinal trouble or subnormal physical conditions. Five were uni-
versity students, two were from the Health Center Clinic. Six were
males, one a boy, and five were females. Ten were whites and one a
negress. Three were natives of California and eight were born else-
where, of whom four had come recently to the state. One patient spent
seven years in Sicily, one had lived six years in Persia, and two had

184 University of California Publications in Zoology [| Vou. 20

lived for some (one and ten) years in the Philippines; one was a re-
turned soldier from France who had received since his return two
treatments with emetine bismuth iodide and had been cured of an
infection by Endamoeba dysenteriae. One patient had lived in China
_and in Panama, and another was born and had lived for many years
in Alabama. These data present possibilities of widespread sources
of infection. In addition to these cases detected in Berkeley, we have
preparations of stools from several overseas soldiers in the Debarkation
Hospitals in New York in 1919 containing cysts of Councilmania.
The cosmopolitan distribution of this infection is definitely proved
by the occurrence in literature of figures of cysts of Cowncilmania
attributed to Hndamoeba coli from widely separated parts of the world.
Thus Walker and Sellards (1913, pl. 1, fig. 4) figure what we regard
as a eyst of Councilmania from Manila, P. I. Prowazek (1911, pl. 17,
fig. 18) figures among his #. williamsi (which is E. coli) a eyst from
Sawai, in Samoa, which appears to be that of Councilmania. It is —
probable that some of the figures of Mathis (1913, pl. 2, figs. 2, 6, 9-14,
and 16) from French Indo-China are referable here. Casagrandi and
Barbagallo (1897, pl. 2, fig. 21) figure from Italy an asymmé¢trical
eyst with bud which may be that of Cowncilmania, while Ciauri (1917,
pl. 1, figs. 11-16) describes and figures an amoeba and its cysts which
is undoubtedly Councilmama from a patient who had been with the
Italian forces in Libya and Tripoli. Mathis and Mercier (1917) de-
seribe the process of budding and recognize two types of cysts in
Endamoeba coli, but their diagrammatic figures, as drawn, appear
more lke #. coli than Councilmama, with the exception of figure 15.
Dobell (1920) notes the resemblance of this figure to that of Mathis
(1913). The sources of the amoebae discussed by these investigators
are not stated. It seems probable also that James (1914, pl. 17, figs.
155-156) may have found Councilmania at Panama. From his ac-
count of the activities and the finding of imgested red blood cor-
puscles in H. coli it seems probable that Schiff (1919) has observed
this species in troops in the German hospital at Haidar Pascha, in
Turkey. This list is not exhaustive and there are other less certain
suggestions of its occurrence in the literature. The data cited will
suffice to indicate a wide distribution of this parasite of man compar-
able with though not as yet so extensive and eritically founded as that
of the other amoebae of man.

1921] Kofoid-Swezy: Councilmania lafleurt 185

CULTURE AND INOCULATION

Attempts to eulture Councilmania in diluted eat’s blood and
diluted human pleuritie exsudate (1 to 10) in Locke’s solution have
failed, as did also attempts at culture in blood clot, in blood agar
(made with the blood of the eat and of the guinea pig), in plain agar,
and in nutrient broth.

Attempts to culture with solid tissue under aerobie and anaerobic
conditions likewise failed. We are indebted to Misses Steding and
Burgis for many of these tests.

Inoculations by feeding nine young rats were negative. Cysts were
discharged in the rat’s faeces for not more than the first forty-eight
hours, but the faeces were negative thereafter for from 33 to 77 days,
and no Councilmania were found in six of them at autopsy. Rectal
injection into four young rats has thus far given negative results. We
are indebted to Mr. J. F. Kessel for these experiments.

VIABILITY OF CYSTS

In our student laboratory Mr. L. Freudenthal made a test of the
viability of the eysts of Cowncilmania, using eosin staining as a test.
On the 17th day 85 per cent of 233 eysts were alive, 5.7 per cent dead,
and 5.3 per cent moribund. On the 31st day 85 per cent were still
alive and 15 per cent dead.

Another heavily infected stool was kept at room temperature in a
glass jar for forty days. The faecal matter was smeared on the sides
of the container and was kept moist but not flooded. In this ease there
was great variation at times in the proportion of live and dead eysts
observed on any given day, indicating that the cysts are not equally
viable in all parts of the stool. On the fortieth day cysts were still
alive in this stool with no convineing evidence that they were dying
out. On the sixty-fifth day about 80 per cent of the cysts were still
alive.

Cysts in human urine appear to die more quickly than in water.
In five different stools the cysts were all dead in five hours in urine,
but not in controls, as tested by Mr. L. Freudenthal in the student
laboratory. In another instance most of them were still living at the
end of forty-eight hours. The variables are so great in these tests that
conclusions should be based only upon repeated experiments.

186 University of California Publications in Zoology

[ Von. 20

DISTINCTIONS BETWEEN COUNCILMANIA LAFLEURI AND
ENDAMOEBA COLI

Since these two species both have eight-nucleated cysts the number
of nuclei can not be utilized to separate the two infections as in the
ease of the separation of H. dysenteriae from the two species under
discussion. There is, however, a group of characteristics which may
serve in the eritical diagnoses of infections by Cowncilmania. The
most reliable one, the distribution of the chromatin in the nucleus, is
fortunately most evident in the commonest stages, the eight-nucleate
eyst. The available diagnostic characteristics are set forth below.

Councilmania lafleuri

Endamoeba coli

FREE STAGE

Very active, pseudopodia thrust out
suddenly, ectoplasm sharply separ-
ated from endoplasm.

Red blood corpuscles ingested readily.

Peripheral chromatin in a thin layer,
karyosome large, excentric, with
halo, or often seen in premitotie
condition with chromatin dispersed
in granules in a sphere, ring, or
skein, without halo and often central.

Sluggish, ectoplasm not sharply sep-
arated from endoplasm.

Red blood corpuscles not ingested
normally.

Peripheral chromatin in a thicker
layer, karyosome small, spherical,
with halo, generally excentric.

ENCYSTED STAGE

Cyst wall very thick.

Spheroidal, ellipsoidal or asymmetri-
cal, less often spherical.

Less readily stained.

Glycogen body more resistant to iodine.

Nuclei with little peripheral chroma-
tin and large, generally central or
but slightly excentric, dispersed
karyosome.

Chromatoidal bodies less acicular in
early stages, fascieulate, massed
centrally in later stages and con-
tributing to chromophile buds.

Chromophile ridge forms a bud through
a pore in the cyst wall, which de-
taches uninucleate amoebulae.

Cyst wall thin.

Generally spherical.

More readily stained.

Glyeogen body stains readily in iodine.

Nuclei with more peripheral chroma-
tin and small, excentric, massed
karyosome.

Chromatoidal bodies more distinctly
acicular, with less central massing
and no relation to segregation of
chromophile cytoplasm.

Budding unknown.

1921] Kofoid-Swezy: Councilmania lafleurt 187

SUMMARY
Councilmania lafleuri, gen. nov., sp. NOv.

Free stage with hyaline pseudopodia, abruptly formed, endoplasm
filled with food vacuoles, and even ingested red blood corpuscles, rest-
ing nucleus with moderately thin zone of peripheral chromatin, karyo-
some generally excentric, often with small halo, in premitotie stages
composed of dispersed particles and more nearly central in location.
Pseudopodia broad, rounded, usually less than diameter of the body
in width, generally single. Cysts with one, two, four, or eight nuclei,
rarely more, thick walled, triple contoured, spheroidal, ellipsoidal or
asymmetrical, less frequently spherical. Nuclei of cysts with little
peripheral chromatin and large, excentric or central, spheroidal, reni-
form, or lobed karyosome often divided into seattered particles, eight
chromosomes, and an intradesmose joining polar masses seen best in
first and second mitoses. The cysts form a chromophile ridge from
which cytoplasm emerges through a pore in the eyst wall as a chromo-
phile bud. A nucleus migrates into the bud which detaches as an
amoebula. The process is repeated till the eyst is emptied of nuclei.
Budding may occur in the bowel. Free stages from 63 by 35 microns
to 20 to 85 microns when rounded up; spheroidal cysts 16 to 20, rarely
8 to 34, microns in diameter. Parasitic in the human intestine. Type
species Councilmania lafleuri.

188 University of California Publications in Zoology — [Vou. 20

LITERATURE CITED

CASAGRANDI, O., and BARBAGALLO, P.
1894. Pntamoeba hominis s. Amoeba coli (Lésch). Studio biologico e clinico.
Ann. Igiene Sperim., n. s., 7, 103-166, pl. 2.
CIAURI, R.
1917, Dissenteria amebica. Giorn. Med. Milit., 65, 934-941, pl. 1.
CouncILMAN, W. T., and LAFLEuR, H. A.
1891. Report in pathology. I. Amoebie dysentery. Johns Hopkins Hosp.
Rep., 2, 395-548, pls. 1-7.
DOoBELL, C.
1919. The amoebae living in man (London, John Bale, Sons and Danielsson).
Pp. vi + 155, 5 pls.
JAMES, W. M.
1914. <A study of the Entamoebae of man in the Panama Canal Zone. Ann,
Trop. Med. and Paras., 8, 133-310, pls. 12-17.

Kororp, C. A., KorNHAUSER, 8S. L, and Swexzy, O.
1919. Structure and systematic relationships of the ‘‘Iodine Cysts’’ from
human faeces. Military Surgeon, 45, 30-43, 25 figs. in text.

Koro, C. A., and Swezy, O.
1921. Councilmania laflewri, a new amoeba of the human intestine. Proc.
Soc. Exp. Biol. and Med., 18 (in press).
Matuis, C.
1913. Recherche des cystes d’amibes dans les selles de 1’homme. Bull. Soe.
Med-Chir. de 1’Indo-Chine, 4, 334-350, pls. 1-4.

Marais, C., and Mercier, L.
1917. La schizogonie chez les entamibes intestinales de 1’homme. Bull. Soc.
Path. Exot., 10, 311-322, 15 figs. in text.

PROWAZEK, S.
1911. Beitrag zur Entamoeba-Frage. Arch: f. Prot., 22, 345-350, pl. 17, 1
fig. in text.
Scuirr, F.
1919. Zur mikroskopischen Diagnose der Amoébenruhr. Arch. Schiffs-Tro-
penhyg., 23, 113-127.
WaLKer, E. L., anu SELLARDS, A. W.
1913. Experimental entamoebic dysentery. Philippine Jour. Sci., Ser. B, 8,
253-331, pl. 1, 3 tables.
WERNER, H.
1911. ‘*Hntamoeba coli,’’ in Prowazek, Handbuch der pathogenen Protozoen,
1, 67-77, pl. 2, 1 fig. im text.

=

EXPLANATION OF PLATES

All figures are of Councilmania lafleuri gen. nov., sp. nov., of drawings from
preparations fixed in hot Schaudinn’s fluid and stained in iron haematoxylin.
The magnification in all cases is 2500 diameters.

PLATE 18
Free stages of Councilmania lafleuri

Fig. 1. Small free amoeba with single, broad, hyaline pseudopodium. Sharp
demarcation between ectoplasm and endoplasm, very. few vacuoles, and very
large nucleus with central karyosome with halo, heavily decolorized. Peripheral
chromatin lobed.

Fig. 2. Free amoeba with clear pseudopodium in early phase of protrusion
and one in last phases of retraction filled with endoplasm at the opposite end of
the body. Small nucleus with fairly heavy peripheral chromatin and large,
ragged, slightly excentric karyosome with narrow halo.

Fig. 8. Free amoeba with small pseudopodium, chromophile margin at boun-
dary of ectoplasm and endoplasm, some food vacuoles with contents, nucleus
with continuous thin zone of peripheral chromatin; excentrie karyosome without
halo.

Fig. 4. Large free amoeba with two pseudopodia (below) in the last stages
of retraction, a broad one (above) in full extension, and one recently formed
(at left); endoplasm vacuolated, ectoplasm more homogeneous; nucleus with
irregularly lobed peripheral chromatin, spheroidal, excentric karyosome with
narrow halo.

[190]

UNIV. CALIF. PUBL. ZOOL. VOL, 20 [KOFOID AND SWEZY] PLATE 18

.
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PLATE 19
Free and precystic phases of Councilmania laflewri

Fig. 5. Free amoeba with food vacuole containing remnant of red blood
corpuscles, nucleus with a thin zone of peripheral chromatin and central, sphe-
roidal karyosome with halo. Three pseudopodia in retraction, one at the right
forming.

Fig. 6. Preeystic stage with some food contents, including a cyst of Chilo-
mastix davainei, in vacuoles. Halo about the nucleus, heavier zone of peripheral
chromatin, very excentric karyosome with halo, intermediate zone with devel-
oping chromatin net, no ectoplasm differentiated.

Fig. 7. Precystic stage with no food contents, enlarged nucleus in premi-
totic phase with no peripheral chromatin zone, but with vague, destained merid-
ional strands, karyosome slightly excentric, dispersed in a ring of granules, no
well defined halo.

Fig. 8. Binucleate free amoeba with no vacuoles, and single, large pseudo-
podium. Nuclei with thin zone of peripheral chromatin and central, dispersed
karyosome suggesting recent or coming mitosis.

Fig. 9. Rounded-up free amoeba crowded with food vacuoles containing red
blood corpuscles, and bacteria, nucleus with thin zone of peripheral chromatin,
slightly excentric karyosome with excentric halo.

[192]

UNIV. CALIF, PUBL. ZOOL. VOL, 20 [KOFOID AND SWEZY] PLATE 19

re)
4

Pees

PLATE 20
Encystment in Councilmania lafleuri

Fig. 10. Rounded-up free amoeba with ectoplasmic zone sharply set off from
the vacuolated endoplasm. Nucleus with nearly uniform zone of peripheral
chromatin, subcentral, spherical karyosome with narrow excentric halo.

Fig. 11. Very small uninucleate cyst with large central glycogen vacuole
crowding the nucleus to the wall. Nucleus with neither peripheral zone nor
karyosomes, its chromatin scattered in peripheral nodes with connecting strands,
some of them double, a few vague chromatoidal masses in the cytoplasm, cyst
wall present.

Fig. 12. Binucleate cyst with central glycogen vacuole, two nuclei in the
peripheral cytoplasm nearing the prophase of mitosis, scattered flakes of chro-
matoidal substance in the cytoplasm, thick cyst wall present.

Fig. 13. Binucleate cyst with nuclei in early anaphase, each with sixteen
chromosomes in the equatorial region and deeply stained polar masses (centro-
somes) with traces of an intradesmose on the nuclear wall connecting them,
small chromatoidal bodies in the cytoplasm.

Fig. 14. Quadrinucleate cyst, with cytoplasm encroaching upon the central
glycogen vacuole, nuclei showing two conjoined polar masses (centrosomes)
connected by strands with about eight chromatin masses on the nuclear mem-
brane, no chromatoidal bodies, cytoplasm chromophile.

Fig. 15. Eight-nucleate, thick-walled cyst with a narrow chromophile ridge
almost encircling the body, scattered chromatoidal threads traversing the eyto-
plasm, and a chromophile mass near the middle of the ridge. Nuclei with no
peripheral chromatin, clear intermediate zone and central karyosome of dis-
persed granules.

Fig. 16. Eleven-nucleate cyst with central chromatoidal fascicle viewed from
the end, several chromatoidal needles. Nuclei generally with subecentral dis-
persed karyosomes, and peripheral chromatin masses in some of the nuclei.

[194]

[KOFOID AND SWEZY] PLATE 20

WNIV. GALIF, PUBL. ZOOL. VOL. 20

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PLATE 21
Bud formation in Councilmania lafleuri

Fig. 17. Hight-nucleate cyst’ with fasciculate chromatoidal body with one
end in a bud in the early stages of formation. Nuclei with dispersed karyo-
somes, and some peripheral chromatin.

Fig. 18. The same stage, with chromatoidal body exhausted in formation
of chromophile ridge. Nuclei with scattered chromomeres.

Fig. 19. EHight-nucleate cyst with fasculate chromatoidal body from one
end of which three narrow chromophile ridges radiate, one of which is emerging
at the pore in the cyst wall at its free end. Nuclei with central karyosomes
of dispersed type and little, if any, peripheral chromatin.

Fig. 20. Hight-nuecleate cyst with narrow, chromophile ridge, and a bud
with deeply chromophile cytoplasm emerging on top of the cyst through the
pore. No nucleus in the bud. Nuclei in prophase for next mitosis. No chro-
matoidal body except small spherical remnant.

Fig. 21. Eight-nucleate cyst with heavy, wide, chromophile ridge, adjacent
to a diffusely chromophile area of the cytoplasm, no chromatoidal body. Nuclei
with central karyosomes of spireme type and no peripheral chromatin. Bud not
yet formed. ‘

Fig. 22. Hight-nucleate cyst with deeply chromophile bud containing one
nucleus, nuclei of spireme type, no peripheral chromatin, small remnant of
chromatoidal body left in cytoplasm.

[196]

[KOFOID AND SWEZY] PLATE 21

UNIV. CALIF. PUBL, ZOOL. VOL. 20

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ee Git

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PLATE 22
Budding in Councilmania lafleuri

Fig. 23. Budding eyst with central, fasciculate chromatoidal body, from one
end of which a chromophile area passes out into the densely chromophile cyto-
plasm in the pore to the chromophile bud, or amoebula which contains a single
nucleus, seven nuclei remaining in the cyst.

Fig. 24. Budding eyst in fresh smear, showing protruding cytoplasm and
three nuclei remaining in the cyst. Reduction of the cytoplasm is indicated
by the outline of the lobe partially filling the cyst.

Fig. 25. Cyst with small, protruding, chromophile bud, no chromatoidal
body, except a nucleus-like remnant (?), three enlarged nuclei in the spireme
stage remaining in the cyst.

Fig. 26. Binucleate cyst with chromophile ridge and remnant of chroma-
toidal body, nuclei enlarged. Pore not detected.

Fig. 27. Budding cyst with remnant of disintegrating chromatoidal body
from which a faint chromophile area leads to the pore filled with deeply chro-
mophile strand connecting with a uninucleate, deeply stained bud or amoebula.
Six nuclei with peripheral chromatin in large granules remain in the cyst.

Fig. 28. Elongated cyst with no chromatoidal body but deeply chromophile
area massed as though for emergence as a bud, four nuclei remaining in the
cytoplasm.

[198]

to

UNIV, CALIF. PUBL, ZOOL. VOL. 20 [KOFOID AND SWEZY] PLATE 2

"UNIVERSITY OF CALIFORNIA PUBLICATIONS— (Continued)

ae ‘A Study of the Races of the White-Fronted Goose (Anser albifrons) Occur-
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-2 figures in text, plate 13. October, L9V7 ann..s niece ec eccesceeeesncendene 15
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STU -Occbes. oh NG, LOLS oS oe eS ORS aa aa ee ee oe een cee 20
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.35
_ 2, An Unustial Extension of the Distribution of the Shipworm in San Francisco
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4, New Species of Amphinomidae from the Pacific Coast, by Christine Essen-
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- convivus Stal, by Irene McCulloch. Pp. 75-88, 35 figures in text. Decem-
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-* 6. On the Orientation of Erythropsis, by Charles Atwood Kofoid and Olive
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7. The Transmission of Nervous Impulses in Relation to Locomotion. in. the
Earthworm, by J: ony F. Bovard, Pp. 103-134, 14 figures in text. . January,
. SDSS SS pi Suet capt oe pa A a1 SE Md Sen See | yA ws MESS 35:
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. $5044," fipure:in’ text: | dandary, LOLS 2 oe Ee ee ee 10
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& Faeces, by William C. Boeck, . Pp. 145-149. December, 19147 2220200 Sao AO
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: 12 figures in text. March, 1918 .25
Pe 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,
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16. The Subclavian Vein and Its Relations in Elasmobranch Fishes, by J, Frank
; Daniel. Pp. 479-484, 2: figures in text. August, 1918 222i -10
17. The Cercaria of the Japanese Blood Fluke, Schistosoma japonicum Katsu-
-. rada, by William W. Cort, Pp. 485-507, 3 figures in text.
"48. Notes on the Eggs and Miracidia of the Human Schistosomes, by William
~ -W. Cort... Pp. 509-519, 7 figures in text. j
Nos.217 and 18 in one cover. January, 1919 22.2... oot eee 35

Index; pp. 521-529,

UNIVERSITY OF CALIFORNIA PUBLICATIONS (Continued)

Vol. 19, 1. Reaction of Various Plankton Animals with Reference to Their Di
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85- 88, 2 figutes.4 in text. May, 1919 i ec Te SE Sea

figures in text. “April, POND 2 SAREE go a a ee aS pace:
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10, A New Morphological Interpretation of the Structure of Noctiluca, : and =

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Kofoid. Pp. 317-334, plate 18, 2 figures in text. February, 1920 ..—

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Pp. 338-388, plates 19-25, .1 figure in text.. May, 1920 ..

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13..Demonstration of the Function of the Neuromotor Apparatus in Ey

“by the Method of Microdissection, by Charles V. Taylor.

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Index in preparation. é. :

‘Vol. 20. 1. Studies on the Parasites of the Termites. I. On Streblomastia stria,.

x Kofoid and Olive Swezy. Pp. 1-20, plates 1-2, 1 figure in text. Jule
2, Studies on the Parasites of the Termites. II. On Trichomitus termitid

by Charles Atwood Kofoid and Olive vere
firuresin ‘text. Juby,- 39196 os a he eg aoe me

nov., Sp. NOv., by Charles Atwood Kofoid oe One Swez)

5, On the Morphology and Mitosis of Chilomastix mesnilt (Wenyon), a ‘Commas
Flagellate of the Human Intestine, by Charles A. Kofoid and Olive
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Gercomonas, Chilomastix, Trichomonas, and Giardia, by Charles A.
Pp. 145-168, 9 figures in text. Jume, 1920 — 2.2... c 1A ee :
7. On the Free, Encysted, and Budding Stages of Councilmania lafleurt, a Para-~
sitic Amoeba of the Human Intestine, by Charles Atwood Kofoid
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River and Its Tributaries in and near Stockton, California, in 191)

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1-470, plates 1-50, 165 figures in text. yoo 1921. Be ie

= - Z00L0GY_ Sar ,
tes 23. 26, as Aguresin text» “March 7, 1922 ce A eee we

BOs! AND FISSION IN THE ACTIVE AND

Pos

> ie Zee liste ee : ~ ie Ee;
eu a a3 : eS oe oe

te ‘see

ions hore ot

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UNIVERSITY OF CALIFORNIA PUBLICATIONS
IN

ZOOLOGY
Vol. 20, No. 8, pp. 199-234, plates 23-26, 11 figures in text March 7, 1922

MITOSIS AND FISSION IN THE ACTIVE AND
ENCYSTED PHASES OF GIARDIA ENTERICA
(GRASSI) OF MAN, WITH A DISCUSSION OF
THE METHOD OF ORIGIN OF BILATERAL
SYMMETRY IN THE ~ POLYMASTIGOTE

FLAGELLATES

BY
CHARLES A. KOFOID anp OLIVE SWEZY

CONTENTS

PAGE

Introduction .................

Material and methods
"CTR ES yg oe ee 202
5 EYGNS) EIQ CS HE TAG ae Ee OP ee 202
The neuromotor system and its relation to the symmetry of the organism 205
WN ERAS RES AY TE EDN OES Se a PE 212
DADS) TELM UE AAS SRST Se ee rE em 215
The origin of bilateral symmetry in the polymastigote flagellates 215
pINHEmen CYSTC Ce pase were tee oe ree | Se Erased cctecedeboceeat decussate 217
Meditferentiationhin encystment 2-22. 0.2 c02s---ck--eosede-cceeeeac-n seececeeesceeeeee eee 22]
PETE GLO Ta O eT CVS VEL ONG ees see on 2 ane Ze cen sce aseazcpaneseeecnecep anes 222
Specific differences between Giardia enterica and Giardia from rodents ............ 222
SUTRITETIIAT cate ped ee el ee pen 224
Mniterature cited: 2.<.-< 22 -c2.--2-c-cesc2cas- SSS So ce 225
BESS Lea TT RT OTTO La [DLA LUGS See eee Ea san cee SF onn cs evceecsentecsaee= 228

INTRODUCTION

Giardia enterica is a flagellate parasitic in the human intestine, and
of general distribution throughout the world. It is widely prevalent
in the tropics, occurs rather frequently in our southern states (Stiles,
1915), and is found more often in children than in adults. It often
attends cases of acute or chronic diarrhea, achylia, duodenal ulcer, and
cancer of the stomach, though clinical evidence that it is an etiological
factor in these diseases is inadequate or lacking.

200 University of California Publications in Zoology [ VoL. 20

Species of the genus Giardia are found in the tadpoles (G. agilis)
of Amphibia, in the blood(?) of the faleon (G@. sanguinis), and in
the intestine (G. muris, G. microti, G. duodenalis, G. enterica) of
mammals, especially of rodents, cats, and man. There are morpho-
logical grounds to support the view (Bensen, 1908; Kofoid, 1920) that
there are several species in the genus, and that the species in rodents
differ from that found in man. This matter is important in the pre-
vention of human infection, for if any of the species in mice and rats
should be identical with that in man, the sources of infection are
greatly increased because of possibilities of contamination of food
supphes in the granary, warehouse, mill, bakery, and store by the
infected faeces of these rodent pests. On the other hand, if the species
in man is distinet from those in rodents, the sources of human infection
are more circumscribed, and are limited mainly to the contamination
of food or water by water-borne and fly-borne cysts from human faeces,
by the dirty hand of the infected food handler, and by contaminated
washbowl or towel.

It is the purpose of this paper to define accurately the morphological
features of the Giardia parasitic in man, and thus to assist in the
clearer definition of the morphological distinctions which may be used
to separate this parasite from related species of Giardia in other
mammals, notably in rodents. The accurate description will also afford
a basis for the accurate microscopical diagnosis of the infection in the
examination of human stools.

We have elsewhere (Kofoid, 1920) set forth the grounds under the
Zoological Code of Nomenclature for using the name Giardia enterica
for the parasite widely designated in medical treatises as Lamblia
intestinalis. Earlier investigators (Grassi, 1881; Grassi and Schewia-
koff, 1888; Metzner, 1901, among others) regarded these flagellates in
man and in rabbits, rats, mice, and cats as but a single species desig-
nated as Megastoma entericum, and later commonly known as Lamblia
intestinalis. In consequence of this fact figures of the Giardia aseribed
to man have often been drawn from specimens taken from rodents and
have been widely copied in textbooks as figures of the human parasites.
Thus, even Fantham, Stephens, and Theobald, in their recent work
on the Animal Parasites of Man (1916), reproduce the widely copied
figures of Grassi and Schewiakoff (1888) as Lamblia intestinalis,
although the latter authors explicitly state that their material came
from rats and mice.

1922] Kofoid—Swezy: Mitosis and Fission in Giardia enterica 201
‘

An added reason for the publication of a critical analysis of the
morphological figures of the parasite found in man is the fact that
some recently published figures in textbooks and in works specially
designed for use in clinical diagnosis or in stool examination (Doflein,
1916; Cammidge, 1916; Hegner and Cort, 1921) publish or copy figures
of the Giardia of man which give an incorrect or at least an atypical
representation of the parabasal bodies, organs most useful in specific
diagnosis in the genus Giardia. Thus Deschiens (1921) in describing
the parabasals states that they. are ‘‘généralement fusionnés et trés
polymorphes,’’ whereas careful focusing with a good binocular micro-
scope with immersion monobjective will almost invariably resolve the

pair of parabasals accurately.

MATERIAL AND METHODS

Our material is all from human faeces and presumably belongs to
a single widely distributed species parasitic only in man. It has
included large and small races and infections in which ellipsoidal as
well as spheroidal, or even elongated cysts were present. In the
absence of other structural distinctions than those of size and propor-
tions of the cyst, we incline to the view that these relatively rare but
divergent forms are at the most but mere races within the variable
species. It is quite within the range of possibilities that the species
parasitic in the rodents may at times establish a foothold in man, but
experimental tests of their capacity to do this and of the degree of
host specificity prevalent in the genus Giardia are as yet lacking,
except in the case of the experimental transfer of the human Giardia
to kittens by Fantham and Porter (1916) and by Deschiens (1921).

Our material came in part from stool examinations made on 576
home service (37 cases, 6.4 per cent) and 2300 overseas soldiers (131
cases, 5.7 per cent) at the Army Laboratory, Port of Embarkation,
New York City, in 1918-1919 (see Kofoid, Kornhauser, and Plate,
1919, and Kofoid, 1919), and in part from over 9000 routine stool
examinations made in the Parasitological Laboratory of the California
State Board of Health of residents of California, including persons
from other states of the Union and from Mexico, Central America,
the Hawaiian and the Philippine Islands, China, Japan, Persia, India,
Siberia, and Armenia. It is thus presumably widely representative of
exposures to human infections, though we have no means of establish-
ing the actual geographical sources of the infections we have studied.

202 University of Califorma Publications in Zoology [Vou. 20

The material has been studied in fresh smears, in iodine-eosin
preparations, and mainly in iron haematoxylin stain following hot
Schaudinn’s fluid (with 4 per cent acetic acid) as a fixer of smears upon
slides, by both the slow and rapid methods of staining. Strong illumi-
nation (100 watt Mazda) and Wratten filters K1 and G and immersion
oil between condenser and slide have been used in determination of the
fibrillar system by the monobjective binocular microscope.

MORPHOLOGY

The fact that the investigation of Giardia enterica is, as a rule,
dependent upon stools rather than flagellates in place in the intestine
of the host, as in the case of Giardia in mice, puts limitations upon
the amount and quality of material of the free flagellates available
for investigation. The free flagellates are rare in the faeces except
occasionally in diarrheic stools, and the moribund condition of many
of them usually limits the material available for critical study of
mitosis. It is quite otherwise in the cysts of this parasite, which find
their normal conditions of hfe in the discharged faeces.

Tue AcTIVE FLAGELLATE

The shape of the body is remarkably uniform in properly fixed
material and is subject to but slight modifications as a result mainly
of the varying degrees of contraction of the cytostome (ef. figs. 4 and
5, pl. 23, and fig. 6, pl. 24), and of the tail which may be more or less
elevated (fig. D; pl. 23, figs. 3, 5) or laterally deflected (pl. 24. fig. 8).
The constriction of the cytostome elevates the dorsal surface, and’ the
tail may be elevated as much as forty-five to ninety degrees (Zabel,
1901) above the horizontal plane when the flagellate is in place on an
epithelial cell of its host. In the faecal smears the tail usually les
horizontally in the plane of the peristomal region.

The body is pyriform in outline in dorsal or ventral view (fig. B),
is flattened ventrally by the shallow, sucker-like cytostome, and is
contracted posteriorly into the tapering tail. In lateral view, it is
convex dorsally and flat ventrally, with the greatest elevation at the
level of the posterior margin of the cytostome (pl. 23, fig. 2). The
greatest transverse diameter of the body is at the level of the posterior
third of the eytostome and slightly exceeds (0.51—0.54) half the length.
The dorsoventral diameter is a little less than half the transdiameter
in stained and presumably contracted material, but appears to be

1922] Kofoid—Swezy: Mitosis and Fission in Giardia enterica 203

relatively greater than this in living individuals. The tail is conical
(25°-30°), concave laterally near the base in dorsal view, tapering to
a blunt point, and is about 0.6 to 0.7 transdiameter in length. Its
ventral surface is flattened and continuous with the flattened area
between the posterolateral flagella. In life it is very mobile. It ter-
minates in the posterior flagella proceeding from the axostyles.

trans. comm. —— --—.
See ants peristte = — -o

DULCMT LZ ao

— ant. lat. fl. —
post. perist. f.

_-post. lat. fl.

Fig. A. Lateral view of Giardia enterica (Grassi). Fig. B. Ventral view.
Fig. C. Cyst with 2-zooid individual. Abbreviations: ant. node, anterior
node; ant. perist. f., anterior peristomal fiber; ax., axostyle; bleph., blepharoplast ;
kary., karyosome; cent., centrosome; cyst., cyst wall; cyt., eytostome; intracyt.
lat, fl., intracytoplasmic lateral flagella; intracyt. post. lat. fl., intracytoplasmic
posterolateral flagella; imtranuc. rhiz., intranuclear rhizoplast; lat. 7l., lateral
flagella; nuc., nucleus; par., paradesmose; par. b., parabasal; par. b. rhiz., para-
basal rhizoplast; post. fl., posterior flagella; post. perist. f., posterior peristomal
fiber: rhiz., rhizoplast; trans. comm., transverse commissure; vent. jl., free ventral
flagella. X 2260.

The structures found in the body (figs. A and B) are two nuclei
(nuc.), the eytostome (cyt.), and the neuromotor apparatus consisting
of the two axostyles (ax.), the two parabasal bodies (par. b.), the
peristomal fiber (perist. f.) around the eytostome, the centrosome
(cent.), the rhizoplasts (rhiz.), blepharoplasts (bleph.) and the eight
flagella (fl.).

204 University of California Publications in Zoology [Vow. 20.

These structures traverse the cytoplasm, which is finely and uni-
formly granular and exhibits no evidence of cell inclusions or food
vacuoles and lacks alveolar differentiation. The peculiar anterior halo
around the blepharoplasts and the triangular halo between the postero-
lateral flagella seen by Kofoid and Christiansen (1915) in Giardia
muris are less sharply set off in Giardia enterica, in which the anterior
halo appears to be little more than the thinner anterior edge of the
body, and the triangular halo to show less contrast in density with the
adjacent cytoplasm than in @. muris. This ‘‘halo’’ or area takes on a
uniformly lighter tone as the plane of foeus reaches it because of the
flattening of the ventral surface between the posterolateral flagella
which bound it.

Fig. D. Giardia enterica from vomit in ease of carcinoma of the stomach.
After Zabel (1901, pl. 2, fig. 41).

Fig. E. Giardia enterica attached to cells of the duodenum, After Miiller
(1888, pl. 2, fig. 2).

There are no glycogen masses or granules in G. enterica, in either
the free or encysted stages. With Best’s carmine (Prowazek and
Werner, 1914) the cytoplasm of the cyst stains a diffuse red, indicat-
ing diffuse paraglycogen.

The cytostome (cyt.) is the concave area on the anteroventral sur-
face encircled by the peristomal fiber. It is a sucker-like organ (pl. 23,
figs. 2, 3) adapted for adhesion to the tops of the epithelial cells (figs.
D, E). Miiller (1890) found the flagellates on the cells of the jejunum
of an executed criminal at autopsy (fig. E) and Zabel (1901) found
them in place on the cells of epithelial fragments (fig. D) in the vomit
from a case of cancer of the stomach. The function of the cytostome
in attaching Giardia enterica to the cells of its host is thus proved by
observation to be similar to that of Giardia in rodents where exami-
nation of the relations of the parasite to the cells of the intestine of
the host is easily made.

1922] Kofoid—Swezy: Mitosis and Fission in Giardia enterica 205

The contractility of the cytostome is suggested by its variable out-
line in fixed material (cf. pl. 23, figs. 3, 4, 5), and by the varying ratios
of the anteroposterior and transverse diameters. The postmargin is
more deeply indented in the contracted state than in the uncontracted,
and the anteroposterior diameter is 0.80 of the transverse in the con-
tracted to 0.82 in uncontracted. In other words, the anteroposterior
grip of the cytostome is greater than the transverse.

There is no evidence of any other function of the cytostome than
that of an organ of adhesion. No gullet leads from it and no food
particles can be found in the cytoplasm. We have not succeeded in
cultivating Giardia in physiological salt solution, in culture media
such as peptone broth, or in human ascitic fluid or diluted blood, in
most of which the human intestinal flagellates (Chilomastix, Tricho-
monas) live for some time if frequently transferred to fresh media.

The fact that Giardia enterica has a powerful organ of attachment
in the cytostome affords an anatomical ground for the persistence with
which infection is maintained in the bowel of man when exposed to
the pressure of passing food and the peristaltic movements of its
substrate. In our experience it attains greater numbers than any other
human intestinal protozoan.

Tur NEUROMOTOR SYSTEM AND ITs RELATION TO THE SYMMETRY OF
THE ORGANISM

The newromotor system ot Giardia enterica is bilateral and consists
of the two centrosomes and the integrated fibrillar structures attached
thereto. These elements, attached to each centrosome or to the
blepharoplast, are as follows: an intranuclear rhizoplast (intranuc.
rluz., fig. B) passing to the central karysome (kary.), while peripherally
a rhizoplast (rhiz., fig. A) runs to the blepharoplast (bleph.) at the
anterior end of the axostyle (ax.), the parabasal body (par. b.), the
peristomal fiber (perist. f.), and four flagella (fl.). Giardia is bi-
nucleate and bilateral, but not diplozoic. The symmetries of the two
cells and their attendant neuromotor systems stand in a reversed rela-
tion, viewed from the standpoint of other unicellular, polymastigote
flagellates, notably Chilomastix. We find the neuromotor system
duplicated as to its constituent parts, but with those of the right side
arranged in the mirror image of those of the left. The constituent
elements of this system are duplicated throughout, except the com-
missures. Joining the two systems of the right and left sides is the
transverse commissure (trans. comm.) between the blepharoplasts,

206 University of California Publications in Zoology [Vou 20

while a second junction is effected between the intracytoplasmic parts
of the anterolateral flagella in the anterior node (ant. node).

The centrosome (cent., fig. A) is a minute granule at the anterior
pole of the nucleus and is found with difficulty, especially in the eysts.
Duplication of the neuromotor system proceeds from it through the
blepharoplast at mitosis, and the paradesmose (pl. 23, fig. 7; fig. A,
par.) is spun out between the daughter centrosomes prior to the meta-
phase and lies on the nuclear membrane as a dark meridional thread.

The intranuclear (intranuc. rhiz., fig. B) and extranuclear (rhiz.)
rlizoplasts are found with difficulty in both free and encysted stages
since they destain quickly. The intranuclear rhizoplast passes to the
central karyosome in the resting stage of the nucleus, and forms the
line along which the longitudinal spireme forms at mitosis (pl. 23,
fig. 5), as Boeck (1919) has shown in G@. microti. The extranuclear
rhizoplast passes obliquely antero-laterally to the head of the axostyle
or blepharoplast.

The blepharoplast (bleph., fig. B) in G. enterica is obscured more
than in G. muris and G. microti by the fact that it has nearly the same
diameter as the axostyle and, unless decolorization is carried on until
the latter organ destains, the small blepharoplast buried in its head
is not distinguishable from the contiguous axostyle. When destaining
is adequate (fig. B; pl. 23, fig. 4), and especially in the prophase, the
blepharoplast is seen as a small ellipsoidal body with its longer longi-
tudinal axis buried in the head of the axostyle. To it converge all of
the elements of the neuromotor system, though the parabasal rhizoplast
par. b. rhiz., fig. A), the ventral and posterolateral flagella, and the
posterior peristomal fiber are generally so obscured by the axostyle
that they cannot be followed separately to any junction with the
blepharoplast. Their trend is toward the blepharoplast as far as they
can be followed. From it passes the rhizoplast (rhiz.) to the centrosome.
We have been unable to find the two granules figured diagrammatically
by Hartmann (1910) in @. muris and by Rodenwaldt (1911) in [2]G.
enterica. It is possible that they were prophetic of the prophase of
mitosis (see our text-figures F—-K).

The transverse commissure (trans. comm., fig. B) is a heavy fibrous
band joining the two blepharoplasts,

The four pairs of flagella (fl., fig. A) all appear to arise from, or
near, the blepharoplasts, in a bilateral arrangement. They all stroke
posteriorly and are typically found in living and fixed material trailing
more or less posteriorly.

1922] Kofoid—Swezy: Mitosis and Fission in Giardia enterica 207

The anterolateral flagellum (ant. lat. fl., fig. B) passes anteriorly
in a laterally convex are to the anterior node (ant. node), which in an
earlier paper (Kofoid and Christiansen; 1915) was called the anterior
chiasma, a laterally extended granule. From this point, it is impos-
sible in the free stage to determine whether on leaving the node the
flagellum crosses over to the opposite side, or bends abruptly laterally
on its own side. Either course is possible. The latter alternative seems
more probable in case Giardia has been derived from Chilomastix by
the morphological reversal of one daughter at mitosis (see Kofoid and
Swezy, 1920). The origin of the node, however, in the prophase (figs.
F to K) with the outgrowth of new anterolaterals indicates unmis-
takably a contact rather than a cross-over of the flagella. We therefore
substitute ‘‘node’’ for ‘‘chiasma’’ to avoid the connotation of a crossing
and to establish in its place one of connection only.

From this node, the flagellum runs laterally dorsal to and within
the anterior peristomal fiber to near the middle of the lateral peristomal
are where it crosses that fiber, uniting with it in a slight angular
prominence in its course, and emerges posterolaterally as a free flagel-
lum. Its intracytoplasmic part stains more lightly than the adjacent,
peripheral, peristomal fiber, except just before mitosis, when it is
noticeably thickened and larger than the peristomal fiber. There is
no distinct basal granule at or near its point of emergence from the
cytoplasm in G. enterica. It is distinct from the peristomal fiber on
careful focusing, though often confused with it by earlier investigators.
The level of emergence of this flagellum appears to be characteristic
in G. enterica, as compared with G. muris and G. microti, in that it is
somewhat more anterior. In twenty-five individuals chosen at random,
it was distinctly anterior to the middle in eighteen (pl. 23, fig. 1),
median in five, and slightly posterior in two only. Obviously, in a
mobile organ such as the sucker, the state of contraction of the peri-
stome and the localization of the contraction modify the relative
positions of the emergence of this flagellum.

The posterolateral flagella (post. lat. fl., fig. B) emerge postero-
laterally at the level of the constriction of the tail. Their points of
emergence are about the same distance from those of the anterolaterals
as the latter are from the apex of the body. Their intracytoplasmic
parts (intracyt. post. lat. fl.) pursue an anteromesad course, appar-
ently uniting with the posterior peristomal fibers as they curve
anteriorly and merge with the axostyles on their way to the blepharo-
plasts. Presumably they join the blepharoplasts of their respective

208 University of California Publications in Zoology | Vou. 20

sides. We have been unable to detect them as separate fibers parallel
to the axostyles as in Hartmann’s (1910) diagrammatic figure of
G. muris. The intracytoplasmic sections destain with difficulty, and,
next to the peristome and axostyles, are the heaviest fibrillar parts of
the neuromotor system. There is no basal granule at or near the point
of emergence of these flagella. Their position indicates that they are
possibly the homologues of the flagellum of the undulating membrane
in trichomonad flagellates.

The free ventral flagella (vent. fl., fig. A) emerge close together at
the level of the posterior margin of the cystostome (pl. 23, figs. 2, 3)
and appear to merge with the axostyles at about their level. We are
not able to trace them forward as distinct fibers to their respective
blepharoplasts. In life, these flagella are extraordinarily active,
moving in unison, and in fixed material usually maintain their
parallelism.

The posterior flagella (post. fl., fig. A) are free extensions of the
axostyles emerging at the tip of the tail and are without basal granules.

The axrostyles (ax., fig. B) are two tapering, closely parallel rods
of deeply staining or grayish fibrillar substance passing posteriorly
from the two blepharoplasts. They and the adjacent parabasals con-
stitute the most striking elements of the neuromotor system in the free
stages, and especially in the cysts in the process of multiple fission.
The blepharoplasts are lodged in the anterior ends of the axostyles and
are not distinguishable from the axostyle except by adequate destain-
ing. The axostyles are continued distally as the free posterior flagella
and are thus morphologically the intracytoplasmie parts of such
flagella. The lively movements of the tail in whose ventral surface
they lie demonstrate the motor function of these axial organs.

The question as to the homology of this organ of the component
cells of Giardia to the axostyles of other polymastigote flagellates is
an interesting one. We regard it as homologous with the slender rod
in Chilomastix davainei which we (Kofoid and Swezy, 1920) called
the parastyle. In Chilomastix, this organ lies in a position with
regard to other organs, notably the cytostome and blepharoplast,
homologous to that of the axostyle in the right cell of Giardia, and in
the left cell to that which it would occupy in the mirror image of
Chilomastix. It does not, however, reach the posterior end of Chilo-
mastix nor form a free flagellum at its tip. It differs from the axostyle
of Trichomonas and related genera in the development of the free

1922] Kofoid—Swezy: Mitosis and Fission in Giardia enterica 209

flagellum at its tip and in being more condensed and more deeply
stainable, though not in contractility and its general morphological
relations.

It is to be expected, in so diverse a group as the polymastigote
flagellates, that there will be a wide range of form, development, and
possibly of modifications of function, in so fundamental an organ as
the axostyle, that these diversities will increase as our knowledge of
the group is extended, and that the evidence for the homology of the
diverse types will be strengthened as more types come to light.

The parabasal bodies (par. b., fig. A) are a pair of comma-shaped
structures lying in the eytoplasm on ‘the dorsal side, just posterior to
the middle of the body and to the posterior peristomal fiber.

Their position in the cytoplasm is in sharp contrast to that of all
other intracytoplasmic structures which trend more or less posteriorly.
The parabasals, on the other hand, generally le transversely or
obliquely to the major axis with their larger blunter ends near the
axostyles and the tapering pointed ends directed laterally, indifferently
to right or left or obliquely dorsally, and reaching, in ventral or lateral
view respectively, about half the distance to the periphery. Their
length in the free stage is equal to or a trifle more than that of the
longer axis of the nucleus. They are curved bodies, shaped like a
comma without a head, and taper from a bluntly rounded, larger end
directed mesially, to a finely pointed end directed peripherally. From
their blunter ends a fine thread, the parabasal rhizoplast (pl. 24,
fic. 9; par. b. rhiz., fig. A), runs anteriorly into the region of the
axostyle, and presumably thence to the blepharoplast. Rodenwaldt
(1912) incorrectly denies that these bodies have any relation to the
fibrillar system. The individuals so oriented as to exhibit the parabasal
rhizoplast are rare, and long searching of abundant material has been
necessary to detect this connection.

The most striking and persistent feature of the parabasals is their
uniform parallelism. This appears in both the free and eneysted stages
and in mitosis when daughter parabasals are formed by longitudinal
splittmg. It is an expression of the bilaterality of the organism which
manifests itself in the mutual relations of the elements of this pair of
organelles, though the pair itself may lie in a great variety of positions
in the body, in but few of which do its members exhibit the definite
right-left position of the other organs.

The parabasals stain uniformly in varying tones of black in iron-
haematoxylin and resist destaining quite as much as the karyosome,

210 University of California Publications in Zoology | Vou. 20

except at mitosis, when they tend to destain. They are the most promi-
nent organelles in the living and in the stained cyst because of their
optical properties and stainability.

The parabasal bodies tend to fade out at mitosis in the free flagel-
lates (pl. 24, figs. 7, 8, 11) and are found at the metaphase only with
the greatest difficulty, if at all, often only as vague clouds in heavily
destained slides. They are not homologues of the chromatoidal bodies
of intestinal amoebic cysts, but their enlargement during encystment
and their dense stainability during mitosis in the cysts of Giardia
strongly suggest their functional analogy to the ephemeral chroma-
toidal bodies of the cysts of the intestinal amoebae.

The homology of the parabasals of Giardia is indicated by their
shape which is that of a curved rod; by their position with relation
to the posterolateral flagella, the homologues of the undulating mem-
brane of the trichomonads; by their connections by a rhizoplast with
the blepharoplasts; and by their behavior in mitosis. They are
homologous with the curved parabasal of Chilomastix (parabasal,
Kofoid and Swezy, 1920) alongside the eytostome with its undulating
membrane; with the curved chromophile rod in the base of the un-
dulating membrane of Protrichomonas, Tritrichomonas, Trichomonas,
Pentatritrichomonas and Trichomitus, which we have ealled (Kofoid
and Swezy, 1915, 1920) the parabasal; and with the chromophile rod
coiled about the axostyle in Parajoema (Janicki, 1911). There are
two of them present in Giardia because this genus is a bilateral,
binucleate organism derived from the trichomonads by duplication of
the nucleus and its attendant neuromotor system, including the para-
basal, and by a reversal of symmetry of the neuromotor system of one
of the two constituent cells.

It is to be noted in this connection that the two parabasals le
asymmetrically in the cytoplasm and that the sagittal plane which
parts them does not coincide in vertical position with that parting the
other organs. In other words, the morphological sagittal plane which
passes between the two parabasals is variously inclined in the dorsal
protoplasmic mass according to the varying positions of this pair of
organs. It is characteristic of the parabasal of some polymastigote
flagellates to vary considerably in its position, as for example, in
Parajoema, although it is fairly constant in its relation to the un-
dulating membrane in the trichomonads.

The parabasals of Giardia enterica present a great variety of
appearances according to their position and their relations to stages of

1922] Kofoid—Swezy: Mitosis and Fission in Giardia enterica 211

mitosis. Typically they have the form above described, but in some
aspects, unless carefully focused out, they appear as a single mass.
For this reason, and also because in a moribund state degenerative
phenomena affect these organs, they have been figured as a single
median structure, a misleading and certainly atypical condition. Thus
Benson’s (1908, fig. 2, see also Doflein, 1916, fig. 642b) figure of
Lamblia intestinalis from man has a single stout clavate parabasal
which probably is only the superposed members of the pair of para-
basals. The same type of parabasal appears in Heener and Cort (1921,
pl. 4, fig. 6) in Simon’s figure of Giardia intestinalis from man. The
single parabasal appears in Grassi’s (1883, pl. 3, figs. 3, 4) original
figures of Megastoma entericum (host not stated but presumably man)
and in those of Grassi and Schewiakoff (1888, pl. 15, fig.8) of Mega-
stoma entericum from rodents as well as in some of Metzner’s (1901,
pl. 15) figures of Megastoma entericum from the rabbit. From the
nature of the figures it appears that failure to focus out and analyze
the pair of parallel parabasals underlies the interpretation or figure
of these organs as a single rather than a paired structure. We find
it possible to analyze the seemingly single bodies into the pair in our
material as a general rule and regard the other interpretations as
erroneous.

The peristomal fiber (ant. and post. perist. f., fig. B) is a deeply
staining thread encircling the so-called eytostome of Giardia. It lies in
the immediate periphery of the coneave depression on the anteroventral
surface of the body. Its anterior half has an almost semicircular
course, while posteriorly it has a recessed reniform one, the lateral ares
incurving anteriorly to the median line, along the inner ends of the
posterolateral flagella. The incurved ares appear to fuse with anterior
sections of the axostyles as the latter curve dorsally over the recessed
eytostome. Along the inner edge of the anterolateral portion runs the
intracytoplasmic section of the anterolateral flagellum closely parallel to
and often obscured by it. The two are often drawn as a single fiber, as
in the figures of Prowazek and Werner (1914) and of Simon in Hegner
and Cort (1921). At the point where the flagellum crosses over the
peristomal fiber to emerge from the cytoplasm, often slightly posterior
to the middle of the lateral arc, there is generally a slight angle in the
otherwise regular curvature of the are, but we have found no granule
there, as in Giardia microti and G. muris (Kofoid and Christiansen,
1915), though one appears in Simon’s figure.

212 University of California Publications in Zoology VoL. 20

The peristomal fiber is continuous across the median line in the
anterior are, though at that point in some individuals it may be reduced
somewhat in size and degree of stainability.

The peristomal fiber of Giardia is the homologue of two peristomal
fibers of the trichomonads. Its dual origin is not shown on the anterior
margin but is preserved in the posterior one. The right half is an
identical homologue of the well-developed, sucker-like peristome of
Chilomastic and Waskia and of the feebly developed one of Tricho-
monas, Tritrichomonas, and Pentatrichomonas. The left half is a
reversed homologue, being a mirror image of the right. As in Chilo-
mastic the right half lies at the right of the axostyle and near a curved
parabasal, and a blepharoplast lies to its left.

The function of the peristomal fiber is unknown. It lies in the
margin of a mobile organ of adhesion in the part in closest contact
with the cells of the host, and in an area of maximum mobility in
contraction in the adhesive functioning of the cytostome. In so far as
its morphological relations indicate its function, it may be either
contractile or conductile (sensory?) or possibly both in a primitive
undifferentiated state.

We have thus seen that the fibrillar system joiming karysome,
centrosome, blepharoplast, cytostome, parabasal, flagella, and axostyle,
and connected across the median plane by the anterior node, trans-
verse commissure, and anterior peristomal fiber, forms a structurally
integrated whole of the organs concerned in locomotion and adhesion.
We will now consider their relations in mitosis.

Mirosis IN THE ACTIVE PHASE

Owing to the fact that active flagellates are discharged in the faeces
under unusual conditions in the bowel from the site of the infection
at some distance up the intestine where multiplication by binary
fission is presumed to take place normally, the normal condition of
the chromatin is subject to modifications due to the moribund state of
the flagellates. The central karyosome is usually rounded up and
mitotic figures are generally rare in the free stages obtained from
stools.

The following facts are established by available data. First, mitosis,
and presumably its culmination in binary fission, takes place in the
free stage in the bowel (pl. 23, figs. 4, 5). Secondly, there are four
chromosomes, appearing as rounded-up spheroidal masses (pls. 23 and

1922] Kofoid—Swezy: Mitosis and Fission in Giardia enterica 213

24, figs. 4-12). Thirdly, the neuromotor system is duplicated at the
time of mitosis (pl. 24, figs. 10,11). The method of duplication is less
certain. This appears to take place by duplication of the centrosomes
and blepharoplasts by division, by the longitudinal division of the
axostyle and parabasals, the former from the anterior end posteriorly,
and by the splitting of the peristomal fiber from the posteromedian
end anteriorly.

The question as to whether these organs split or are duplicated by
distal outgrowth of a new element whose growing distal end alongside
the old lies near or in contact with the older structure, is largely one
of interpretation. In the absence of free ends of new outgrowths and
of young or small axostyles and parabasals, the morphological evidence
favors the interpretation of splitting for these organs, though either
is possible. The equality of size and peripheral contact of the two
elements support the interpretation of splitting.

There is, however, clear evidence in support of the view that the
flagella are duplicated by the outgrowth of one new set rather than
by splitting of the old. There is a new outgrowth of a pair of antero-
laterals at the prophase shortly after the division of the centrosome
and formation of the paradesmose (pl. 24, figs. 7-10, and text figs.
F-K). The sprouting flagella when first seen (fig. F) form an inverted
V-shaped, anterior projection from the blepharoplasts, the apex of
which is the anterior node. It appears to involve the whole of the
transverse commissure. This structure is figured by Simon (see
Heener and Cort, 1921) but not interpreted as here stated. It is
typical of the prophase only. The anterior outgrowth continues until
the node is carried out near the parental one (fig. H) and the growing
flagella extend from it beyond the node in the laterally arched processes
which come ultimately to be dorsal to the older flagella. At no time
until the completion of the anterior progression of the node is there
even the semblance of origin by splitting. It is clearly a case of origin
by outgrowth of one new flagellum duplicating the parental one which
persists. The homologous flagellum of Chilomastix and the tricho-
monads also arises by new outgrowth.

There is a suggestion of a similar sprouting process in the ventral
and posterolateral flagella in the fact that at the prophase there appear
deeply stained enlargements of the intracytoplasmic parts of the
posterolaterals near their junctions with the axostyles and on the axo-
styles at the poimts where the ventrals appear to join them. These
enlargements we interpret as local growth phenomena preparatory to

214 University of California Publications in Zoology — [ Vou. 20

2

the outgrowth of new flagella. It is probable that ‘‘basal granules’’
oceasionally seen at the points of emergence of the flagella are prophase
phenomena suggestive of the emergence of new flagella.

Rodenwaldt (1912, fig. 4) in Prowazek, Handbuch der Pathogenen
Protozoen, has given an entirely different, and in the light of our
evidence, an incorrect interpretation of the nature of this outgrowth.
His figures 4-I to 4-V fail to show the progressive changes in the

@
®P

Figs. F—K. Several stages in the origin of the new anterolateral flagella from
the blepharoplasts and transverse commissure by new outgrowth establishing the
basis for the fission of the organism in the longitudinal frontal plane. X 2400.

Fig. F. Slight anterior projection of the transverse commissure. Karyosome
elongating in nuclear axis along the intranuclear rhizoplast.

Fig. G. Transverse commissure carried anteriorly in the form of an inverted
VY. Paradesmose formed, karyosome transforming into longitudinal skein.

Fig. H. New anterolateral flagella diverging from the tip of outgrowth.
Skein resolved into chromosomes.

Fig. I. Continued outgrowth of anterolateral flagella. Posterior peristomal
fiber divided. Nuclei approaching metaphase.

Fig. J. Outgrowth of anterolaterals reaches the periphery. Nuclei in anaphase.

Fig. K. Outgrowth of free ends of new anterolaterals. Mitosis completed.

V-shaped process and he incorrectly connects the axostyles with the
tip of the process, whereas they retain their connections with the
blepharoplasts from which the parental anterolateral flagellar arch
springs. His figures also incorrectly leave these blepharoplasts
stranded on the rhizoplasts. Wenyon and O’Connor (1917, pl. 2,
figs. 1-5) figure correctly but do not diseuss the significance of several
stages in the origin of the daughter anterolaterals. They separate the

1922] Kofoid—Swezy: Mitosis and Fission in Giardia enterica 215

two blepharoplasts at the head.of each axostyle; in our material the
destaining was not carried far enough to detect the separate graunles,
if such exist.

THE PLANE OF FISSION

The plane of fission (pl. 24, fig. 12) is a longitudinal frontal one
rather than a sagittal one. In consequence of this the parent nuclei
of the right and left sides and their attendant neuromotor systems
give rise by division or outgrowth to homologous parts of the daughters,
whereas if the organs of the two sides each gave rise to a daughter,
each lateral half of the neuromotor system must then necessarily give
rise to its supplemental half by a reversal of symmetry of the added
half, that is, the nucleus and neuromotor system of the right side
would at mitosis have one daughter cell retaining the parental
symmetry and the other assuming its mirror image. The telophase
figured (pl. 24, fig. 8) shows clearly that this does not take place, but
rather that the symmetry of the parental nucleus-neuromotor system
of the whole organism is reproduced in the daughters by the frontal
division.

The paradesmose arises between the daughter centrosomes in the
prophase. The centrosome divides and one daughter migrates on the
outside of the nuclear membrane to the posterior pole of the nucleus,
spinning out a deeply staining strand, the paradesmose, between the
daughter centrosomes. This has no constant position, can be found
only when favorably located for observation (pl. 24, fig. 7), and soon
fades out as the metaphase passes.

Tue ORIGIN OF BILATERAL SYMMETRY IN THE POLYMASTIGOTE
F'LAGELLATES

The polymastigote flagellates present two unique types of multi-
cellular somatellas, distinct in organization from the dendritic (Dino-
bryon), and radial (Synura) types evolved in the lower orders of the
Euflagellata, and from the geometrical types of plate (Gonium),
ellipsoid (Pandorina), sphere (Volvor), and flattened, spirally bilateral
and anteroposteriorly differentiated ellipsoid (Platydorina), evolved
among the Volvocidea.

These two unique types of somatellas are alike anteroposteriorly
differentiated in the location both of nuclei and of their attendant
neuromotor systems; but in the number of nuclei and in their grouping
and in the interrelations of their attendant neuromotor systems, they
present two distinct types of symmetry, the spiral and bilateral.

216 University of California Publications in Zoology — [Vou. 20

The spiral type is represented by Stephanonympha and probably
by Calonympha, multicellular parasites of the termites, in which the
nucleus-neuromotor units are spirally grouped in the anterior end of
the asymmetrical body of the organism. The direction of the spiral
is unfortunately not clearly established in Janicki’s (1911) figures.
It is obvious that this type of organismal symmetry is dominated by
the spiral organization typical of the body of the unicellular flagellates.
There is, in so far as is known, no fibrillar connection between the
cellular units of these somatellas beyond that achieved by the proximity
of axostyles.

The bilateral type found in the Hexamitidae is, on the other hand,
a very great morphological departure from the fundamental primi-
tive, spiral, organismal organization so widely expressed among the
Mastigophora. It involves two fundamental departures from the
organization of these spiral somatellas. The first is the existence of
the transverse commissure and its derivative, the anterior node, which
unite the cells of the right and left sides of the body. The second is
the reversal of symmetry of the nuecleus.and neuromotor system of
the one side so that it constitutes a mirror image of these organs of
the other side. Given the spirally asymmetrical, uninucleate flagellate,
representing the right half of Giardia, this morphological reversal
affords the only method by which a binucleate, bilateral organism
(Giardia) might have been evolved from the uninucleate spiral one
(Chilomastix). We do not mean to postulate that Giardia itself was
evolved from Chilomastix, since these two genera are highly differ-
entiated representations of their respective groups, but assume that
the divergence of the binucleate group occurred in some more primitive
condition of ancestral representatives.

The morphological contrasts involved in the evolution of sinistral
and dextral, spiral uninucleate flagellates, as illustrated in certain
parasitic flagellates of the termites, such as Dinenympha gracilis
Leidy and Personympha flagellata Grassi, are comparable in type with
those reversals in stereometric relations which exist between organic
compounds such as laevulose and dextrose and between sinistral and
dextral albuminoids. Should organ-forming substances be in the
control of symmetry in flagellates, their potency in establishing the
sinistral and dextral types of organisms is to be sought in their own
sinistral and dextral molecular structure.

The morphological modifications of symmetry involved in the evolu-
tion of the Hexamitidae from the Trichomonadidae, or of Giardia from

1922] Kofoid—Swezy: Mitosis and Fission in Giardia enterica 217

Chilomastix, involve not only the origin of reversed symmetry, but
the additional factor of the union in one individual, not of two indi-
viduals of Chilomastix, but of Chilomastix and its mirror image. The
fibrillar union of the neuromotor systems of the two into one integrated
system perfects their functional codrdination in one organism.

Since the bilateral Hexamitidae combine in one organism the
sinistrally and dextrally organized halves, we may infer (Kofoid and
Swezy, 1920) that the sinistral half might have arisen in evolution by
a reversal of symmetry at one pole of the nucleus at mitosis, resulting
in one daughter organism being the mirror image of the other, a
relation permitting union of the two in a single bilateral organism.
That such a reversal is dependent upon a stereometric reversal of an
organ-forming substance is an inference inviting further investigation
on other than morphological lines.

Tue ENcYSTED PHASE

Cysts of Giardia enterica are abundant at irregular intervals in the
stools of infected persons. In the intervals between the periods of
abundance they decline in numbers or disappear entirely for periods
of several days to several weeks. The numbers in which they may
appear are large. Porter (1916) reports a case in which a stool of
950 ce. contained, on computation, 14,440,000,000 cysts or 15,200,000
per cubie centimeter. In a ease of giardiasis under daily examination
by us in New York for forty-two days, cysts were found on haemo-
cytometer count on seventeen of the forty-two with a maximum record
of 3,925,000,000 Giardia in a stool of 365 grams, or 21,216,216 per gram.

The cysts are found in all stages of multiple mitosis, that of the
single zooid similar to the free stage (pl. 24, fig. 13) up to the 12- and
presumably the 16-nucleate, 8-zooid stage (pl. 26, fig. 31). The single
zooid and the 8-zooid stages are, however, very rare in our material.
The period of eneystment in the bowel is one of multiple fission, but
apparently not of plasmotomy. The presence of a considerable pro-
portion of 2-zooid cysts in the faeces suggests the possibility that some
eysts do not proceed beyond binary fission. .On the completion of
plasmotomy such cysts might give rise to so-called copulation cysts
with two individuals in end-to-end, back-to-back position.

Multiple fission gives rise successively to 2-, 4-, and 8-zooid stages
with 4, 8, and 16 nuclei respectively, and attendant, but often lagging
neuromotor systems lacking the free flagella, with cytoplasm as yet

218 University of California Publications in Zoology [| Vou. 20

undivided. The pairs of curved parabasals are especially prominent
and paired axostyles are evident, so that the cyst appears to have a
tangle of overlapping, stained strands from which the constituent
pairs of nuclei and their attendant neuromotor systems are dis-
entangled with increasing difficulty as the divisions progress within
the as yet undivided cytoplasm. The increased size of the deeply stain-
ing parabasals, the curvature of the axostyles in the confinement of
the cyst, and the shifting of the nuclear pairs as mitosis proceeds, add
to the variety of relations presented in the successive stages of multiple
mitosis (pls. 25 and 26).

The cysts are ellipsoidal bodies of hyaline, bluish, highly refractive
optical properties. They are quickly and easily discerned among the
faeeal débris and are at once distinguished from all other cysts in the
faeces by their ellipsoidal form and the prominent parabasals and
axostyles which, in the fresh state, in Lugol’s solution, in iodine-eosin,
and in iron haemotoxylin staims, stand out even more clearly than do
the nuclei.

The shape of the cysts varies considerably in different races.
Figures on plates 25 and 26 were chosen to represent these variations
as well as those of a fluctuating nature.’ The most abundant race is
that represented in the ellipsoidal rather than spheroidal or elongated
cysts. These are symmetrically ellipsoidal, with the major axis 1.3
to 1.7 times the transverse in length. In some eysts (pl. 25, fig. 18)
the anterior end tapers slightly, giving an ovoidal form to the eyst,
thus reversing the proportions of the free stage. There is a stout race,
the frée stage of which is as yet not studied, which has a cyst approach-
ing the spheroidal form with an axial ratio of 1 to 1.11 to 1.22 (pl. 25,
fig. 16; pl. 26, fig. 23). This is not based on end views of ellipsoidal cysts.

This stout race was found together with the ellipsoidal in one
instance of infection but it was the sole or predominant one in several
others, supporting the inference that this type of cyst may represent
a form-size race. The elongated type (pl. 25, fig. 21; pl. 26, fig. 24) is
probably not a form-race, but occurs in small numbers among the
ellipsoid cysts. In some instances in stained eysts the cytoplasm has
withdrawn locally from the cyst wall as though shrunken by artefact.
These lacunae also appear in some cysts in fresh smears as though the
cyst might under some circumstances contain more space than the
volume of the protoplasmic body. A few eysts (pl. 25, fig. 19; pl. 26,
fig. 30) are irregular in outline.

The size of the cysts varies from 8.5 by 11.8 to 11 by 19u.
Spheroidal races are 11.1 by 11.7 to 10.8 by 13.4n.

1922] Kofoid—Swezy: Mitosis and Fission in Giardia enterica 219

The cyst wall is a hyaline, homogeneous structure of uniform thick-
ness throughout, less than 0.54 in thickness. Its formation evidently
begins as a secretion about the as yet uncontracted 1-zooid body. In
figure 13, plate 24, an individual is shown in which the cyst is in the
process of formation, while the tail is not, as yet, fully retracted
within the ellipsoidal mass characteristic of the encysted phase, and
the cyst wall shows two separated layers posteriorly, the inner one of
which has the contour of the free stage.

The following structures only can be identified as taking part at
some stage at least in multiple mitosis: the nuclei, centrosomes,
blepharoplasts, parabasals, axostyles, intracytoplasmic parts of the
posterolateral and the anterolateral flagella including the anterior
node. Free flagella and the peristomal fiber disappear and the rhizo-
plasts are found with difficulty, if at all.

The sequence of events within the cyst is as follows: Eneystment
of the 2-nucleate, 1-zooid flagellate occurs within the bowel, presum-
ably at or near the locus of infection. Since eysts are discharged in
fresh stools in various stages of multiple fission, it is certain that suc-
cessive mitoses proceed as the cysts are in transit through the bowel,
but that the rate and stage attaimed vary among the cysts. Many of
them are in the 4-nucleate, 2-zooid and 8-nucleate, 4-zooid stages, few
are discharged in the 2-nucleate, 1-zooid, and 16-nucleate, 8-zooid stages.

The individuality of the zooids formed by mitosis in the period
prior to plasmotomy, which is so marked in the struggling zooids of
the 8-celled somatellas of Trichomonas (Kofoid and Swezy, 1915),
is manifested in Giardia at the close of the first mitosis when the two
pairs of nuclei and their attendant neuromotor systems may, in some
cysts, shift to opposite poles of the cyst (pl. 25, fig. 21; pl. 26, figs. 24,
30). That they do not always attain or retain this position is indicated
by the occurrence of 4- and 8-nucleate cysts with the nuclei all at one
end (pl. 26, figs. 23, 28). This variability in position suggests some
mobility and functional and structural individuality of the constituent
zooids of the cysts of the as yet common cytoplasmic mass. In other
words, the individuality of the binucleate zooids is maintained through-
out the life of the eyst and the common cytoplasm appears to be merely
the containing medium. The cyst of Giardia is not comparable with
a cleaving egg but rather with a multiple cysticercus or chain of
planarians. It is asexual reproduction of the Giardia zooid which
progresses in the cyst.

Plasmotomy does not often occur within the cysts, in so far as we
have observed them, though seen in G. mwris, and duplications of the

220 University of California Publications in Zoology — [Vou. 20

neuromotor system or parts of it often lag behind nuclear mitosis.
The duplication of parabasals (pl. 26, fig. 24) proceeds most nearly at
the same rate with that of the nuclei, though it may be accelerated or
delayed, the axostyles and posterolateral flagella follow closely there-
after, and the other elements are less active. There is no constant
uniformity in the relative rates ci the nuclei (pl. 25, fig. 22), some,
singly or in pairs, dividing before others. Likewise the axostyles may
remain undivided (pl. 26, fig. 31) while the nuclei have proceedéd to
the 8 (at one end)—16 (at the other end) cell stage. Since the rate
at which eysts of Giardia die varies among cysts of the same and of
different stools, this variability in fission rate of different organelles is
an index not only of viability but of the extent of the pathological
conditions present among the eysts. Abnormal rates and irregularities
of mitotic and fission phenomena are thus indications of pathological
conditions in Giardia which are comparable with pathological irregu-
larities in cleavage and in fission in the metazoan individual.

Nuclear conditions in the cysts throw some light on the nature of
mitosis in Giardia. The chromatin elongates in the axis of the nucleus
along the intranuclear rhizoplast (pl. 26, fig. 25), as in G. muris
(Boeck, 1916). Prior to the metaphase the four chromosomes assume

a parallel and later, in the equatorial plate, an end-to-end position.

(pl. 26, fig. 26). There is also an increase in the amount of granular,
peripheral chromatin on the nuclear membrane in the prophase (pl. 23,
fig. 5) and a rounding up of the nucleus. The volume of the nuclei
decreases somewhat as the mitoses progress, but varies considerably
during the different phases of the process. The nuclear membrane
remains intact at all times. A paradesmose joining daughter nuclei is
sometimes seen (pl. 26, fig. 30). :

One of the most remarkable features of the encysted stage is the
very considerable increase in mass, 100 to 400 per cent, of the para-
basal bodies. In this regard they behave in close analogy with the
chromatoidal bodies of the amoebie cysts of the human intestine. In
these cysts the chromatoidal bodies develop as the glycogen mass
decreases and disappear after mitoses have been completed. There is
no glycogen body in Giardia but the parabasals increase greatly during
the first two mitoses and seem to be reduced somewhat in the older
eysts (pl. 26, figs. 30, 31). There are occasionally small chromatoidal
granules (pl. 25, fig. 17) lyimg free in the cytoplasm, but they are
ephemeral and rare. They may in some instances represent remnants
of the peristomal fiber.

1922] Kofoid—Swezy: Mitosis and Fission in Giardia enterica 221

DEDIFFERENTIATION IN ENCYSTMENT

During the encysted period those parts of the organism functioning
in locomotion and attachment dedifferentiate, namely, the free ends
of the four pairs of flagella, the sucker-shaped cytostome, and per-
istomal fiber, all of which disappear promptly when the body rounds
up within the cyst wall, except the posterior peristomal fiber, which
lingers for a time. A similar dedifferentiation does not occur in the
mitosis of the free stage. Even the newly formed intracytoplasmic
parts of the anterolateral flagella stain feebly and lag in formation
in the cyst. Similar parts of the posterolaterals, however, stain heavily
and are more evident. A similar phenomenon appears in somatellas
of Trichomonas, in which the homologue of the posterolateral of
Giardia, the marginal fiber of the undulating membrane, is deeply
stained during multiple fission.

The posterior peristomal fiber shifts in location with the disappear-
ance of the cytostome and the anterior peristomal fiber. Its median end
moves posteriorly and the two sides form a V-shaped structure (pl. 25,
fig. 18) which gradually fades out. In this condition the peristomal
fiber tends towards the more elongated condition seen in Trichomonas
and Chilomastix. The connection of this V-shaped structure with the
anterior part of the peristomal fiber persists for a time (pl. 25, fig. 22),
but the former soon disappears and only the latter can be found. It
is not to be confused with persistent intracytoplasmic parts of the
posterolaterals or the parabasals.

On the other hand, the axostyles and parabasals are enlarged and
stain more deeply in the cysts than in the free stages and their
duplication proceeds in step with the mitoses. The axostyles are
important organelles in the separation of the zooids at plasmotomy of
the somatella, and the parabasals appear to be concerned with meta-
bolism, perhaps in a fashion analogous to that of the chromatoidal
bodies in amoebic cysts in stools, and are therefore not dedifferentiated
as are the functionless, free flagella.

222 University of California Publications in Zoology | Vou. 20

FUNCTION OF ENCYSTMENT

The encystment of Giardia has the same dual functions as in that
of the intestinal amoebae. It provides, in the first place, a resistant
stage discharged in the faeces of the infected host, which survives under
favorable conditions of temperature and moisture and may serve to
infect a new host by the oral route. It serves a second function in
providing for the period of multiple fission of the encysted Giardia
as Wenyon (1915) proposed. It results in the formation in the common
cytoplasm of the organs of 2, 4, and 8 individuals which presumably
separate at encystment. We find no evidence whatever to support the
opinion of Schaudinn (1903, p. 550) and later defended by Penfold,
Woodcock, and Drew (1916), that there are copulation eysts in Giardia,
or that of Hartmann (1910, p. 47) that autogamy occurs in the eysts,
and that there is an Octomitus stage. It is to be noted that Hartmann
and Schilling (1917, p. 169) admit the uncertainty of sexual processes
in the cysts. We find neither maturation, fertilization, sexual behavior,
nor zygotes. We regard the so-called copulation eysts as eysts in
which binary fission and plasmotomy are completed and the two indi-
viduals are sister zooids, separated and awaiting escape, not gametes
to fuse into a zygote. We have not found such binary eysts in G.
enterica from man, though we did find them (Kofoid and Christiansen,
1916) among the cysts of G. microti and G. muris. Furthermore, the
opinion hazarded by Penfold ef al., that only a single individual will
be liberated from the cyst of Giardia finds no support in our analysis
of multiple fission in the cyst.

SPECIFIC DIFFERENCES BETWEEN G. ENTERICA AND GIARDIA FROM
RopENTS

Giardia enterica of man differs from G. muris (Grassi) of the house
and albino mouse and Peromyscus maniculatus, from G. microti
(Kofoid and Christiansen), from Microtus californicus, and from G.
cumcult (Bensen) of the rabbit, in a number of important. and readily
recognized morphological characters. The Giardia of the rat requires
further elucidation before conclusions can be drawn regarding it. The
following table summarizes the known differences.

1922] Kofoid—Swezy: Mitosis and Fission in Giardia enterica 223

CHARACTERISTICS OF GIARDIA FROM MAN AND RODENTS

Organ enterica murs microti cuniculi
Kofoid and Kofoid and Metzner (1901)
Christiansen Christiansen Benson (1908)
(1916) (1916)
Shape Medium Stout Slender
slender
Lateral contour Concave Convex More nearly Concave
of tail straight
Level of emergence Somewhat Somewhat Median Anterior
of anterolateral anterior posterior
flagellum
Shape of Curved Stout, Slender, Stout
parabasals comma- rounded slightly comma-
shaped bodies, eurvedcomma- shaped
often fused shaped
Shape of Head of Head of Head of ?
blepharoplasts axostyle not axostyle axostyle
expanded expanded expanded
by them 3
Anterolateral None Present Present 2

basal granule

Posterior basal None Present Present Present
granule

The morphological differences here noted justify the specifie dis-
tinction of these parasites and raise the question as to the extent, if
any, to which the rodent hosts of other species than G. enterica can
normally serve as carriers and disseminators of the human parasite.
We regard Deschien’s (1921) conclusion that Giardia muris and G.
enterica are identical species as unsupported by the evidence presented
and his morphological analysis as quite inadequate.

It is highly desirable that experiment should determine the extent
to which transfer of species among these hosts is feasible and permanent
and that the effect of the new hosts upon the structures of the parasites
above enumerated should be carefully analyzed before final conclusions
as to the status of these species be drawn. The morphological evidence
clearly indicates their specific distinction. The status of cross immuni-
ties, if any exist, should also be determined.

224 University of California Publications in Zoology [Vou. 20

SUMMARY

Giardia enterica has the typical structures of the genus. It lacks
basal granules on the anterolateral and posterior flagella, its blepharo-
plasts are not enlarged so as to expand the head of the axostyle. The
parabasals are rather slender, curved, comma-shaped, tapering, deeply
staining structures lying as a pair parallel to each other in various
positions posterior to the cytostome in the dorsal region. It has the
structurally integrated neuromotor system typical of the genus. The
eytostome is an organ of adhesion. There is no evidence of the
ingestion of any formed food particles.

There are four chromosomes; a paradesmose forms between parting
centrosomes outside of the nuclear membrane which remain intact
during mitosis. Daughter parabasals, axostyles, and peristomal fibers
are formed by contact outgrowth of the daughter structure or by
distad splitting of the parent structures. Anterolaterals are certainly
duplicated by new outgrowth.

Mitosis in the eyst resembles that in the free stage except that free
flagella are lacking and duplication of the neuromotor elements lags
unevenly after nuclear division. Dedifferentiation of free flagella,
eytostome, and the entire peristomal fiber occurs in the cyst.

Eneystment provides for dispersal of the parasite to new hosts and
affords opportunity for multiple mitosis to the 8-zooid, 16-nucleate
somatella. Plasmotomy was not seen in the cysts. Binary fission in
the cysts was not observed. Interpretations of the cysts as provision
for conjugation and for autogamy are erroneous. There is no evidence
that there is an Octomitus stage (Hartmann, 1910) in the life-cycle of
riardia.

Giardia is bilaterally symmetrical. Fission is in the longitudinal,
frontal plane. It is asexual reproduction. There is no evidence of a
transition through or a return to a one-cell stage. The symmetry of the
right side of Giardia is that of the one-celled Chilomastix and the
organs of that side are homologous with those of a Chilomastix. The
organs of the left side of Giardia are those of a Chilomastix, but they
are those of a morphologically reversed Chilomastiz, i.e., they form a
mirror image of those of the right side. Giardia may be derived from
Chilomastix by a morphological reversal of one daughter nucleus and
its attendant neuromotor system at mitosis and the persistence of a

a |

1922] Kofoid—Swezy: Mitosis and Fission in Giardia enterica 22

union between the centrosomes via the blepharoplasts forming the
anterior commissure. Bilateral symmetry in this instance is brought
about by the formation and persistent union of two structural units of
opposite symmetries, dextral and sinistral, whose stereometric relations
recall those of dextrose and laevulose, and of analogous albuminoids.
Giardia enterica differs in structural details, shape of body, contour
of the tail, points of emergence of the anterolateral flagella, shape of
the head of the axostyle, absence of basal granules on the flagella, and
in shape of the parabasals, from species of Giardia in rodents. Rodent
species of Giardia are therefore not sources of human. infection.
Experiment is needed to determine the host toleration and cross
immunities of Giardia in cross infections, the effect of transfer upon
the morphological characters, and practicability of rodents serving as
temporary or persistent carriers of Giardia enterica of man,

ZOOLOGICAL LABORATORY,
UNIVERSITY OF CALIFORNIA.

Transmitted November 14, 1921.

LITERATURE CITED

Benson, W.
1908. Bau und Arten der Gattung Lamblia. Zeitschr. f. Hyg. u. Infekt.,
61, 109-114, 6 figs. in text.
BoEck, W. C.
1917. Mitosis in Giardia microti. Univ. Calif. Publ. Zool., 18, 1-26, pl. 1,
1 fig. in text.
CAMMIDGE, P. J. ;
1914. The faeces of children and adults, their examination and diagnostic
significance with indications for treatment (New York. Wood),
viii + 576 pp., 15 pls., 96 figs. in text.
DESCHIENS, R.
1921. Les entérites 4 Giardia (Lamblia). Inaug. Diss., Paris, ii + 83 + ii
pp-, 3 pls.
DOoFLEIN, F.
1916. Lehrbuch der Protozoenkunde (ed. 4) (Jena. Fischer), xv + 1190 pp.,
1198 figs. in text.
FANTHAM, H. B., STEPHENS, J. W. W., AND THEOBALD, F. V.
1916. The animal parasites of man (London. John Bale, Sons & Daniel-
son), xxxii + 900 pp., 324 figs. in text.
GRASSI, B.
1881. Di un nuovo parasite dell’ uomo Megastoma entericum (mihi). Prima
nota. Gazetta degli Ospitali, 2, 575-580.
1882-1883. Sur quelques protistes endoparasites appartenant aux classes des
Flagellata, Lobosa, Sporozoa, et Ciliata. Arch. Ital. Biol., 2, 402—
444; 3, 23-37, pls. 1-4.

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GRASSI, B., AND SCHEWIAKOFF, W.
1888. Beitrag zur Kenntnis des Megastoma entericum. Zeitschr. f. wiss.
Zool., 46, 143-154, pl. 15.

HARTMANN, M.

1910. ‘‘Protozoologie’’ in Kisskalt und Hartmann, ‘‘Praktikum der Bakteri-
ologie und Protozoologie’’ (ed. 2). Zweiter Teil, vi + 106 pp., 76
figs. in text.

HARTMANN, M., AND SCHILLING, C.

1917. Die pathogenen Protozoen und die durch sie verursachten Krankheiten
zugleich eine Einfiihrung in die allgemeine Protozoenkunde. Hin
Lehrbuch fiir Mediziner und Zoologen (Berlin. Springer), x +
462 pp., 337 figs. in text.

HEGNER, R. W., AND Cort, W. W.

1921. Diagnosis of Protozoa and worms parasitic in man (Baltimore. Johns
Hopkins University School of Hygiene and Public Health), 1-72
pp., 8 pls.

JANICEI, C.

1911. Zur Kenntnis des Parabasalapparats bei parasitischen Flagellaten.

Biol. Centrbl., 31, 321-330, 8 figs. in text.
Koroip, C. A.

120. Hookworm and amoebiasis in California. Calif. State Jour. Med., 18,
329-332.

Koror, C. A., AND CHRISTIANSEN, E. B. :

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Publ. Zool., 16, 30-54, pls. 5-8, 1 fig. in text.

Koro, C. A., KoRNHAUSER, S. I., AND PLATE, J. T.
1919. Intestinal parasites in overseas and home service troops of the U. 8.
Army with especial reference to carriers of amoebiasis. Jour.
Amer. Med. Assoe., 72, 1721-1724.

Kororp, C. A., AND Swxzy, O.
1915. Mitosis and multiple fission in trichomonad flagellates. Proc. Amer.
Acad. Arts and Sei., 51, 289-378, pls. 1-8, 7 figs. in text.
1920. On the morphology and mitosis of Chilomastix mesnili (Wenyon), a
common flagellate of the human intestine. Univ. Calif. Publ. Zool.,
20, 177-174, pls. 15-17, 2 figs. in text.
1921. The flagellate infections of the human digestion tract in Nelson’s
Loose-leaf Encyclopaedia of Living Medicine, 5, chap. 20, pp. 365—
398, 6 pls. December, 1920.
METZNER, R.
1901. Untersuchungen an Megastoma entericum Grassi aus dem Kaninchen-
darm. Zeitschr. wiss. Zool., 70, 299-320, pl. 15.
Miuurr, E.
1890. Ein Fund von Cercomonas intestinalis im Jejunum des Menschen. Biol.
For. Forh., Stockholm, 2, 42-54, pl. 2.
PENFOLD, W. J., Woopcock, H. M., AND Drew, A. H.
1916. The excystation of Entamoeba histolytica (tetragena) as an indication
of the vitality of cysts. British Med. Jour., 1916, I, 714-715 8 figs.
in text.

bo
=)

1922] Kofoid—Swezy: Mitosis and Fission in Giardia enterica 2

Porter, A.
1916. An enumerative study of the cysts of Giardia (Lamblia) intestinalis
in human dysenterie faeces. Lancet, 1916, I, 1166-1169.

PROWAZEK, 8., AND WERNER, H.
1914. Zur Kenntnis der Flagellaten. Arch f. Schiffs-und Tropenhyg., 18,
Beiheft 5, 155-170, pl. 10, 1 fig. in text.

REULING, F., AND RODENWALDT, E.
1921. Giardia-Lamblia. Arch f. Prot., 42, 336-346, 2 figs. in text.

SCHAUDINN, F.
1908. Untersuchungen iiber die Fortpflanzung einiger Rhizopoden (Vor-
laufige Mittheilung). Arb. Kais. Gesundh., 19, 547-576,

Simon, C. E.
1921. Giardia enterica; a parasitic intestinal flagellate of man. Amer. Jour.
Hyg., 7, 440-491, pls. 22-24, 2 figs. and 12 curves in text. Dated
July, published in November.*

STILES, C. W. :

1915. Intestinal infections. The school grades attained by 2166 white
school children (1062 boys, 1104 girls) in the city of X, classified
by age, sanitation and intestinal parasites. U. 8S. Publie Health
Reports, 30, pt. 2, 2060-2067.

WENYON, C. M.

1915. Observations on the common intestinal Protozoa of man. Lancet,

1915, IT, 1173-1183, 1 pl.

WENYON, C. M., anp O’Connor, F. W.
1917. Human intestinal Protozoa in the Near East (London. Wellcome Bur.
Sei. Research), 1-218 pp., 4 pls., 5 figs in text.

* Dr. C. E. Simon’s article on ‘‘ Giardia enterica, a parasitic intestinal flagel-
late of man,’’ in the July number of the American Journal of Hygiene (pub-
lished in November) was received while this article was in press, so that it has
not been feasible to incorporate herein a discussion of points raised by his
divergent interpretations. A paper dealing with these will appear later.

EXPLANATION OF PLATES

All figures were drawn with camera lucida from specimens of Giardia enterica
from smears of human stools, fixed in hot Schaudinn’s fluid and stained with
iron haematoxylin by the wet film method. Magnification of all figures 3200
diameters.

PLATE 23

Fig. 1. Typical active vegetative phase viewed from dorsal surface, showing
axostyles, parabasals, eytostomal fibers, intra- and extranuclear rhizoplasts, centro-
somes, and full complement of flagella.

Fig. 2. Obliquely lateral view of free flagellate, showing the concave,
sucker-like cytostome on ventral face. Note parabasal rhizoplasts extending
to the axostyle from the ends of the parabasals.

Fig. 3. Ventrolateral view of active flagellate with cytostome somewhat
expanded.

Fig. 4. Dorsal view. Prophase with four chromosomes within the nucleus.

Fig. 5. Ventral view. Prophase with chromatin arranged in an axial
spireme; paradesmose present on nucleus at left with the daughter centrosomes
at its ends. Note thickened intracytoplasmic flagella.

23

PUBL. ZOOL. VOL. 20 [KOFOID AND SWEZY] PLATE

UNIV, CALIF.

PLATE 24
All figures of Giardi enterica (Grassi) from human stools. X 3200.

Fig. 6. Dorsal view. Nuclei in prophase with four chromosomes in each;
centrosomes at opposite poles, each pair connected by a lateral paradesmose.
New anterolateral flagella with new anterior node appearing as an anterior out-
growth from the two blepharoplasts and anterior node.

Fig. 7. Ventral view. Further outgrowth of new anterolateral flagella with
new cytostomal rim formed posteriorly; equatorial plate in left nucleus while
right nucleus is still in prophase; paradesmose visible on each nucleus; para-
basals fading out.

Fig. 8. Ventral view. Nuclei in telophase; new intracytoplasmic organelles
completed; parabasals indistinct.

Fig. 9. Dorsolateral view from the right showing nuclei in telophase;
paradesmose still persisting. Note rhizoplasts running anteriorly from the blunt
ends of the parabasals along the dorsal sides of the axostyles. The parabasals
are somewhat foreshortened and enlarged.

Fig. 10. Ventral view. Nuclear division completed; new flagella not yet
appearing; axostyles, parabasals, posterior peristomal fiber, and nuclei all
duplicated.

Fig. 11. Ventral view. Parabasals indistinct; new anterolateral flagella
completely formed; posterior peristomal fiber and nuclei duplicated; region of
union of axostyles, posterolateral flagella, ventral flagella, and posterior per-
istomal fiber greatly thickened. Longitudinal fission in frontal plane indicated.

Fig. 12. Dorsal view of two sister individuals in late phase of plasmotomy
in frontal plane not yet sepArated posteriorly. Duplication of organelles com-
pleted. Parabasals indistinct.

Fig. 13. Individual in early phase of encystment. The forming cyst has not
yet attained its typical ovoidal contour. Cyst wall forming about the partially
contracted tail, bilamellate posteriorly.

[230]

UNIV. CALIF. PUBL. ZOOL. VOL. 20 [KOFOID AND SWEZY] PLATE 24

11 12

oon ene ine: 9 ag tt

;

PLATE 25
All figures of Giardia enterica (Grassi) from human stools. X 3200.

Fig. 14. Cyst containing degenerating(?) flagellate. Note persistence of
neuromotor system after loss of most of the cytoplasm.

Fig. 15. First division of encysted flagellate; one nucleus still in prophase;
parabasals and axostyles duplicated; remnants of intracytoplasmic parts of the
flagella persisting.

Fig. 16. Cyst of the spheroidal type. Nucleus on the left showing parades-
mose and equatorial plate of the first division. Parabasals greatly enlarged.

Fig. 17. Ellipsoidal cyst. Somewhat abnormal telophase of the first divison
in central nucleus; cytoplasmic inclusion at the right (remnant of peristomal
fiber?).

Fig. 18. Ovoidal eyst. Flagellate with first division completed; all nuclei
anterior; V-shaped posterior peristomal fibers fading out, their proximal ends
shifting posteriorly.

Fig. 19. Asymmetrical irregular cyst. Second division approaching with one
nucleus in telophase, one in late prophase, and the others early prophase; four
sets of parabasals present.

Fig. 20. Characteristic cysts with four anterior nuclei.

Fig. 21. Greatly elongated cyst with sister flagellates at opposite poles of
the common protoplasmic mass and of the cyst.

Fig. 22. Cyst showing the extreme phase of the spheroidal form. Both
zooids anterior, parabasals greatly enlarged.

[232]

UNIV. CALIF. PUBL. ZOOL. VOL. 20 [KOFOID AND SWEZY] PLATE 25

‘

PLATE 26
All figures of Giardia enterica (Grassi) from human stools. X 3200.

Fig. 28. Early prophase of the second division; paradesmose visible on two
nuclei; second division of parabasals and axostyles completed; both zooids
anterior.

Fig. 24. The daughter zooids diverging to opposite poles in the as yet un-
divided protoplasm of the cyst. Note coincident divergence of nuclei and
neuromotor system in the absence of external free flagella.

Fig. 25. Prophase of the second division with spireme forming in upper two
nuclei; one parabasal detached from its mate.

Fig. 26. Prophase of second division with chromosomes doubled in each
nucleus, but in pairs, either in the early phase side by side, or in the later one,
swinging into an end-to-end relation.

Fig. 27. Second division, two enlarged nuclei in telophase. Note heavy
parabasals.

Fig. 28. Second mitosis; division nearly completed. One nucleus still in
prophase with doubled chromosomes, the other three nuclei having completed
their division. Note variation in size of nuclei, anterior location of all nuclei,
and lagging of division in the neuromotor system behind that of the nuclei.

Fig. 29. Second division of nuclei completed. Division of nuclei and neuro-
motor systems (axostyles and parabasals) synchronous.

Fig. 30. Large asymmetrical cyst with third division in progress showing
the nuclei widely divergent in size. Two zooids with larger nuclei, at opposite
poles of the cyst.

Fig. 31. Third division partly completed, with twelve nuclei but only a
single pair of axostyles.

[234]

[ KOFOID AND SWEZY] PLATE 26

UNIV. CALIF. PUBL. ZOOL, VOL. 20

UNIVERSITY OF CALIFORNIA PUBLICATIONS—(Continued)

= 12, A Synopsis of the Bats of California, by Hilda Wood Grinnell. Pp, 223-
ate _ 404, plates 14-24, 24 figures in text. January, 1918 22. eee
\ +18. The Pacific Coast Jays of the Genus Aphelocoma, by H. 8. Swarth. Pp.
5 405-422, 1 figure in text, February 23, 1918 20220o..kl eects
_- 14, Six New Mammals from the Mohave Desert and Inyo Regions of California,
by Joseph Grinnell. Pp, 423-430.
be. Notes on Some Bats from Alaska and British Columbia, by Hilda Wood
Grinnell. Pp. 431-433.
Nos, 14 and 15 in one cover. April,’ 1918-2200... eee
16. Revision of the Rodent Genus Aplodontia, by Walter P. Taylor. Pp, 435-
Nao 504, plates 25-29,.16 figures in text. May, 1918 2.0.2 2co 2. ee
17. The Subspecies of the Mountain Chickadee, by Joseph Grinnell. Pp. 505-
eG Seb Lge cy ie ROS IN DOR GS >) Wea eck Od se eB i eee ecu mee patos
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
MPR OSs o Ve TIE LOL Oo a ga eer eae Lh ei ee ee Se ha
Ba Index, pp. 537-545,
See .

wis 1. poesle in Giardia microti, by William C. Boeck. Pp. 1-26, plate 1.. October,
: 5 OS Gate nT RY Sen REE TELNT SASS CE 2S CAR ep Bee NERD oo ae at RUPAA =A Scone EE MS Ca 8 =A

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

8. Description of Some New Species of Polynoidae from the Coast of Cali-
is fornia, by Christine Essenberg. Pp. 45-60, plates 2-3, October, 1917 ....
~~. 4, New Species of Amphinomidae from the Pacific Coast, by Christine Essen-
berg. Pp. 61-74, plates 4-5. October, 1917 oo ee

6. Crithidia euryophthalmi, sp. nov., from the Hemipteran Bug, Buryophthalmus
convivus Stal, by Irene McCulloch. Pp. 75-88, 35 figures in text. Decem-

TION A ge eens RCA ee a Se ee AN a aed ee wane eae

6. On the Orientation of Hrythropsis, by Charles Atwood Kofoid and Olive
Swezy. Pp. 89-102; 12 figures in text. December, 1917 ~..2....0.0. ck.

7, The Transmission of Nervous Impulses in Relation to Locomotion in the
Earthworm, by John F. Bovard. Pp. 103-134, 14 Aeures in text. January,
ALY) SS eer ae Oe Sea ROR MERA, SPC SU veloc Le 2 PALS PPB IAN SStn -taats SP ANTE A Re Nr ne Sa Pate

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

Si ed 00-144, # fiptre in texts: January, OVS so See es ceacloclsgectuarecdeoneute
~. 9, A Rapid Method for the Detection of Protozoan Cysts in Mammalian
Faeces, by William C, Boeck. Pp, 145-149. December, 1917 222.022...

10. The Musculature of Heptanchus maculatus, by Pirie Davidson. Pp. 151-170,
U2tienres:instext: March, 1918 oe as ae eS OS

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,

m PP ULOS 1 GOKU co TAR CIN GER ee oe a Soo san goatncnsee seat gptnasesen tarsus esaapkaorck
- 12. Differentials in Behavior of the Two Generations of Salpa democratica
Relative to the Temperature of the Sea, by Ellis L. Michael. Pp. 239-298,
plates 9-11, 1 figure in text. March, 1918 -22o.02...200 cee

a 13, A Quantitative Analysis of the Molluscan Fauna of San Francisco Bay, by
: E. L. Packard. Pp. 299-336, plates.12-13, 6 figures in text. April, 1918 ....

14. The Neuromotor Apparatus of Fuplotes patella, by Harry B. Yocom. Pp,
337-396, plates 14-16.~ September, 1918 ~.......2 in oe

--- 15, The Significance of Skeletal Variations in the Genus Peridinium, by A. LL.
ane Barrows. Pp. 397-478, plates 17-20, 19 figures in text. June, 1918 -..)...
16. The Subclavian Vein and Its Relations in Elasmobranch Fishes, by J. Frank
5 Daniel, Pp. 479-484, 2 figures in text. August, 1918 .200. ee.
17. The Cercaria of the Japanese Blood Fluke, Schistosoma japonicum Katsu-
rada, 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 ...2 2.0 ee
». Index, pp. 521-529.
Vol.1 19, 1, Reaction of Various Plankton Animals with Reference to Their Diurnal
Migrations, by Calvin 0. Esterly, Pp. 1-83. April, 1919 0 oN.
2. The Pteropod Desmopterus pacijicus (sp. nov.), by Christine Essenberg. Pp.
: Bb-Op, 2 heares-in textr< May, 1919 = 28: eh ie aS ea
oe: Studies on Giardia microti, by William C. Boeck. .Pp. 85-136, plate 1, 19
eaelg Vey Lee a eeey 0) 3 1 ale S08 h: Be app meena teeny crt eet Miya Lined ec Tlen p in eae

$2.00

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UNIVERSITY OF CALIFORNIA PUBLICATIONS—(Continued)

4. A Comparison of the ‘Life Cycle of Crithidia with that of Trypanosoma in
_ the Invertebrate Host, by Irene McCulloch. © Pp. FRS-1 00) plates 2-6, 3
figures in text. October, 1919 s 3
5. A Muscid Larva of the San Francisco: Bay Region Which Sucks the Blood.
of Nestling Birds, by 0. HE. Plath. Pp, 191-200... February, 1919...
6. Binary Fission in Collodictyon triciliatum Carter, by Robert Clinton Bho es,
. Pp. 201-274, plates 7-14, 4 figures in text. - December, -1919 22...
7. The Excretory System of a Stylet Cercaria, by William W. Cort. z
281, 1 fieure:in text. — August, 3919.0 ee ee ee a ee
8 A New Distome from Rana aurora, by Williem W.. Cort. Pp. 288-296, 6
figures in text..-November,- 1919 2255233). see See ee
9. The Occurrence of a Rock-boring Isopod along the Shore of San Francisco
Bay, California, by Albert L. Barrows. Pp. 299-316, plates 15-1’
cember, 1919 S65 soe ke ere See ee
10. A New Morphological Interpretation of the Structure of Nocti ca,
Bearing on the Status of the Cystoflagellata (Haeckel), by Charles -
Kofoid. Pp. 317-334, plate 18, 2-figures in text. - February,-1920 ...
11. The Life. Cycle of Echinostoma revolutum (Froelich), by John C, Johnson.
Pp. 338-388, plates 19-25, 1 figure in text.. May, 1920-2002. B
12. On Some New Myriopods Collected in India in 1916 by C. A. Kofoid, by S
Ralph V. Chamberlin. Pp. 889-402; plates 26-28. August, 1920 .. 3
13. Demonstration of the Function of the-Neuromotor Apparatus in E g
by. the Method of Microdissection, by Charles V. Payiors Pp. 403-470,
plates 29-33, 2 figures in text. October, . pS 1 Sites se op.
Index in preparation. :
Vol. 20. 1. Studies on the Parasites of the Termites, I. On ‘Sireblomastic: striz, a Po :
mastigote Flagellate with a Linear Plasmodial Phase, by Charles Atwood ~~
Kofoid and Olive Swezy.. Pp. 1-20, plates 1-2, 1 figure in text. July, 19
2, Studies on the Parasites of the Termites. IL. On Trichomitus termitidis,
Polymastigote Flagellate with a Highly Developed Neuromotor ria
by Charles Atwood: Kofoid and Olive Swezy. Pp. 21-40, pees 3-4, 2
figures-in text, dilly, 1929 32 a i eee

sp. nov., by Charles Atwood Kofoid and Olive Bogs Pp. 41-98, plates a
5-12, 4 figures in-text..* duly; 31939-° 8 ae
4, Studies on the Parasites of the Termites. IV. On ‘Leidyop sphaeriea gen.
nov., sp. noy., by Charles Atwood Kofoid and Olive Swezy. Pp. 99-116,
plates 13-14, 1 figure in text.” July; 1919 i208 2 A aS st eee ee
5, On the Morphology and Mitosis of Chilomastix mesnili (Wenyon), a Common
Flagellate of the Human Intestine, by Charles A. Kofoid and Olive Swezy.
Pp. 117-144, plates 15-17, 2 figures in text. April, 1920 222220 ..
6. A Critical Review of the Nomenclature of Human Intestinal Flagellates,
C€ercomonas, Chilomastiz, Trichomonas, and Giardia, by Charles A. Kofoid;
Pp. 145-168, 9 figures in text. June, 1920 z
7. On the Free, Encysted, and Budding Stages of Councilmania lajleuri,
sitic Amoeba of the Human Intestine, by Charles Atwood Kofoid and
Olive Swezy. . Pp. 169-198, plates 18-22, 3 figures in text. June, 1921 0.
8. Mitosis and Fission in the Active and Encysted Phases of Giardia enterica :
(Grassi) of Man, with a Discussion of the Method of Origin of Bilater:
Symmetry in the Polymastigote Flagellates, by Charles A. Kofoid and
Olive Swezy. Pp. 199-234, plates 23-26, 11 figures in text. Ma‘
1922

Vol.21. 1. A Revision of the Microtus californicus Group of Meadow Mice, by Reming-
ton Kellogg, Pp. 1-42, 1 figure in text.. December, 1918 2.02.
2. Five New Five-Toed Kangaroo Rats from California, by Joseph Grinnell.
Ep. -43-47,- Maren, TO19 oa es ee esetee e
8. Notes on the Natural History of the Bushy-tailed Wood Rats of Californ
by Joseph Dixon. Pp. 49-74, plates 1-3, 3 figures in text. December, 1919
4, Revision of the Avian Genus Passerella, ‘with Special Reference to the Dis-
tribution and Migration of the Races in California, by H. 5. ‘Swarth. Pp.
75-224, plates 4-7, 30 figures in text. September, 1920 02a
5A Study of the California Jumping Mice of the Genus Zapus, by A. Brazie
Howell. Pp. 225-238, .1 figure.in text. May, 1920-2000 k
6. Two New. Rodents (Genera Thomomys and Marmota) from the Bastern
Border of California, by Joseph Grinnell. Pp. 239-244, 6 figures in text.
Noveniber, “1925 -o.0 0 sae Se ae i et et kOe at
Vol. 22. 1. A Quantitative and Statistical Study of the Plankton of the San. Joaquin iY
River and Its Tributaries in and near Stockton, California, in 1913, by -
Winfred Emory Allen. Pp. 1-292, plates 1-12, 1 figure in text. June, 1920. $3
Vol. 23.. The Marine Decapod Crustacea of California, by Waldo L. Schmitt. Pp. oR
1-470, plates 1-50, 165 figures in text. _ May, aS mae SSS Shade eee aay

1922 |

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Vol, 17. 1. Diagnosis of Seven New Mammals from East-Central California, by J oseph -
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2. A New Bat of the Genus Myotis from the High Sierra Nevada of California, rey
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4. A New Spermophile from the San Joaquin Valley, California, with Notes ~~

on Ammospermophilus nelsoni nelsoni Merriam, by Walter P. Taylor. Pp.
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6. Description of Bufo canorus, a New Toad from the Yosemite National Park, eT
by Charles Lewis Camp. Pp. 59-62, 4 figures in text. November, 1916
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UNIVERSITY OF CALIFORNIA PUBLICATIONS

IN
ZOOLOGY
Vol. 20, No. 9, pp. 235-242 April 12, 1922

THE MICRO-INJECTION OF PARAMAECIUM

CHAS. WM. REES

The development of the technique of isolation and micro-injection
by Barber (1914) and of micro-dissection and micro-injection by
Chambers (1918) and Taylor (1920) is of fundamental importance.
Barber applied this technique to studies in bacteriology ; Chambers to
studies of the physical changes that occur in living protoplasm, and
Taylor demonstrated the presence of conductile fibers in Huplotes.
The present paper is a report of some studies of the effect of a nema-
tode toxin on Paramaecium. The results of feeding Paramaecium
on Ascaris toxin are here contrasted with those of injecting this toxin
into the living cytoplasm.

The work was done under the direction of Dr. Charles A. Kofoid
of this laboratory and Dr. Walter R. Bloor of the Department of
Biochemistry. An attempt was made to induce transmissible changes
in the stock of Paramaecium. At first, the ciliates were fed on solu-
tions and suspensions of the ground up body wall, of the reproductive
organs, and of the digestive tract of Ascaris from the pig. The
ciliates were also placed in diluted solutions of the body fluid of
Ascaris. The results in all these tests were negative in so far as toxic
symptoms or other noticeable effects are concerned. Paramaecium
thrived in all the solutions as well as or better than in the controls.
An attempt was then made to get more concentrated extracts of the
toxins by repeating the work of Shimamura and Fujii (1917).

Shimamura and Fujii extracted from Ascaris a proteose toxin
which they named askaron. The toxin was obtained as follows. The
worms, preserved in alcohol, were dried and pulverized. The fats
and lipoids were extracted with ether and alcohol, and the fat free

236 University of California Publications in Zoology (Vou. 20

powder treated with about eight times its volume of water for three
two-hour periods in a shaking machine. The water extract was
evaporated at 60° C to about one-thirteenth of its volume and two
parts of 95 per cent aleohol added to one part of the concentrated
extract. A copious precipitate was obtained, giving the phenyl hydra-
zine reaction and reducing Fehling’s solution, but failing to give the
iodine test for glycogen. After twenty-four hours the precipitate
was filtered out and the filtrate evaporated to a thick syrup at about
50° C. An excess of absolute aleohol added to this syrup produced
a precipitate. This precipitate, a white powder, was soluble in water
and dilute alcohol. It reacted positively to nearly all of the protein
tests. Very small doses of 0.8 mg. injected intervenously into guinea
pigs caused respiratory disturbances similar to those of anaphylactic
shock and usually produced fatal results within about ten minutes.
The post-mortem findings showed inflation of the lings which is char-
acteristic of anaphylaxis. Horses were even more sensitive than
euinea pigs, 0.0004 mg. per kg. of body weight being the lethal dose.
Rabbits and dogs were less sensitive, and rats and mice were immune.
Similar results were produced by the injection of very large doses
interperitoneally into guinea pigs, but in this case the symptoms were
not manifest for about six hours. No definite results could be obtaimed
by subeutaneous injection, and the powder was non-toxie when given
by mouth.

A substance was prepared by us answering to the deseription of
askaron and giving the same chemical reactions. This substance pro-
duced in guinea pigs the chewing movements, gasping for breath,
cramps, and partial paralysis described by Shimamura and Fujii,
but in none of the five animals tested was the result fatal. The char-
acteristic temperature curve described by these authors was produced
in animals surviving the operation. These symptoms were not pro-
duced in the three control animals injected with normal salt solution.

Paramaccium thrived in our solutions of askaron. But the follow-
ing review of the literature on the anatomy and the digestive processes
of Paramaccium led us to expect this ‘result, since askaron is non-
toxic to the experimental mammals when fed to them by mouth.
Paramaecitum has a mouth or eytostome, an elongated cytopharynx,
and an anal aperture. According to Nirenstein (1905 and 1920) the
food vacuole is produced by an invagination of the lamella which
lines the blind caudal end of the ceytopharynx, so that the vacuole
thus formed is surrounded by a membrane.

|

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1922 | Rees: The Micro-Injection of Paramaecium 237

The vacuole is filled with bacteria and other food materials which
are wafted in by the eytopharyngeal membranelles and is then de-
tached and passed into the endoplasm by a process which resembles
a swallowing movement. The food vacuole first becomes tear-shaped
by being forced out to a point opposite the attached end. It is then
rotated through an angle of 180°, being in the meantime drawn out
at the attached end, so that when detached it is spindle shaped, but
quickly becomes spherical.

There is a forward streaming of the endoplasm and its inclusions
along the animal’s left side from the posterior to the anterior end
and caudad along the right side. The food vacuole begins this cireuit
but goes only halfway, crossing over at a point opposite the posterior
end of the macronucleus and returning in the posteriorly streaming
protoplasm of the right side to the starting point. It usually repeats
this cireuit several times.

It was demonstrated by the use of very dilute solutions of neutral
red that the reaction of the vacuole when first released from the eyto-
pharynx is acid. During this time endoplasmic granules, which at
first adhere to the vacuolar membrane, pass through the latter into
the vacuole. They are also stained red. These granules and the
ingested organisms are then aggregated into a compact mulberry
mass, surrounded by a clear fluid within the vacuole. The fluid is
gradually resorbed, so that the vacuole becomes smaller until its
membrane comes into immediate contact with the mulberry mass.
Then the vacuole begins to get larger, the mulberry mass is disrupted,
and the endoplasmic granules and the bacteria become again sharply
differentiated. This marks the close of the first period.

The microdrganisms within the vacuole and the vacuolar fluid now
become colorless. The granules retain their color until finally dis-
solved. These changes characterize a transition from an acid to
an alkaline reaction. No further changes were recorded until the
vacuoles become aggregated in the region of the anal aperture and
are defaecated.

Nirenstein concluded, in agreement with Greenwood (1894), and
Greenwood and Saunders (1894), and in opposition to Metchnikoff
(1889), that no digestion takes place during the time of the acid
reaction. But Metalnikow (1912) found that the acid reaction con-
tinues longer and is more pronounced when protein is fed to Para-
maecium than when carbohydrates are fed. Lund (1914) proved that
in Bursaria protein digestion occurs during the acid reaction.

238 University of California Publications in Zoology (Vou. 20

In 1910 Nirenstein demonstrated by the use of Sudan III that
fats are digested, in Paramaeciwm, during the time of the alkaline
reaction. Fat-containing vacuoles appeared in organisms that were
first starved, then placed in dilute solutions of glycerol and sodium
oleate. Mcissner (1888) demonstrated that starch is digested and
stored as glycogen in the Protozoa.

The above account shows that food of Paramaecium is separated
from the endoplasm by a membrane just as truly as is the food in
the intestine from the adjoining protoplasm in the Metazoa, where
the digestive tract has a continuous lumen. Furthermore, the diges-
tive processes of the Protozoa are in fundamental respects similar to
those of the Metazoa. It was thought, therefore, that askaron might
be found toxie to Paramaecium when injected into the living ecyto-
plasm.

In order to accomplish micro-injection, a special method of isola-
tion was devised (Rees, 1921). Barber’s miecro-injection pipette
(Barber, 1914) was used.

One of the surprising observations of this work was the exceeding
toughness of the pellicle of Paramaectwm. Organisms were frequently
bent double on the point of the pipette and the pellicle still not
pierced. It was always easy, however, to determine whether or not
the injection was successful. A characteristic flash (gelation?) is
seen in the cytoplasm, caused by the disturbance of the protoplasmic
granules and other inclusions. If this disturbance extends to the
anterior end of the body, the animal disintegrates. It may extend
over the entire posterior half of the body, as it usually does in suc-
cessful injections, and then the organism recovers.

At first the askaron used in injection was dissolved in normal salt
solution as it is when injected into mammals. But the controls showed
that injection of the normal salt solution alone was fatal to Para-
maecium. It was then dissolved in tap water, and this solution Para-
maccium survived.

The results of the injections and controls are recorded in the fol-
lowing tables. Table I shows that, of fifteen organisms successfully
injected with normal salt, seven were dead in fifteen minutes and
the other eight in thirty minutes. As is shown by Table II ten organ-
isms successfully injected with tap water were all alive and normal
at the end of three hours but they dried out overnight. Hight others
injected with tap water, as shown in Table III, were normal at
the end of one hour. The injections with askaron are recorded in

1922 | Rees: The Micro-Injection of Paramaecium 239

Table IV. Of the nine successfuly injected, one was dead in thirty
minutes, eight were normal at the end of one hour, and four at the
end of twelve hours.

The injected fluid comes in contact not only with the endoplasm
but also with the ectoplasm. The dose was not constant but was equal
in volume to about one-fifth of the body of Paramaccitum. The path
followed by this injected fluid was, therefore, in part different from
that taken by fluid entering the food vacuoles through the cyto-
pharynx. Unlike injected droplets of mercury which were soon cast
out of the body, the injected fluids were retained at least during the
five to fifteen minutes of careful observation immediately after the
injection.

TABLE I
InsEcTION witH NORMAL SALT SOLUTION

No. of the Result of Effect on Organism

Organism Injection 15 minutes 30 minutes 12 hours
1 Unsuccessful Normal Normal
2 Successful Dead
3 Successful Dead
1 Successful Very feeble Dead
5 Successful Dead
6 Control Normal Normal
7 Successful Dead
8 Successful Dead
9 Successful Normal Dead

10 Successful Normal Dead
11 Successful Normal Dead
12 Successful Very feeble Dead
13 Successful Very feeble Dead
14 Successful Very feeble Dead
15 Successful Lost

16 Successful Dead

17 Successful Dead

18 Control Normal Normal
19 Suc:essful Dead

20 Successful Dead

240 University of California Publications in Zoology [Vor. 20

TABL

BH II

INJECTION witH Tap WATER

eS Result of Effect on the Organism
Organism Injection 15 minutes 30 minutes 1 hour 3 hours 12 hours
1 Successful Normal Normal Normal Normal Dried out
2 Successful Normal Normal Normal Normal Dried out
3 Successful Normal Normal Normal Normal Dried out
4 Successful Normal Normal Normal Normal Dried out
5 Successful Normal Normal Normal Normal Dried out
6 Disrupted by
too heavy dose Dead
7 Successful Normal Normal Normal Normal Dried out
8 Successful Normal Normal Normal Normal Dried out
9 Successful Normal Normal Normal Normal Dried out
10 Successful Normal Normal Normal Normal Dried out
11 Successful Normal Normal Normal Normal Dried out
12 Control Normal Normal Normal Normal Dried out
TABLE III
INJECTION WITH TAP WATER
No. of the Result of —————_— Condition of the Organism
Organism Injection 15 minutes 30 minutes 1 hour 12 hours
1 Successful _Normal Normal Dried No record
2, Successful Normal Normal Normal No record
3 Successful Normal Normal Normal No record
4 Disrupted by too
heavy dose Dead
5 Disrupted by too
heavy dose Dead ;
6 Successful Normal Normal Normal No record
7 Successful Normal Normal Normal No record
12 Successful Normal Normal Normal No record
Successful Normal Normal Normal No record

1922 | Rees: The Micro-Injection of Paramaecium 241

TABLE IV

INJECTIONS WITH ASKARON 4 MG. PER CC. IN Tap H,O

No. of the Result of —————————Condition of the Organism— —__—_—_———
Organism Injection 12 minutes 30 minutes 1 hour 12 hours
1 Disrupted by too
heavy dose Dead
2) Disrupted by too
heavy dose Dead
3 Successful Normal Normal Normal Normal
4 Successful Normal Normal Normal Normal
5 Successful Normal Normal Normal Normal
6 Successful Normal Normal Normal Dead
7 Successful Normal Normal Normal Dead
8 Disrupted by too
-heavy dose Dead
9 Successful Normal No record
11 Successful Lost
12 Successful Lost
13 Control Normal Normal Normal Dead
14 Control Normal Normal Normal Dead
15 Control Normal Normal Normal Dead
16 Control Normal Normal Normal Normal
CONCLUSIONS

1. Micro-injection has been used in a comparative study of the
effects of the toxins of Ascaris on Paramaecium when taken in through
the eytostome and when introduced directly into the living cytoplasm
by the pipette.

2. It has been found that the solution of askaron is not fatal to
Paramaecium when ingested through the eytostome, and it is no more
fatal when injected into the living cytoplasm than is its solvent.

242 University of California Publications in Zoology | Vou. 20

LITERATURE CITED
BarsBer, M. A.
1911. Technique for inoculation into living cells. Jour. Inf. Diseases, 8,
348-360, 5 figs. in text.
1914. The pipette method in the isolation of single microdrganisms and in
the inoculation of substances into living cells. Philippine Jour Sci.,
9, Ser. B, 307-385, 19 figs. in text.

CHAMBERS, R.
1918. The micro-vivisection method. Biol. Bull, 34, 120-136, 8 figs. in text.

GREENWOOD, M.
1894. On the constitution and mode of formation of food vacuoles in Infusoria
as illustrated by the history of the process of digestion in Carchesium
polypodium. Phil. Trans. Roy. Soc. London, 185, B, 355-883, pl. 1.

GREENWOOD, M., and SAuNDERS, E. R.
1894. On the réle of acid in protozoan digestion. Jour. Physiol., 16, 441-467,
pl. 15.

Lunp, E. J.
1914. The relation of Bursaria to food. Jour. Exp. Zool., 17, 1-43, pls. 1-2.

MEISSNER, M.
1888. Beitrige zur Ernahrungsphysiologie der Protozoen. Zeitschr. f. wiss.
Zool., 46, 498-516, pl. 1.

METALNIKOW, 8.
1912. Contributions 4 1’étude de la digestion intracellulaire chez les Proto-
zoaires. Arch. de Zool. Exp. et Gen. (5), 9, 373-499, pls. 21-22.

METCHNIKOFF, E.
1889. Recherches anatomiques et physiologiques sur les infusoires cilies. Ann,
Inst. Pasteur, 3, 25-29.

NIRENSTEIN, E.
1905. Beitrige zur Ernahrungsphysiologie der Protisten. Zeitschr. allg.
Physiol., 5, 435-510, pl. 1.
1910. Ueber Fettverdauung und Fettspeicherung bei Infusorien. Zeitschr. f.
allg. Physiol., 10, 137-149, pl. 1.
1920. Ueber das Wesen der Vitalfiirbung. Pfliigers Arch. f. ges. Physiol.,
179, 233-337, pl. 1.

REEs, C. W.
1921. The technique of isolation of Paramaecium with notes on micro-
injection and micro-dissection. (In press.)

SHIMAMURA, T. and Fugu, H.
1917. Ueber das Askaron, einen toxischen Bestandteil der Helminthen be-
sonders der Askariden und seine biologische Wirkung. Jour. Coll.
Agr. Imp. Univ. Tokyo, 3, 189-257.

TAYLOR, C. V.
1920. Demonstration of the function of the neuromotor apparatus in Huplotes
by the method of micro-dissection. Univ. Calif. Publ. Zool., 19,
403-470, pls. 29-33, 2 figs. in text.
1920. An accurately controllable micro-pipette. Science (n.s.), 51, 617-618,
1 fig. in text.

~ _ UNIVERSITY OF CALIFORNIA PUBLICATIONS— (Continued)

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

'. «15. Notes on Some Bats from Alaska and British Columbia, by Hilda Wood
> Grinnell, “Pp. 431-483.

Nos. 14 and 15 in-one cover. April, 1918 ..220cigc ae 15

16. Revision of the Rodent Genus Aplodontia, by Walter P. Taylor. Pp. 435-
504, plates 25-29,°16 figures in text.. May, 1918 22.002 ee “75

17. The Subspecies of the Mountain Chickadee, by Joseph Grinnell. Pp, 505-
GIS, ME ULES Rete R Garey Wea POL as a ie ee 8 SS a ie SG ~15

18. Excavations of Burrows of the Rodent dAplodontia, with Observations on
the Habits of the Animal, by Charles Lewis Camp. Pp. 517-536, 6 figures
DECOR Bac: PIO, EOL fas oe ey EN ae A a ee 20
Index, pp. 537-545.

‘Vol. 18. 1. Mitosis in Giardia microti, by William C. Boeck, Pp. 1-26, plate 1. October,

G2] Sy GC? Sak AOE Ta TAs De NaN RR OCC ne Rl ER A Og A Oa NN AE MOMS ENC c Bbo reg 35

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

3. Description of Some New Species of Polynoidae from the Coast of Cali-
fornia, by Christine Essenberg.. Pp. 45-60, plates 2-8, October, 1917 ..... +20

4, New Species of Amphinomidae from the Pacific. Coast, by Christine Essen-
berg. Pp. 61-74, plates 4-5. October, 19V7 esc. eect cee eeeceeeeee 15

5. Crithidia euryophthalmi, sp. nov., from the Hemipteran Bug, Buryophthaimus

convivus Stal, by Irene McCulloch. Pp. 75-88, 35 figures in text. _Decem-
: POE AOL Bice Se Reet ay Ieee re eek ON RES Ae TGS ES one 15

' 6. On the Orientation of Lrythropsis, by Charles Atwood Kofoid and Olive
Swezy. Pp. 89-102, 12 figures in text. December, 1917 222.00..200002cu. 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,
SS Ai a IER aE SE See EARN CSIP AEN eR Sy eh pe YS ne eit tallest 335

8; The Function of the Giant Fibers in Rarthworms, by John F. Bovard. Pp,
135-144, "1 -figure-in:text:: January, 1918 ws. a en ee 10

9. A Rapid’ Method for the Detection of Protozoan Cysts in Mammalian
Faeces, by William C. Boeck. Pp. 145-149. December, 1917 22.00 05

10, The Musculature of Heptanchus maculatus, by Pirie Davidson. Pp. 151-170,
i? fepresin texts Wlarch, 2O%8o1 ore 3 ee Se eee «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, i918 “75

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

Relative to the Temperature of the Sea, by Ellis L. Michael. Pp. 239-298,
plates: 9-11;-E fenre intext. March; 1918 tr Fk Se ee .65

13. A-Quantitative Analysis of the Molluscan Fauna of San Francisco Bay, by
BE. L. Packard. Pp, 299-336, plates 12-13, '6 figures in text, April, 1918... .40

14. The Neuromotor Apparatus of Euplotes patella, by Harry B. Yocom. Pp.
337-396, plates 14-16. “September, 1918: 0.222 a ccieoete Nit -70

15, The Significsnce of Skeletal Variations in the Genus Peridinium, by A. L.
Barrows. . Pp.-397-478, plates 17-20, 18 figures in text. June, 1918 2.0... 90

16. The Subclavian Vein and Its Relations in Elasmobranch Fishes, by J. Frank
; Daniel.. Pp. 479-484, 2 figures in text. August, 1918 coo 10

17. The Cercaria. of the Japanese Blood Fluke, Schistosoma japonicum Katsu-
rada. 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 020.0 eccce 35
Index, pp. 521-529.

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

: Migrations, by Calvin O, Esterly. Pp. 1-83. April; 1919 2.20. 85
2, The Pteropod Desmopteris pacificus (sp. nov.), by Christine Essenberg. Pp.
86-88;,2-figures-in text. “May, A919 ot A ee .05
8. Studies on Giardia microti, by Wiliam C. Boeck. Pp. 85-136, plate 1, 19
Hei ved In texts. April: F910 ee eee ee oe ee 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
fipnres-in- texts: OCto Ber. 21910 ao enon ak ye Oh cca 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 2.000... 10
6. Binary Fission in Collodictyon triciliatum Carter, by Robert Clinton Rhodes.
Pp. 201-274, plates 7-14, 4 figures in text. December, 1919 ......:.2000 io. $1.00
7. The Exeretory System of a Stylet Cercaria, by William W. Cort. Pp. 275-
28151 -fienre. intext: -Aurust; 1999 oo ae ae eS 10

8..A New Distome from Rana aurora, by William W. Cort. Pp. 283-298, 5
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UNIVERSITY OF CALIFORNIA PUBLICATIONS—(Continued) —

9. The Occurrence of a Rock-boring Isopod along the Shore of San Francisco
Bay, California, by Albert L, Barrows. Pp. 299-316, plates 15-17. ~
centuer, 1919 oe ee Re reg ea ee ee n

10. A New Morphological Interpretation of the Structure of Nocti a
Bearing on the Status of the Cystoflagellata (Haeckel), by Charles A
Kofoid. Pp. 317-334, plate 18, 2 figures in text. February, 1920 -_o.:

. The Life Cycle of Echinostoma revolutum (Froelich), by John C. Johnson,
Pp. 338-388, plates 19-25, 1 figure in text. May, 1920 ..
12. On Some New Myriopods Collected in India in 1916 by €. ws ‘Kofoid, by
Ralph V. Chamberlin. Pp. 389-402, plates 26-28. August, 1920
18. Demonstration of the Function of the Neuromotor Apparatus in Euplotes
by the Method of Microdissection, by Charles V. onayior, Pp, bie as
plates 29-33, 2 figures in text. October, 1920: aise eases a
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Vol. 20. 1. Studies on the Parasites of the Termites. I On Btteblomastiz striz, a Poly.
mastigote Flagellate with a Linear Plasmodial Phase, by Charles Atwood
Kofoid and Olive Swezy. Pp. 1-20, plates 1-2, 1 figure in text, J: uly, 1919

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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 s
$.. Studies-on the Parasites of the Termites. Tit. ‘On Trichonympha campanula.
sp. nov., by Charles Atwood Kofoid and Olive Swezy. Pp. 41-98, plates
5-12, 4 figures in: ftexbo. POLY A919 | ec a eee
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noy., sp. nov., by. Charles Atwood Kofoid and Olive Swezy. Pp. 99-116,
plates 13-14, 1 figure in-text. July,- 1999.0 ss a ee
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Fiagellate of the Human Intestine, by Charles A. Kofoid and Olive Swezy. =
Pp. 117-144, plates 15-17, 2 figures in text. April, 1920 %
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Cercomonas, Chilomastiz, Trichomonas, and Giardia, by Charles A. Kofoid.
Pp. 145-168, 9 figures in text.. June, 1920 <2 nen hee
7. On the Free, Encysted, and Budding Stages of Councilmania tafleurt, a Para:
sitic Amoeba of the Human Intestine, by Charles Atwood Kofoid and
Olive Swezy. Pp. 169-198, plates 18-22, 3 figures in text. June, 19217 __..
8. Mitosis and Fission in the Active and Encysted Phases of Giardia ‘enterica.
(Grassi) of Man, with a Discussion of the Method of Origin of Bilateral
Symmetry in the Polymastigote Flagellates, by Charles A. Kofoid and
Olive Swezy. Pp. 199-234, plates 23-26, 11 figures in < Seext ‘March,
1922 zs
9, The Micro-Injection of Paramaccium, by Chas. Wm. Rees. Pp. 235- Z
AE A OOO nN ee ti SOS A ee een ae ee Se geht eee aa ae a

Vol. 21. 1, A Revision of the Microtus californicus Group of Meadow Mice, by Reming-

ton Kellogg. Pp. 1-42, 1 figure in text. December, 1918 2.00.0 Bore

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

Pp. 48-47. March, 1919 ar

8. Notes on the Natural History of the Bushy-tailed Wood Rats of California, —

by Joseph Dixon. Pp. 49-74, plates 1-3, 3 figures in text. December, 1919

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Howell, Pp, 225-238, 1 figure in text. May, 1920-2. c.2 uk. ees,

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Meadow Mice, by Remington Kelloge. Pp. 245-274, 25 figures in text.

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UNIVERSITY OF CALIFORNIA PUBLICATIONS

IN
ZOOLOGY
Vol. 20, No. 10, pp. 243-300, pls. 27-28, 15 figures in text May 8, 1922

ON BALANTIDIUM COLI (MALMSTEN) AND
BALANTIDIUM SUIS (SP. NOV.), WITH
AN ACCOUNT OF THEIR NEURO-
MOTOR APPARATUS

BY
J. DALEY McDONALD

CONTENTS pia

Thnaetriteys PRION ea os onl entero cee eee crea ee tees 244
Acknowledgments.................. see aree ; : 245
Material and technique... H: : d : 245
Occurrence and Seaman diserbubion : i 246
Studies of living organisms................0....0.... 5 246
Systematic position of genus and epeciee Bed , 248
VES TILOUIUELORUUTIL COLL Neenaty ee. teeta see eoeee neces ee 249
Balantidium suis sp. nov. fei LCP eS 3 F 250
Method and use of shexneraTeite : 254

Other specific characters...................0.0000+ 258
Balantidiwm from man............................... oro : . 261

Mion polo pyar tite Rete rotors cesenceccsrt aes = 262
Ectoplasmic structures Poet ieltcd tes ier . 263
Relliiele terse sees eee ceca s tan ce ae 263
Ectoplasm.. 266
(Giligeiree tee eae As, 7. ae eco YAY)

Basal apparatus of cilia...............0....04. Ree : 271
C@iliamy movements e.siccc--- desserts a 218
Cytostome =Ebe Ede eee Leap ee eee 4 eG.

(Oy gail 0) Ite atece eeeeee eee eee aoe coe eee ise 280
@ontrachilesvacnolessee. veer cece vedas eee o39 PRY)
Endoplasmic structures) ...........ccecceeseeeseneeeeess eR errors san PAPA
ind oplasmiy sees... et 282

Hoo dav a CUOleS raster taeaet tare ee Tee Sh eecseses te ae,

Mia cromueleus:2ec...c.cccccceceseteers-- bce : oe 282
Micronucleus........ Pe etait 283
Neuromotor apparatus...............c:ccccccceeeeeeee ate : nt . 284
IVE GTONTU yee Seen wed hee St re esiccs ype ee sestehes wa: 280
Circumoesophageal fiber.................... : ne 285

Adoraliieniary fibers. .22...s..<csn+-sceseveze: ne a hates Sao

244 University of California Publications in Zoology [Vou. 20

PAGE

Adoral ‘ciliary rootlets): cacckc cece e eRe ED Ts
Riadialltibersissesccs- EE eR ry ON OREO al sham -eomeronee onteaaexn DAO
DISCUSSION! 7.0-c-s-e2cs caneoeete toy ale tec Pe i rekon oortceneaicke ieee 288
Summary.... rect ; Serer PPE CRS co PAPERS: 293
Literature cited Sree ees : EERE SCRE OEE diy Caer rene reece 294
MXxplanation of platesscs.-c2ssorcacsceanstcovessatass ae cesea ds sts es ee 298

INTRODUCTION

The earliest observation of Protozoa of the genus Balantidium has
in several instances been accredited to Antony von Leeuwenhoek
(1708). During an attack of dysentery he detected motile organisms
in the discharges. At that time no discrimination had been made
between ciliated and flagellated protozoa and his account of his obser-
vations is not sufficiently complete to make possible the classification
of the organisms which he found. However, he stated that they were
about the size of red blood corpuscles, which would indicate that they
were intestinal flagellates and not Balantidiwm, which is very much
larger.

Malmsten (1857) was the first to describe Balantidium coli. This
species has become better known than the other species of the genus,
due to its being the cause of a specific dysentery known as balantidiasis.
Two persons suffering from this disease came to Malmsten for medical
attention during 1856-57. He was assisted in the study of protozoans
which he found in the excreta from these two patients by the zoologist
Loven who believed that the parasites were new to science and so pre-
pared a careful description of them accompanied by figures. For the
organism they suggested the name Paramoecium (2) coli. Since that
time infections with Balantidiwm coli have been reported in increasing
numbers and some cytological studies have been made, though much
more attention has been given to the problems of prophylaxis and
treatment of the disease which this species causes than to the parasite
itself.

The first record of Balantidium coli as a parasite of pigs was made
by Leuckart (1861). Stein (1862) also studied these forms from pigs
and he was the first to assign them to the genus Balantidium. The
genus had been established by Claparéde and Lachmann (1858) with
Balantidium entozobn from the frog as the type species. More recently
Strong (1904), Brumpt (1909), Walker (1913), and others have ear-
ried on investigations on this parasite of pigs in order to become
acquainted with the problems involved in the infection of man.

1922] McDonald: On Balantidium coli and Balantidium suis 245

ACKNOWLEDGMENTS

It has been my privilege to study the morphology of Balantidiuwm
coli and Balantidium suis (sp. noy.) under the direction of Professor
Charles A. Kofoid, to whom I am indebted for helpful suggestions and
for oversight of the entire work. Acknowledgment is due Professor
William W. Cort for many valuable criticisms. I also take this oppor-
tunity to express my appreciation of the courtesy of Mr. R. B. Brown,
superintendent of the Oakland Meat and Packing Company, who
kindly granted me permission to work in the company’s abattoir and
also facilitated the work in every possible way.

MATERIAL AND TECHNIQUE

The material for these studies was obtained almost exclusively from
pigs killed by the Oakland Meat and Packing Company, Stockyards,
California. At their abattoir I was permitted to work in the room
where the pigs were dressed, which made it possible to obtain the
material from the intestine before it had cooled below the normal body
temperature. To determine the presence of the balantidia a small slit
was made in the caecum and a drop of the contents withdrawn with
a pipette. This drop was quickly placed on a warm slide and exam-
ined with a microscope. If the animals were present they would be
detected very readily for they are exceedingly active; in most cases
they occurred in numbers sufficiently large that from one to ten could
be seen in every field when a 16 mm. objective was used. This method
was rapid enough to allow all pigs to be examined as fast as they were
killed and dressed.

A sample from the caecum was not relied upon as eritical in the
determination of infection until examination of the entire length of
the intestine had been made in several instances. In order to discover
the normal distribution throughout the intestine it was removed entire
and taken to the laboratory of the abattoir. Incisions were made every
one or two feet, beginning with the duodenum and continuing to the
rectum, and samples examined from each of these incisions. In no
cases were balantidia found more than three feet above the ileocaecal
valve, and only in two or three instances were any at all present in

246 University of California Publications in Zoology [Vou. 20

the small intestine. In the caecum and first three or four feet of the
colon the balantidia were always more active and more numerous than
elsewhere. Posteriorly from this region they were found in progressive
stages of encystment until in the rectum the majority were completely
encysted.

OccCURRENCE AND GEOGRAPHIC DISTRIBUTION

Approximately 200 pigs were examined. They had been raised in
the Sacramento Valley, except for one-lot from Los Banos, California,
and a lot from the state of Nevada. Of the 200 pigs examined 68
per cent were infected. The examinations were made at nine separate
times between September, 1913, and May, 1918, ten to sixty individuals
being examined each time. In five of the nine lots every pig was
found to be infected. The lowest percentage of infection was 13 per
cent, in the lot shipped from Nevada. This indicates a very general
infection of pigs with Balantidiwm in this region of the United States.

Stiles (cit. Strong, 1904), Bel and Couret (1910), and others have
previously found the organisms in pigs in the United States. Leuckart
(1861), working in Germany, was the first to find Balantidium in pigs.
Since then Stein (1862), Eekecrantz (1869), and Prowazek (1913)
have reported them from the same country. In 1871 Wising noted
their occurrence in pigs in Sweden. Grassi (1882) and Calandruccio
(1888) have found the parasites in swine in Italy. Rapchevski (1882)
reported the occurrence of balantidia in Russia. In France they have
been found in pigs by Railliet (1886), Neumann (1888), and Brumpt
(1909). Strong (1904), Walker (1913), and several others have
noted the occurrence in pigs in the Philippine Islands. Similar reports
from China have been made by Maxwell (1912), and Mason (1919) ;
from Cuba by Taboadela (1911) ; and from South America by Bayana
(1918). These citations indicate that Balantidium coli is probably as
widely distributed geographically as is its host, the pig.

Strupres or Living ORGANISMS

The balantidia are very sensitive to changes of temperature. When
the medium in which they are swimming is cooled a few degrees they
slow up their movements very decidedly. After a time they become
almost perfectly spherical, in which form their activitiy is restricted
to a rotary motion with little or no progression. In this condition they
will live for six or eight hours at ordinary room temperature.

1922] McDonald: On Balantidiwm coli and Balantidium suis 247

In order to avoid the deleterious effects caused by cooling and by
increased bacterial action, most of the studies on living organisms were
made at the abattoir. When continuous observation over a long period
was desired, however, the material was conveyed to the laboratory in
thermos bottles and kept in the incubator at 37.5° C. In this manner
material could be kept for three days. Ultimate degeneration of the
organisms seemed to be due more to the increase in the bacterial con-
tent of the medium than to any other cause. During observation either
an electric warm stage or the microscope warm oven designed by Long
(1912) was employed. Of the several vital stains used, neutral
red proved most satisfactory in the differentiation of the neuromotor
apparatus.

Fixation and staining. —The following fixatives were used: Schau-
dinn’s fluid, Zenker’s fluid, formalin, osmie acid, and picromercuric
fluid (according to the formula by A. D. Drew, used by Yocom, 1912).
Quick action was one of the most important factors in the fixation,
and was usually obtained by having the killing fluid hot (60—80° C.)
and using an amount at least equal to the amount of the material to
be fixed. Frequently the action was so nearly instantaneous that the
cilia on the killed animals retained their exact relative position (see
fig. N). After fixation the material was thoroughly washed, iodine
alcohol being used if mercurial salts were present. Material was pre-
served in 70 per cent alcohol.

Before staining, the preserved material was usually concen-
trated by elimination of lighter débris by centrifuging and the heavier
by sedimentation. Water was found to be a more satisfactory medium
for these operations than either alcohol or salt solutions. In case see-
tions were to be made, additional care was taken in the concentration
process and then the material was handled according to the methods
employed by Metealf (1909) and by Sharp (1914).

Tron haematoxylin gave uniformly the best results in staining. For
eysts, however, on account of their imperviousness, it was necessary to
use Delafield’s haematoxylin to which had been added a small amount
of acetic acid. In addition to the first mentioned stain, Mallory’s con-
nective tissue stain was used on sections.

248 University of California Publications in Zoology

[ Vor. 20

SYSTEMATIC POSITION OF GENUS AND SPECIES

Claparéde and Lachmann (1858) removed Bursaria entozoén from
the genus in which it had been placed by Ehrenberg (1838) and

ereated for it the new genus Balantidiwm.

This genus was of the

family Bursaridae and the order Heterotricha. The twenty-two species
of the genus that have been described to date are listed below.

KNOWN SPECIES OF THE GENUS BALANTIDIUM

Species
Balantidium entozoén

Balantidium

Balantidium
Balantidium

Balantidium

Balantidium

Balantidium
Balantidium
Balantidium
Balantidium
Balantidium

Balantidium

Balantidium
Balantidium
Balantidium
Balantidium
Balantidium
Balantidium
Balantidium
Balantidium
Balantidium
Balantidium

coli

duodeni
elongatum

medusarum
amphictenides

gyrans
viride
minutum
giganteum
helenae

gracile

rotundum
faleiformis
ovale
hyalinum
littorinae
testudinis
hydrae
piscicola
caviae
orchestia

Original description by

Ehrenberg, 1838
Malmsten, 1857

Stein, 1862
Stein, 1862

Mereschkowsky, 1879

Entz, Sr., 1888

Kellicott, 1889
Willach, 1893
Schaudinn, 1899
Bezzenberger, 1903
Bezzenberger, 1903

Bezzenberger, 1903

Bezzenberger, 1903
Walker, 1909
Dobell, 1910
Dobell, 1919
Chagas, 1911
Chagas, 1911

Entz, Jr., 1913
Entz, Jr., 1913
Neiva et al., 1914
Watson, 1916

Hosts
Rana esculenta
Rana temporaria
Sus scrofa
Homo sapiens
Rana esculenta
Triton cristatus
Triton alpestris
Triton marmoratus
Rana esculenta
Rana temporaria
Bougainvillea, Obelia,
Eucope, Broda sp.?
Amphictenis,
Turbellaria marina
Aquatic worm
Columba sp.?
Homo sapiens
Rana esculenta
Rana cyanophlyctis
Rana tigrina
Rana limnocharis
Rana hexadactyla
Rana cyanophlyctis
Rana hexadactyla
Rana esculenta
Rana palustris
Rana tigrina
Rana tigrina
Littorina
Testudo graeca
Hydra olygactis
Piarectus brachypomus
Cavia aperea
Orchestia agilis
Talorchestia longicornis

1922] McDonald: On Balantidium coli and Balantidium suis 249

The wide diversity of hosts, ranging from hydroids and crustaceans
to the warm-blooded vertebrates, including man, must demand a wide
versatility on the part of the parasite. Considerable structural varia-
tion is apparent even on cursory examination, and some of these
structural differences might be sufficiently marked to serve for generie¢
differentiation. A new generic division would seem desirable, but the
suggestions of Biitschli (1884) and Schweier (1900) in this direction
have not been generally accepted.

BauaNntipium cout MAuMsSTEeNn (1857)
SYNONOMY:
Paramoecium (?) coli Malmsten, 1857.
Plagiotoma coli, Claparéde and Lachmann, 1858.
Leucophyra coli, Stein, 1860.
Holophyra coli, Leuckart, 1861.
Balantidium coli, Stein, 1862.

Up to the present time only one species, Balantidium coli, has been
described as parasitic in pigs. It was first described by Malmsten
(1857) who, noting its hkeness to Paramoecium colpoda (Ehrenberg),
suggested the name Paramoecium (?) cok. During the following year,
Claparéde and Lachmann (1858) reproduced one of Malmsten’s orig-
inal figures and after considering his description transferred the
species to the genus Plagiotoma. In 1860, Stein, using the description
by Malmsten (1857), pointed out that the organism was not a Para-
moecium and believed that it properly belonged in the genus Lewco-
phyra. Leuckart in 1861 discovered a ciliate in the intestine of pigs
which he coneluded was identical with the one already described, but
he was not satisfied with the genus to which it had been assigned by
Stein (1861) and believed that its closest relation was with Holophyra
in which genus it should be placed. In 1863 he still retained this view
but suggested the appropriateness of the establishment of a new genus.
But Stein (1862) had already recognized those characters of the
species which showed its close relation to Balantidiwm entozodn and
had placed it in the genus Balantidium.

During the present investigation the following specific character-
istics have been found very constant. The individuals of the species
Balantidium coli are ovoid in form, the more pointed end being an-
terior; length varies from 30 to 150; breadth varies from 25, to
120; in the majority of individuals the length is 1.3 times the breadth ;
the greatest transverse diameter intersects the longitudinal axis poster-

250 University of California Publications in Zoology [Vou. 20

ior to its midpoint; the adoral zone is approximately terminal, and-
the anterior tip of the body les within it; the plane of demarcation
between the apical cone of the ectoplasm and the endoplasm is approx-
imately at right angles to the long axis of the body; the macronucleus
is elongate but the length usually does not exceed three times the
breadth; two contractile vacuoles are present, a smaller one located
anteriorly and a larger one located posteriorly ; a posterior cytopyge
is usually distinetly visible.

BALANTIDIUM SUIS SP. NOV.

Early in the work of examining pigs for Balantidiwm coli it became
evident that this protozoan showed extreme variation in shape. In
many instances the diversity occurred among individuals from the
same host. Further observations led me to believe that there were
two fairly distinet types, the one, longer and more slender as compared
with the other which was distinctly ovoid. Measurements have been
recorded by various writers of Balantidiwm coli from man. Malmsten
(1857) in the original description gave the length as 60-100; breadth,
50-70u. Solojew (1901) recorded the length as 65n, the breadth as
40n. Wising (1871) states that the length varies from 50-100n, while
the breadth varies from 40-50. Prowazek (1913) gave the length as
5271p, the breadth, 40-58. Leuckart (1861) measured balantidia
from swine and found the length to be 75-110» and the breadth 70.
Still others give dimensions, but all are inadequate for the determina-
tion of the occurrence of types with distinet proportions. First, with
one or two exceptions all dimensions have been taken of balantidia
found in man, and from these it might not be safe to draw conclusions
regarding diversity among those found in pigs. In the second place,
the measurements given are either averages or else represent extreme
limits. In either case they are practically useless in determining indi-
vidual variations, for even in the case of extreme types the range
between limits is so great that two, or even more, distinct types, based
on proportions of breadth to length, might be included. Nowhere
has there been found a series of individual measurements which would
make it possible to determine whether variations were continuous or
discontinuous. To obtain such a series of measurements was the pur-
pose of the phase of the work about to be described.

Material which was to be used in taking the measurements was
killed and preserved with all possible care. Hot Schaudinn’s fluid
was used in all cases, the material being quickly and thoroughly mixed

1922] McDonald: On Balantidium coli and Balantidiwm swis 251

into a large quantity of it so that action would be as nearly instan-
taneous as possible, thus avoiding distortion. Osmic acid vapor was
tried but Schaudinn’s fluid gave equally good results and was more
convenient for manipulation. Material was never allowed to cool
before fixing, for on cooling the individuals tend to become spherical.
Several attempts were made to measure living organisms but their
ceaseless activity at normal temperature (37.5° C) made this almost
impossible and slowing them up by the use of Irish moss or by cooling,
as mentioned above, caused them to become distorted. If there were
changes due to fixation, the logical expectation would be that the error
would be on the side of conservatism for such changes would tend to
obliterate rather than accentuate the division into two groups; for the
shape of: the elongate forms would be more changed by the fixative,
the tendency being for them to shorten and broaden and thus approach
the ovoid type. However, in the method of fixation used, I am sure
that distortion was so slight as to be negligible.

Following fixation the material was carefully washed and carried
slowly through the lower grades of alcohol to 70 per cent in which
the material was kept for measuring. A drop of the material from
which measurements were to be taken was placed on a slide, covered
with a coverglass, the excess of fluid removed, and the edges sealed
with vaseline to prevent evaporation. Just enough fluid was removed
from under the coverglass to reduce the depth of the medium so that
the majority of the animals would lie flat, and yet not enough to allow
the coverglass to exert any pressure. The exertion of pressure on the
animals, however, would ordinarily be prevented by the presence of
large particles of foreign material. The object of having animals lie
flat on the slide was to avoid the error which would otherwise be caused
by foreshortening. A slight elevation of one end would make con-
siderable error in the determination of the length of the animal.

The slide was then placed on the microscope and systematically
examined by the use of the mechanical stage. Beginning at the upper
left-hand corner and progressing as one would in reading a book, every
individual encountered in the survey was measured. The only excep-
tions made were in ease the animal was not lying flat or showed marked
signs of distortion. This procedure avoided selection which might
unconsciously be made by the observer. For making the measurements
a 4mm. objective was used in combination with an ocular-micrometer
inserted in 9x compensating ocular. With the magnification given by
this combination the limit of error did not exceed one micron.

252 University of California Publications in Zoology [Vou. 20

The longitudinal axis and the longest transverse axis of each indi-
vidual were measured, and the ratio of length to breadth computed
(see Table I). c

TABLE I

CoMPARATIVE MEASUREMENTS OF ONE HUNDRED INDIVIDUALS EACH OF
BALANTIDIUM COLI AND BALANTIDIUM SUIS

INDIVIDUALS FROM PiG No. 1 INDIVIDUALS FROM PiG No. 4
(Bal. suis, WaLhyive exceptions) (Bal. coli, with one exception)
Ratio of Ratio of
Length in Breadth in length to Length in Breadth in length to
microns microns breadth microns microns breadth
114 42 2.71 99 75 1.32
108 51 2.11 96 81 1.19
63 36 1.75 126 75 1.68*
90 45 2.00 118 87 1.36
93 48 1.94 111 87 1.28
72 39 1.85 87 75 1.16
126 69 1.83 90 66 1.36
87 48 1.82 105 75 1.40 .
108 57 1.90 78 66 1.18 |
102 54 1.89 90 63 1.43
117 51 2.30 87 72 1.21
120 57 2.10 87 72 1.21
96 42 2.29 99 75 1.32
84 39 2.12 105 75 1.40
120 48 2.50 89 75 1.19
117 48 2.42 81 63 1.28
93 51 1.83 81 66 1.23
96 51 1.89 75 63 1.19
72 57 1.26* 84 69 1,22
75 54 ilsshy) 69 60 1.15
111 51 2.14 90 66 1.36
111 54 2.03 93 75 1.23
84 36 2.31 87 69 1.26
69 39 USA 90 81 iat
78 51 1.52* 105 90 1.16
81 45 1.80 87 69 1.26
111 45 2.42 84 72 1.17
81 45 1.80 66 58 1.14
84 45 1.86 75 60 1.25
90 45 2.00 81 63 1.29
84 42 2.00 81 60 1.35
90 42 2.12 99 78 1.27
117 57 2.03 87 69 1.26
60 33 1.82 78 63 1.24
108 57 1.90 81 60 1.35
108 57 1.90 105 75 1.40
96 48 2.00 90 60 1.50
75 42 179 93 78 1.18

* Other characters showed that these individuals were of the other species rep-
resented in the table.

1922] McDonald: On Balantidium coli and Balantidiwm suis 253

TABLE I—(Continued)

INDIVIDUALS FROM P1G No. 1 INDIVIDUALS FROM PiG No. 4
(Bal. swis, with five exceptions) (Bal. coli, with one exception)
2 —

Ratio of Ratio of
Length in Breadth in length to Length in Breadth in length to
microns microns breadth microns microns breadth
105 57 1.85 69 60 1.15
99 57 1.74 81 o4 1.50
105 54 1.95 87 66 1.32
54 27 2.00 84 69 1.22
57 30 1.90 78 60 1.30
36 24 1.50* 66 51 1.29
81 31 2.23 66 54 1.22
75 42 1.78 96 66 1.45
66 33 2.00 81 54 1.50
78 36 2.08 84 66 1.28
93 39 2.39 114 87 1.31
63 36 1.74 87 69 1.26
102 48 2.06 93 72 1.29
78 39 2.00 84 72 ala ly
81 42 1.93 102 65 1.58
87 48 1.82 87 63 1.38
93 45 2.03 99 66 1.50
84 42 2.00 81 72 1.13
81 39 2.04 78 63 1.24
75 39 1.93 102 75 1.36
78 45 1.74 66 48 1.38
75 37 2.01 75 57 1.32
100 45 2.11 84 66 1.27
81 39 2.04 93 66 1.40
72 39 1.85 72 58 1.24
84 40 2.06 96 72 1.33
90 42 2.07 90 68 1.32
78 36 2.08 60 39 1.54
102 48 2.06 87 69 1.26
81 37 2.10 69 54 1.28
66 37 1.78 90 69 1.31
87 36 2.41 69 54 1.28
78 42 1.86 72 60 1.20
84 42 2.00 75 57 1.47
90 38 2.37 75 62 1.21
66 35 1.89 72 63 1.14
78 42 1.86 90 75 1.20
97 44 2.20 74 66 1.12
79 37 2.07 72 54 1.33
98 42 2.32 60 50 1.20
90 45 2.00 84 58 1.45
87 49 1.78 84 64 1.31
90 36 2.50 88 60 1.47
81 35 2.31 99 75 1.32
69 48 1.44* 75 54 1.39

* Other characters showed that these individuals were of the other species rep-
resented in the table.

254 University of California Publications in Zoology (Vou. 20

TABLE I—(Continued)

INDIVIDUALS FROM PiG No, 1 INDIVIDUALS FROM PiG No. 4
(Bal. suis, with five exceptions) (Bal. coli, with one exception)

Ratio of Ratio of
Length in Breadth in length to Length in Breadth in length to
microns microns breadth microns microns breadth
90 54 1.68 81 69 1,18

66 33 2.00 66 52 1.27

76 38 2.00 93 63 1.48

78 42 1.86 81 75 1.08

90 57 1.58 96 75 1.28

66 40 1.65 114 90 1.27

81 39 2.04 75 60 1.25

96 39 2.43 87 72 1.21

75 38 1.98 84 60 1.40

84 39 2.18 87 63 1.38

51 30 1.70 84 63 1:33

75 39 1.92 72 60 1.20
84 40 2.05 90 66 1.36
116 47 2.48 84 72 1.16
84 36 2.32 93 72 1.29

81 39 2.04 87 58 1.50

75 45 1.68 93 72 1,29
Aver- a Ane wt, > ora
age 86 43 1.99 86 66 1.30

While taking the measurements of each individual, observations were
made regarding the position of the mouth, the type and size of macro-
nucleus, number and location of contractile vacuoles, and any other
characters which might aid in differentiation.

In the handling of the data on dimensions I have followed in a
general way the method used by Jennings (1908) in differentiating
races of Paramaecium. For the purpose of this work, however, the
results seemed more lucid if, instead of plotting length and breadth
along separate axes, the ratio of length to breadth was computed for
each individual and if these ratios were then plotted on the abscissa
while the numbers of individuals having each of these ratios were
plotted on the ordinate. In computing the ratios the quotient was
carried to the second decimal place. But in the construction of the
curves only intervals of tenths (or first decimal place) were used;
thus, for example, all ratios occurring between 1.25 and 1.34 inclusive
were grouped as if they were 1.3. This had two advantages: first, it
produced a smoother, steeper curve than would result if smaller inter-
vals were taken, using the same number of individuals measured, and
emphasized group rather than individual variations. Second, this
grouping reduced any error which might result from the observer
showing a preference for one graduation of the micrometer when

1922] McDonald: On Balantidium coli and Balantidium suis 255

an individual measured more than one but less than another whole
division of the seale; e.g., such a preference might result in the tabu-
lation of several individuals having a length of 72, and of none with
a length of 71y, though in reality all lay within these two limits and
as many were as near to one as to the other.

As previously mentioned, the graduations along the ordinate
represent the number of individuals, each small interval representing
one individual. In Jennings’ (1908) work these intervals represent
percentages of the total number of individuals. But it happens that

Tele eoaweleoues doled Gh dz LS 9) 2t0) 2a 22) 283, 2.4, 25) 2:6

Fig. A. Graphie representation of the variation in the ratio of length to
breadth among 200 Balantidium chosen at random from samples of material taken
from several different pigs. The number of individuals is measured on the ordi-
nate, the ratios on the abscissa. The dotted line is the curve resulting from the
combination of the two curves shown in figure B, superimposed here to facilitate
comparison.

in the graphs shown in figures B and C, the number of individuals
showing a certain ratio is identical with the percentage of the total,
for in these cases the total is 100 individuals.

Figure A represents graphically the result of the first attempt
to determine the existence of different types. Measurements were
made of 200 individuals. At least ten slides were used in getting
these measurements and they were prepared from samples taken from
nearly as many different pigs. It will be noted that the curve pro-
duced by plotting the ratios of these individuals is decidedly bimodal.
One mode represents those individuals which are approximately 1.2
times as lone as wide, while the other represents those which are 1.6
to 1.8 times as long as wide.

These findings seemed to fully justify my early suspicions that
there were two very different types of balantidia parasitic in pigs.

256 University of California Publications in Zoology [Vou. 20

But it was decided to conduct one more experiment, for corroboration,
under slightly different conditions and with especial care in fixation
of the material. It had been noted that though often both types
oceurred in the same pig (which was the case in most of the samples
used in the first measurements), still one type might be greatly in
excess, or there might be only one type present. In getting material

Ved 12) AB 4 SRG 78) AG) 20) 25) eee 2 oe

Fig. B. Graphic representation of the difference in ratio of length to breadth
between two carefully selected lots, of 100 individuals each, of Balantidium from
separate hosts. The continuous line represents those from one pig (nearly all are
Balantidium coli) ; the broken line, those from the other pig (nearly pure infection
with Balantidium suis). ,

Fig. C. This graph shows the variation in the ratio of length to breadth among
100 Balantidium secured from a case of balantidiasis in man.

for this second set of measurements, it seemed best to take it from pigs
which had, as nearly as could be determined, pure infections of the
respective types. Fortunately these requirements were fulfilled in
the next lot of pigs examined. Both samples of material, the one con-
taining the ovoid and the one containing the elongate type, were
treated in exactly the same way. They were killed at the same time

1922] McDonald: On Balantidium coli and Balantidium suis 257

with the same fluid (in separate containers) and in the same water
bath. Thence to 70 per cent alcohol the treatment continued identical.
One hundred individuals from each sample of material were measured.

From these measurements the graphs shown in figure B were con-
structed in the same manner as the previous one, except that the
curves of the separate samples were plotted separately on the same
axis. The continuous line represents the individuals from one pig and
the broken line those from the other. The mode of the first curve
oceurs at 1.3. If the ratios 1.2 and 1.4 be included with 1.3, it is found
that 80 per cent came within these limits. Of the entire number of
individuals in the lot only one showed a ratio of 1.6 and one as high
as 1.7, while there were none with a higher ratio. The second curve
reaches its highest point at 2.0, while 71 per cent of the entire number
measured is included between 1.8 and 2.2. A number of individuals
have ratios above 2.2, while one had a ratio as great as 2.7. Five indi-
viduals have ratios below 1.6, at which point the curves begin to over-
lap; but in practically every one of these individuals there were
observed characters (which are discussed below) that made it quite
evident that they were really of the type represented by the other
curve.

Upon comparing figures A and B their likeness is very striking,
the second being corroborative of the results shown by the first. That
both are bimodal is evident. However, the low points, the point of
demarcation of the two groups, do not occur at the same place;
in the first it is at 1.4, while in the second it occurs at 1.6. Also the
median of the first mode in figure A occurs at 1.2 while in figure B
it is at 1.3, and the median of the second mode in figure A is at 1.7
while in figure B it occurs at 2.0; that is, in figure A the entire curve
is shifted to the left, meaning that all ratios are decreased or that all
individuals approach nearer to the spherical shape. This shifting is
greatest in the case of the second mode. This in conjunction with
the greatest breadth of the second curve in figure B is what would
be expected if the premise in regard to the effect of fixation discussed
above (page 251) is correct. Extra care was used in the fixation of
the latter lot of material whereas in the former only the ordinary pre-
cautions were taken. At any rate these differences between the two
groups do not detract from the evidence which they offer that there
are two distinct types of balantidia parasitic in pigs.

The value of these curves in showing race or species differentiation
is directly proportional to the extent to which any other factors which

258 University of California Publications in Zoology [Vou. 20

might produce a bimodal curve are non-operative. Factors involved
in faulty technique were eliminated as far as possible. Over the effect
of growth or age variation, however, the observer has no control. The
possibility of these variations producing such curves as the one above
is precluded by reference to the data from which the curves are con-
structed. Among the individual measurements recorded in Table I
it will be noted that there are small individuals measuring 27 X 50n,
and others measuring 42 & 50x; and that among the larger individ-
uals, some measure 50 X 120, and some, 90 X 150. Further study
of Table I shows that the two types are found among all sizes of
individuals; consequently the variation of body proportions repre-
sented by the graphs is not correlated with variations of size, and
probably not with the age or growth of the individuals.

That the variation could be accounted for by the occurrence of
fission seems unlikely. Individuals might continue to elongate until
binary fission occurred, and then by this process they might be
shortened and the body proportion changed. Two considerations
oppose this explanation. In figure A nearly equal numbers are of the
respective types; in figure B each example contained almost exclusively
one type of individual. In the former material very few dividing.
individuals were found, while in the latter not a single individual was
seen in fission in either sample of material. But to accord with the
above explanation one would expect to find many dividing forms
among the elongate individuals represented by the broken line. In
the second place, if this explanation were valid one would expect
curves showing variation of body proportions to be continuous. Such
is not the case, as is shown in figures A and B where the curve is
bimodal due to a decided decrease of individuals having proportions
intermediate between the two types.

The possibility of any effect from gametic variation, was eliminated
by the study of conjugating forms, through which it was determined
that isogamy was the rule. Likewise the possibility of influence of
the quality of intestinal content of the host was eliminated by the
frequent occurrence of both types in the same host. Other factors it
would seem must be of minor importance and should give way for
more positively corroborative evidence which may be found in cor-
related morphological difference.

Other specific characters.—In addition to the differences in relative
lengths of the axes, one notes a distinct difference in the points of
intersection, due to the variation in the shape of the types as pictured

1922] McDonald: On Balantidium coli and Balantidium suis 259

in figures D, HE, F, G, and H. The one form resembles very closely
a hen’s egg, the small end being anterior. In this case the longest
diameter crosses posterior to the midpoint of the longitudinal axis.
In the elongate type, usually the posterior end is as much drawn to

H

7,

Figs. D-H. Camera lucida drawings of Balantidiwm suis sp. nov. (figs. D
and E), and Balantidiwm coli (figs. F, G, and H), showing specific differences.

a point as is the anterior, and often more so. In these cases the longest
diameter intersects the longitudinal axis at or anterior to its midpoint.

Coincident with the taking of measurements a careful search was
made to detect other morphological differences which might occur
between the two forms and be of aid in distinguishing one from the
other. The earliest difference to be noted related to the macronucleus.
The macronucleus in the ovoid type is relatively short, being approxi-
mately 14 the length of the entire organism. It is customarily bean-
shaped in appearance, but may be almost straight or so sharply bent

260 University of California Publications in Zoology [Vou. 20

at its middle as to form a short V (fig. 1), and its width averages about
0.4 to 0.5 of its length. In the slender types the macronucleus is rela-
tively long and slender, being approximately 14 of the entire length
of the organism. It is ordinarily sausage-shaped, but it may also be
in the form of a straight rod shghtly enlarged toward the ends, or
it may be so curved as to form an almost complete ring. In contrast
to the form described above, the width in this case is about 0.2 to 0.3
of its length. Differences so great as these, viz., a length 2 to 3 times
the breadth in one case and 4 to 5 times the breadth in the other are
easily recognizable without actual measurement. These figures rep-
resent the average and do not mean that the limits of the two never
overlap. This difference in nuclei serves as one of the easiest and
surest ways of distinguishing the two types, for though the organism
during locomotion may modify its proportions tremendously this does
not noticeably affect the nucleus. It has been impossible to determine
any difference between the micronuclei of the two types.

A very noticeable difference concerns the relative position of the
cytostome. In the ovoid type the eytostome is almost, though never
quite, terminal (see figs. F, G, and H). As mentioned previously, the
anterior end is ordinarily drawn out to form a fairly decided point
which lies within the area enclosed by the adoral cilia. In the slender
type the eytostome is more laterally placed. The posterior limit of the
right lip of the eytostome may extend ventrally to a point 14 of the
length of the animal, in which case the dorsal portion of the adoral
ecirclet of cilia may pass approximately through the terminal point
of the body, but in normal form this point will never be within the
adoral area. The parts which make up the adoral region of the
animals show no fundamental differences except variation in the rela-
tive position of parts due to the lateral displacement of the eytostome.
For example, the plane of demarcation between ectoplasm and endo-
plasm, which is approximately vertical to the long axis of the animal
in the ovoid form, is at a decided angle, the ventral edge lying farther
posterior in the elongate forms (see figs. D and E).

In the opinion of some authors the ventral displacement of the
mouth is very significant (Delage and Hérouard, 1896; Minchin,
1912). These authors believe that the ventral displacement of the
eytostome is progressive with evolution of the organism; i.e., that in
the more primitive types the cytostome is terminal while in advanced
groups it is suecessively displaced farther ventrally. Viewed in this
light, the position of the mouth is here of considerable significance as

1922] McDonald: On Balantidium coli and Balantidium suis 261

a specific character. Attempts to discriminate between the two forms
on the basis of eytopyge or vacuoles were without result.

The specific differences just discussed have seemed to indicate a
sufficient degree of separation of the two types to warrant the division
of the ciliates of the genus Balantidiwm which occur in the pig into
two distinet species. The deseription by Malmsten (1857), in con-
junction with the figures (Malmsten, pl. 1, figs. 1-6) which he pub-
lished, make it practically certain that the ovoid type is the one
originally deseribed by him and to which he gave the name Para-
moecium (?) coli.

So far as I have been able to determine, the elongate species above
described has never before been distinguished from Balantidiwm coli.
For this new species I suggest the name Balantidium suis. As a sum-
mary of the specific characters discussed above I give the following
description :

Balantidiuwm swis sp. nov.—Body elongate; length approximately
twice the breadth and varies from 35 to 120; breadth from 20 to 60;
usually tapers more posteriorly, is blunter anteriorly, longest diameter
transects longitudinal axis anterior to its midpoint; adoral region ven-
trally placed, eytostome 15 of way posteriorly along ventral surface ;
nucleus rod or sausage-shaped, at least one-half the length of the entire
organism, its width about one-fourth of its length; the species is para-
sitic in the pig.

The specific name, Balantidium swis, seemed fitting since it indi-
cated the common host, Sus scrofa. Whether or not this species occurs
in man it has not been possible to determine conclusively. A review
of published case records of balantidiasis seems to show that it does
not, but only a few of these records are accompanied by figures or
descriptions of the organisms which are adequate for making positive
discrimination. Fortunately, I have been able in two cases to make
some direct observations.

Through the kindness of Mr. W. H. Barnes, of the Department of
Pathology of the University of California, I was permitted to study
sections of the human intestine which he had obtained at an autopsy
following a fatal attack of balantidiasis. Jmbedded in the serous and
subserous layers were numerous balantidia. Measurements to show
proportions were of little value under the conditions, for the form
of each organism was largely determined by pressure, exerted by sur-
rounding tissues. But from other characters, the type of nucleus
especially, it was conclusively determined that the species there present
was Balantidium coli. No individuals of Balantidiwm swis were found.

262 University of California Publications in Zoology [Vou. 20

Measurements of balantidia as they occur free in the human intes-
tine were made possible through the kindness of Dr. E. L. Walker,
of the Hooper Institute of Medical Research, who loaned me several
slides which he had prepared while in the Philippine Islands. This
material had been stained with haematoxylin. Using the same pre-
cautions as in previous work, a total of 100 individuals was measured.
The data were handled as before and the resulting graph is shown in
figure C. In comparison with previous graphs it will be noted that
this graph closely approaches coincidence with the curves representing
Balantidium coli, for its mode is at 1.3 while the extreme portion of
length and breadth is 1.5.

In addition to slides of human material, there was also loaned
material from one pig and from one monkey. It is interesting that
each showed a pure infection with Balantidiwm coli. The monkey
(Monkey No. 10, Table I; Walker, 1913) had been experimentally
infected by feeding it cysts from a pig, but not the pig from which
the above-mentioned material was taken. Therefore this material
yields no evidence regarding the validity of the specific differentiation
nor the possibility of Balantidium suis becoming established in mon-
keys or in man.

There is no likelihood of confusing the new species, Balantidium
suis with Balantidium minutwm (Schaudinn, 1899). The differences
are very marked. The body of the latter is oval, pointed anteriorly,
more like Balantidiwm coli. The peristome reaches to the equatorial
plane. There is but a single vacuole while there are two in each of
the species considered here. The macronucleus is spherical, whereas
it is elongate in both Balantidium coli and Balantidium suis.

MORPHOLOGY

Balantidium coli (Malmsten) and Balantidiuwm suis sp nov. are
ciliated protozoans, barely visible to the unaided eye, and are in a
general way sac-shape (balantidium, little bag). Viewed through the
microscope they appear grayish green in color. The homogeneity of
the cell contents is broken by the presence of the nuclei, the contractile
vacuoles, the food vacuoles, and sometimes by the presence of highly
refractile bodies, the paramylum bodies. The entire surface of the
body, except that of the oral plug, is covered with fine cilia. The cell
contents are retained by a thin transparent pellicle which is protective

1922] McDonald: On Balantidium coli and Balantidium swis 263

in function. The cytoplasm is distinctly differentiated into ectoplasm
and endoplasm. The former constitutes a thin layer just underneath
the pellicle and in it is situated the basal apparatus of the cilia. The
layer of ectoplasm thickens greatly at the anterior end of the animal,
to form, as it were, a matrix for the cytostome and its accessory appa-
ratus. Within the ectoplasm, but not set off from it by a sharp line
of demareation, is the endoplasm. In the endoplasm are numerous
food inclusions, often present in the form of starch or paramylum
bodies. The macronucleus and the micronucleus are also within it,
but seem to have no constant position in the cell. The macronucleus
is either bean-shaped (as in Balantidiwm coli) or elongate and sausage-
shaped (as in Balantidiwm suis). There are two contractile vacuoles,
the larger being situated anteriorly and the smaller posteriorly. They
he closely beneath or may be entirely surrounded by ectoplasm, thus
belonging really within that layer.

So far as can be determined the animals show no modification with
respect to a substratum, yet the lateral and posterior displacement of
the cytostome has lead to the designation of that side, toward which
displacement occurs, as ventral, and the opposite surface as dorsal.
This terminology is very nearly universal in the literature on Balan-
tidiwm, and the correlated terms of right and left are used in the
original description of the family Bursaridae (Stein, 1867) and the
genus Balantidium (Claparéde and Lachmann, 1858). The dorsal
side may be somewhat more convex than the ventral; this occurs not
infrequently in Balantidium swis, though, due to the plasticity of the
organism, this is by no means constant. No part of the body is differ-
entiated for skeletal purposes. The anal aperture or cytopyge is at
the posterior tip and may be present as an actual aperture or only
as an extreme thinning of the ectoplasm and pellicle at this pomt. In
connection with it there is usually a rectal vacuole which serves as a
storage reservoir for solid waste awaiting extrusion.

EcropLasMIc STRUCTURES

Pellicle—The entire body is covered by an extremely thin but
resistant pellicle (pel., figs. I and L). The pellicle seems to be some-
what thickened, as shown by a higher degree of refractibility, along
the margin of the lips of the eytostome where it turns in to form the
lining of the oesophagus and the groove in which the oral cilia are set ;
otherwise, it is nowhere noticeably specialized. It shows alternating

264 University of California Publications in Zoology (Vou. 20

Oa Cie ee Ok 1 ee ee oe Mee ae Cera te ae OBE
a cir. oes. f.
bg. ad. cil. _____---------------~--------------, | At alo th Sa st ot te or. pl. f.
ict ites eens ar ne
feecltat

a tea SNe

E memb
TOL ES) [ee Rea eer a é i ‘
< SN SSNS, moe.
enl. ad. cil. 7. —----------<3f64------- a \ ba
~$ \\
O(G acres SS ae ea) + By
a eI le ANN ee YS Rn gr.b

ant. ¢c. v.— =

mice membr.

Fig. I. Balantidiuwm coli. Ventral view showing principal structures. The
adoral cilia, with exception of one at either side, are indicated by basal granules
only. The adoral membranelles are also represented solely by the basal apparatus.
Ketoplasm is shaded, and ciliary rootlets are shown on one side only. X 1250.
ad. cil., adoral cilia; ad. cil. f., adoral ciliary fiber; ad. memb., adoral membranelles ;
ant. c. v., anterior contractile vacuole; bg., basal granule; bg. ad. cil., basal granules
of adoral cilia; cil., cilia; cil. r., ciliary rootlet; cir. oes. f., cireumoesophageal fiber ;
cytpg., eytopyge; ect., ectoplasm; enl. ad. cil. r., enlargement of adoral ciliary root-
let; end., endoplasm; gr. b., granular band of ectoplasm; long. f., longitudinal
fibers; mac., macronucleus; mic., micronucleus; mot., motorium; nue. memb.,
nuclear membrane; oes., oesophagus; or. pl., oral plug; or. pl. f., oral plug fibers;
pel., pellicle; per., margin of peristome; post. c.v., posterior contractile vacuole;
rad. f., radial fiber; rect. v., rectal vacuole.

1922] McDonald: On Balantidium coli and Balantidium suis 265

ridges and grooves, the cilia passing through the bottom of the latter,
but this condition is not due to longitudinal thickenings in the pellicle
itself, but to the fact that it is closely applied to the eetoplasm which
is thus furrowed. It can often be separated in ‘‘blisters’’ from the
ectoplasm by tannic acid or weak alcohol, and when thus removed it
shows regular longitudinal rows of perforations through which the
cilia pass out from the ectoplasm. In this condition its transparency
is very evident. The pellicle is not extremely impervious. For
instance, when the active animals are introduced into normal salt

ad. memb. —

CUR MOGSe finn eas
OTD Uh ayeetoe Na NE eee a once
DCT ngs Ge eS

Fig. J. The neuromotor apparatus of the adoral region, anterior view. X 2000.
ad. cil. f., adoral ciliary fiber; ad. memb., adoral membranelles; bg. ad. cil., basal
granule of adoral cilia; cir. oes. f., cireumoesophageal fiber; cytst., eytostome;
mot., motorium; or. pl., oral plug; or. pl. f., oral plug fibers; per., margin of peri-
stome.

solution plasmolysis takes place very quickly, and they present a
grotesque appearance as they swim about with several huge depres-
sions in their surfaces due to the shrinkage. Intra vitam stains such
as neutral red and Janus green also penetrate very quickly. Resist-
ance to pressure and mechanical change, however, is very marked, and
is due to its tenacity and flexibility, both of which qualities are shown
when the animal forces itself through an opening much smaller than
the normal diameter of its body (fig. K) and also by the extreme flat-
tening which it withstands under the increasing pressure of the cover-
glass when evaporation of the preparation is allowed. As previously
mentioned, these qualities indicate that the pellicle is protective and
retentive in function rather than supportive or skeletal.

266 University of Califorma Publications in Zoology [Vou. 20

These organisms show remarkable mobility when observed under
conditions as nearly normal as possible. I have attempted to depict
something of this plasticity in figure K. As they travel amid the
débris in the intestinal contents, which has been removed with
them, a tendeney to penetrate is much more noticeable than any
avoiding reaction. Instead of reversing the ciliary action, backing
away and taking a new direction as would paramaecia, the balantidia,
when they come in contact with a solid objeet, rather apply themselves
to the surface, round up, and seem to roll along it. After a moment
of such slow contortion, they may swim away in a new direction,

Fig. K. Diagrammatic illustration of the plasticity of the organism, resulting
in ability to pass through remarkably small openings.

determined by the direction of the anterior end. They avail them-
selves of the slightest opportunity to foree their way through or
between any obstacles. The anterior end, especially the thickened
ectoplasmie portion, becomes at such times decidedly elongate and
conical (fig. K, b). The cilia of this region beat spirally producing a
boring action as this anterior tip is protruded into any slight opening.
This action has in many instances been observed to cause two obstacles,
either of which was larger than the organism itself, to separate
sufficiently to allow it to pass between. The aperture need not be
one-half of the diameter of the animal for the latter will constrict
(fig. K, c) and the fluid contents flow through anteriorly as it
progresses, resembling the process of putting a bag of beans through
a small hole in a board. Throughout observations of the activity of
these organisms, one is impressed with their fitness for penetrating
the mucous lining of the intestine and the underlying tissues. Its
thigmotropie response, its boring action, and its extreme plasticity,
all seem to be adaptations for the function of penetration.
Ectoplasm.—Immediately underneath the pellicle, the eytoplasm
is differentiated to form the ectoplasm (ect., fig. I; pl. 27, fies. 1-7).
The ectoplasmie layer, except at the anterior end, does not exceed two

1922] McDonald: On Balantidiwm coli and Balantidium suis 267

microns in thickness. The anterior end of the animal, that is, all
anterior to a transverse plane which would transect the body at a
point 14; to YS of the way to the posterior end, is composed entirely
of ectoplasm. In this cone-shaped area is located the cytostome and
a large part of the neuromotor apparatus. The protoplasm of this
region seems to be homogeneously granular in fundamental structure.
It stains very deeply with haematoxylin; so deeply in fact, that in
differentiation it is necessary to destain other parts of the body almost
completely before this part reaches a degree of transparency suitable
for study. With Mallory’s connective tissue stain this region also
stains very densely, taking on both the brilliant red and the deep blue
elements of the stain, in different structures, as will be explained below.
This extensive thickening of the ectoplasm at the anterior end is clearly
shown in the figures by Leuckart (1861) and has been noted by nearly
all who have studied the animal more recently, but I have failed to
find any discussion of its significance. This same phenomenon occurs
in the Ophyroscolecidae, as pointed out by Sharp (1914) in his work
on Diplodinium and by Braune (1913) in Ophyroscolex. In these
cases the change seems to be correlated with the high degree of activity
and specialization of the anterior end of the animal; in Diplodinium
in connection with its selective feeding, and in Balantidium in con-
nection with both feeding and activity of this entire region in pene-
trating the mucosa of the intestine. The centering of the neuromotor
apparatus in this region gives additional evidence in regard to this
question which will be diseussed further in connection with the de-
seription of that apparatus.

Throughout the entire investigation of the minute structure of this
animal, I have been unable to demonstrate the presence of any definite
plane of demarcation between endoplasm and ectoplasm, such as the
‘“eetoplasmie boundary layer’’ described by Sharp (1914) in Diplo-
diniwm, and shown in plate IV, figure 3 of his paper. Many of the
fixed preparations used in the search for such a layer were sections
of the animal, treated as nearly as possible according to the technique
used by him and stained, as were his preparations, with Mallory’s con-
nective tissue stain. So far as it was possible for me to determine,
any sharp boundary line between ecto- and endoplasm is lacking. On
the contrary, they merge into one another and only in a general way
can it be said where one terminates and the other begins.

Prowazek (1913, fig. 2) describes, in Balantidiwm coli, ‘‘eim Art
von Zwischenmembran’’ which appears as wavy lines in optical sec-

268 University of California Publications in Zoology [Vou. 20

5)

tion. According to his interpretation this ‘‘Querlinie’’ separates the
protoplasm of the cell body into two regions, the ‘‘apical zone,’’ which
I have described above as a thickening of the ectoplasm, and the rest
of the cell protoplasm. The extent of this ‘‘Zwischenmembran’’ he
does not note, but his text figures (1 and 2) do not show it as extending
quite to the pellicle, but instead as stopping short of the pellicle at a
distance about equivalent to the thickness of the ectoplasm at that
point. This is significant in my interpretation of this region, namely,
that what appears as a continuous line or plane when viewed from
the side is in reality, as shown in cross-sections (pl. 27, figs. 6 and 7),
a set of diverging fibers. These fibers take origin from dark-staining

“ye

Fig. L. Portion of the peripheral region of a cross-section of Balantidium coli,
showing the structure of the ectoplasm and arrangement of cilia, somewhat dia-
grammatic. X 1500. b.g., basal granule; cil., body-cilia; cil. r., ciliary rootlet;
end., endoplasm; gr. b., granular band of ectoplasm; hy. b., hyaline band of ecto-
plasm; pel., pellicle.

enlargements on the longitudinal fibers in the wall of the gullet and,
diverging, pass peripherally until they turn posteriorly at the very
inner edge of the thin layer of ectoplasm which covers the remainder
of the body (pl. 28, figs. 9-12). Even in lateral view careful focusing
will often show that the apparent ‘‘membrane”’ is really discontinuous,
showing breaks and irregularities as one focuses on different levels
and hence can not be considered as a true membrane. The arrange-
ment of these fibers will be deseribed more exactly under the discussion
of the neuromotor apparatus.

The ectoplasm which constitutes a layer less than 8 microns in
thickness around the remainder of the periphery of the cell shows
a definite and somewhat complex structure. In tangential sections
of the surface, which are so thin that they do not include much of the
underlying endoplasm, one detects alternating light and dark longi-
tudinal spiral bands (gr.b., hy.b., fig. N). These bands are parallel
to the rows of cilia and very nearly equal in width. In cross-sections
(fig. L) they are seen to extend nearly, if not quite the full depth of

1922] McDonald: On Balantidium coli and Balantidium swis 269

the ectoplasm. As one follows these bands (or ‘‘stripes,’’ as they are
named by Johnson (1893, in his work on Stentor) anteriorly they seem
to lose their distinctness when they become continuous with the apical
cone. In some individuals, however, one can follow them some distance
into this cone, but never is there the same degree of differentiation of
the two areas in this region.

In the living animals, which are often quite opaque due to inelu-
sions, it is nearly impossible to distinguish these longitudinal light and
dark bands. With neutral red the dark or granular band stains
faintly. With the haematoxylin stains used in thin sections of fixed
material the dark band seemed to be finely granular in fundamental
structure. The granularity in this case must be determined largely
by the general appearance and stainability, for the individual granules
are so small as to defy identification. There is no indication, however,
of alveolar structure, so that the term granular is probably the more
applicable and will be used to distinguish this from the hght band.
The latter takes only faintly the stains used and seems hyaline in
structure. The granular bands lie directly beneath the ridges in the
cuticle which occur between the rows of cilia. Or more correctly,
the ridges on the surface of the animal are produced by the projection
of these granular bands outwardly beyond the hyaline bands. These
latter are directly beneath the grooves of the surface where the cilia
pass through’ the cuticle and attach with the basal granules. which he
in longitudinal rows; a single row in each hyaline band. The ciliary
rootlets (cil. r., figs. I and L) extending in from the basal granules
proceed diagonally inward and pass into the interior margin of the
granular band.

Stein as early as 1876 pointed out these alternating dark and light
stripes in Stentor. To the granular and bright stripes, Biitschl
(1889) gave the names ‘‘Riffenstreifen’’ and ‘‘Zwischenstreifen,’’
respectively. Johnson (1893) gives a careful description of these
bands as they occur in Stentor coeruleus. He notes that they vary
greatly in width, and this is true in Balantidiwm; but both bands are
much narrower than in Stentor, the combined width of the two not
exceeding two microns. In Stentor coeruleus Johnson (1893) gives
the width of the granular band as 22 and that of the bright band
as 7, these measurements being taken just under the adoral zone. It
is interesting to note that in Balantidiwm coli the granular band is also
slightly wider than the bright band. These bands become narrower
from the region of the greatest circumference of the animals toward

270 University of California Publications in Zoology [Vou. 20

each end, which is comparable with the arrangement in Stentor,
though in the latter the narrowing must necessarily take place in the
posterior direction only. The above author mentions the branching
of stripes, but this does not occur in Balantidiuwm so far as I have been
able to determine. As the bands pass posteriorly, however, they
become less distinctly differentiated and are hard to follow, and it
might be that further study with more intensive stains would reveal
a union in the region of convergence at the posterior end. More recent
work has added to the number of Heterotricha that show this sort of
differentiation of ectoplasm. Maier (1903) shows the striped nature
of this layer in Prorodon and Spirostomwm, while Neresheimer (1903)
confirms the structure found by Johnson (1893) in Stentor. Schuberg
(1887) also indicates a comparable plan of structure in Bursaria.
The granular ridges of ectoplasm between the furrows in which the
anal cirri are situated in Euplotes patella, discovered by Yocom
(1918), may be comparable with the bands which oceur in Hetero-
tricha.

Cilia.—The entire surface of Balantidium coli, with the exception
of the oral plug, is thickly beset with cilia (cil., ador. c., fig. I). These
are of two kinds, viz., those which make up the adoral row of cilia
and which measure from 8 to 12” in length, and those covering the
body, which vary from 4 to 64. Those covering the apical cone, form
an intergradation between the two. On this surface the cilia which
occur immediately posterior to the adoral row are only slightly shorter
and slightly more slender than the adoral cilia themselves. Passing
posteriorly they gradually become shorter and less cirrus-like until
they reach the base of the apical cone. Thence posteriorly they retain
the uniform size.

The body cilia are comparatively short and very slender. So small
are they in fact that to observe a single one is nearly impossible. In
slides prepared by the usual methods no stain remains in the cilia if
destaining is carried sufficiently far to differentiate other structures.
Iodine (Weigert’s solution) gives a fairly satisfactory stain for tem-
porary mounts. The arrangement of cilia may be determined most
readily by using a heavier stain and then observing the distribution
of basal granules. Neutral red proves very satisfactory for this pur-
pose. The cilia oceur in longitudinal, slightly spiral rows, following
the grooves between the ridges in the pellicle. These rows originate
immediately posterior to the groove in which the adoral cilia are set
and for a very short distance pass almost meridionally; very soon,

1922] McDonald: On Balantidium coli and Balantidium suis 271

however, they turn toward the left, that is, in a counter-clockwise
direction when the animal is viewed from a point exactly in front.
They continue their spiral direction until almost to the posterior end
when they again follow a meridional path to their termination. In
passing the entire length of the body any single row of cilia twists to
the left approximately 120°, or one-third the entire circumference.
Whether some rows terminate or become continuous with contiguous
rows before reaching the posterior tip of the animal, I have been
unable to determine, for both basal granules and granular bands
become very indistinct in this region even in the best preparations.
The number of rows was counted with difficulty in several cross-
sections from the equatorial regions of different animals, and it was
found to vary from about 60 in small individuals to 120 in larger ones.
No correlation between the variation in the number of rows of cilia
and the species of the animal could be determined, but this is possibly
due to the limitation of observation.

Basal apparatus—The cilia perforate the pellicle and attach to
the basal granules which he immediately underneath (fig. L). The
latter are small and apparently spherical or oval. They stain very
deeply black or blue with haematoxylin. In the living animal they
are readily emphasized by the use of neutral red, and less so by Janus
green. In preparations stained with Mallory’s connective tissue stain
these granules show brilliantly red with the acid fuchsin, as do the
other parts of the neuromotor apparatus and also the micronucleus.
Longitudinally the granules are so closely placed that it is impossible
to observe whether they are actually connected by a fibre. Cross-
sections show that the cilia of one row have no transverse connection
by any sort of stainable fiber with those of the next row. The rows
of basal granules lie close beneath the depression in the pellicle in
the hyaline or bright band of the ectoplasm. A ciliary rootlet (cil. r.,
figs. I and L) extends from each basal granule centrally toward the
endoplasm. It does not proceed in an exact radial line but rather
diagonally toward the right until it enters the granular band near
the inner surface of the latter. The ciliary rootlets in some cross-
sections appear to have an exactly radial direction. In such cases,
however, the granular band is somewhat diagonal in the opposite
direction. This variation is probably produced either by torsion of
the animal or by the direction of the effective beat of the cilia at the
instant of fixation. The diagonal direction of the rootlets is readily
detected in tangential sections. In focusing down through such a see-

272 University of California Publications in Zoology [Vou. 20

tion, the rootlet is invariably seen to run from the basal granule
toward the observer’s right into the contiguous granular band on that
side. Thus, to avoid any confusion that might arise from the terms
’ right and left, in a cross-section of the animal viewed from the anterior
surface (such a view is shown in fig. Li) the ciliary rootlets swerve in
the counter-clockwise direction and enter the granular band lying
immediately in that direction.

As the rootlet enters into the granular band it apparently enlarges
thus forming a secondary basal granule. In some eases this may stain
even more deeply than the basal granule itself, and appear as a definite
body somewhat elongated in the direction of the circumference of the
animal. It was thought at first that this might be the cross-section of
a longitudinal fiber or myoneme. But the study of numerous tan-
gential sections has failed to show the presence of any longitudinal
fiber within the granular band. The stainability varies greatly and
in preparations which have stained lightly the ciliary rootlets appear
to fray out and merge into the granular band, while still retaming
deeper color than the rest. Which interpretation is correct it is
difficult to say, but the latter seems the more probable, especially in
view of certain relations with the neuromotor apparatus which will
be discussed later. f

Piitter (1903) reproduces a figure from Studnicka (1899) showing
in a schematic way five types of attachment of the cilia with their
basal apparatus. Of these, two, at least, represent cases in which two
basal granules or a diplosome are present. Saguchi (1917) in his
studies on ciliated cells of Metazoa says in part regarding the basal
granules of certain ciliated cells from amphibian larvae, ‘‘ With favor-
able staining the basal corpuscles appear as diplosome or dumbbell
. Shaped granules. One of these is situated at the upper the other at
the lower border of the euticle.’’ In Balantidiwm coli the arrange-
ment with respect to protoplasmic layers is quite different, though
the cilia seem to follow somewhat the same plan of structure even to
the presence of rootlets.

The most fruitful comparison may be made with the basal appa-
ratus of cilia in Jsotricha prostoma as described and pictured by
Braune (1913). In this organism he deseribes diplosomie structure
of the basal apparatus, in which the basal granule lies directly beneath
the pellicle. The cilia, however, extend beyond the basal granule into
the underlying layer—the ‘‘Zwischenschicht’’ of Eberlein, and ter-
minate in the ‘‘Grenzschiecht’’ with a second granule, which upon

1922] McDonald: On Balantidium coli and Balantidium suis 273

maceration remains attached to the basal end of the cilium. So the
basal apparatus in Balantidiwm coli even to the relative location of
the basal granules is almost identical with that in Jsotricha prostoma.
In Balantidiwm, however, as mentioned above, the ‘‘Grenzschicht’’
seems to be lacking. The comparison becomes more significant in view
of the fact that both ciliates are parasitic in the digestive tract of
mammals, and both are in much the same state with reference to
the degree of specialization and degeneration correlated with habits
of the parasitic mode of life. So that in general morphology they
seem to be more alike, though they are in separate orders, than do
Diplodinium ecaudatum and Balantidiwm coli, which are in separate
‘suborders only.

Ciliary Movements.

Locomotion is the chief function of the cilia except for those of
the adoral zone. The balantidia swim in approximately a straight
line and not in a spiral course as do paramoecia. They do, however,
rotate on their axis as they progress. This rotation is generally from
left to right, that is, in a counter-clockwise direction when viewed from
a point in front of the animal. A few instances of reversal of the
direction have been seen, but it is not at all common. The direction
of rotation, i.e., from left to right, seemed at first inexplicable, since
this was not compatible with the direction of the rows of cilia. The
rows of cilia, as described above, are comparable to the threads of a
left-hand screw. In order to penetrate, such a screw must be turned
in a clockwise direction (when viewed from the point, not from the
head). Such, also, is the direction of rotation of balantidia which one
would expect to find if the arrangement of the cilia were the con-
trolling factor, but the rotation is in the reverse direction. In the
further study of the problem, I fortunately obtaimed some very thin
tangential sections of animals.on which the fixative had acted so
quickly that the cilia were stopped instantly and left in the relative
positions assumed in normal ciliary action.

Figure N is a camera lucida drawing of such a section. By analysis
of the position of the cilia on this and other like sections, it was
possible to determine the complete cycle of a single cilium. This cycle
is diagrammatically represented in figures M and O. Figure N
includes about two and one-half cycles of action as represented by
the waves. The dark portions are produced by the prostrate position
of the cilia at the termination of the effective stroke. The lighter

274 University of California Publications in Zoology [Vou. 20

areas between are due to the fact that the cilia are recovering their
vertical position and hence are viewed very nearly endwise. The
position of consecutive cilia in any single row, from one point in a
wave to a similar point in a following wave will fairly represent the
suecessive positions taken by a single ciliuam in making one complete
eyele. Figure M was made in this way. The arrow represents the
long axis of the animal. From this diagram it is seen that the cilium
at the end of the effective stroke lies rather close to the surface of
the body and not along the row but decidedly to the left from it. In

Fig. M Fig. N Fig. O

Fig. M. Diagrammatic representation of the successive steps in one complete
beat of a cilium of Balantidium coli. It also illustrates the positions of the respec-
tive cilia of a single row between the points a and b in fig. M, at which points the
cilia are in a prostrate position at the end of their effective stroke. The arrow
indicates the long axis of the animal.

Fig. N. Tangential section of Balantidium coli. The cilia still retain respec-
tive positions which they had in the normal swimming movements of the organism.
x 1500.

Fig. O. Diagram illustrating the effect of the ciliary action in the rotation of
the organism. The arrow represents the long axis of the animal; ab., the direction
of the rows of cilia; ed., the direction of effective stroke of a cilium attached
at the point of intersection of the three lines; the ellipse is described by the tip
of the cilium.

recovery it straightens up and passes anteriorly, thence to the right,
crossing the row, of which it is a part, at right angles. At this point
the cilium leans anteriorly only slightly from the vertical. The
effective beat is produced by the quick stroke of the cilium posteriorly
and to the left, and continues until the cilium has crossed the row again
and les close to the surface and extends to the left as represented
by the position of the last cilium shown in figure M. According to
the classification given by Piitter (1903), the movement of the cilia
of Balantidiwm would be called infundibular. As will be noted from

1922] McDonald: On Balantidium coli and Balantidium suis 275

figure O, the funnel described by the complete beat of the cilium
is somewhat irregular, the rim outlined by the tip of the cilium is
elliptical, and the base of the cilium, i.e., the neck of the funnel, is
not central but is situated below the anterior focus of the ellipse.
The ellipse described by the tip of the cilium lies in a plane which is
not parallel to the surface of the animal, but which approaches it
much more closely posteriorly. It was possible to corroborate the
action of the cilia in observations on the living material. The cilia of
the apical cone are somewhat larger than the other cilia of the body,
and are closely codrdinated in their action. In balantidia which were
allowed to cool until action had slackened considerably, these cilia
were observed to make their effective stroke at a decided angle to the
rows of cilia. For instance, in the boring action in connection with
the process of penetration described above, these cilia will beat in an
almost exactly transverse direction, always from right to left. In
further corroboration of this interpretation of the movements of the
cilia is the fact that it explains the rotation of the animal during
progression. It will be seen from figure O that the effective stroke
of any single cilium will be in the direction cd. This line crosses both
the arrow, representing the long axis of the animal, and the line ab,
representing the rows of cilia, making an angle of approximately
20° with the latter. That is, the line cd forms an angle with the arrow
on one side about equal to the angle on the opposite side made by the
line ab. It clearly follows that if the effective stroke is in the direction
ed then rotation will be from left to right and not vice versa as would
be the case if the cilia beat in the direction of the rows in which they
are arranged. The structure of the basal apparatus is significant in
view of the direction of the effective stroke of the cilia, viz., posteriorly
and to the left. The cross-section shown in figure L is viewed anter-
iorly and shows that the ciliary rootlet from each basal granule passes
to the right and enters the granular band on that side of the hyaline
band in which the basal granule lies. Without giving to the ciliary
rootlets any motor or skeletal function, it still seems logical that they
conform in a general way to the axis of the cilium, for the latter during
the greater part of its movement inclines to the posterior and left of
the row of which it is a constituent.

The peristome (per. fig. J) may be said to comprise all that part
of the organism which lies within the row of adoral cilia. In the
active animal it is variable in shape. At times it is almost round
while at other times it may become a mere slit or groove. In what

276 University of California Publications in Zoology [Vou. 20

seems to be its more normal proportions, however, it is approximately
pear-shaped with the stem end of the pear directed ventrally. In
Balantidium coli the adoral zone commonly includes the most anterior
point or apex of the animal—at least its dorsal margin passes through
this point. In Balantidium suis, the anterior tip of the animal hes
wholly outside of this zone, the latter having migrated too far ventrally
to inelude it.

The cytostome (cytst., fig. J) does not occupy the whole interior
of the peristome, but is situated at its ventral end. There is some
indication that this aperture may be completely closed by the oral
plug (or. pl., fig. 1) which comprises the rest of the peristome within
the adoral row of cilia. This oral plug bears no cilia. It is exceed-
ingly mobile, adapting itself readily to the almost constantly changing
shape of the apical cone. It lies dorsal to the eytostome and is not
exactly bilaterally symmetrical since it is pushed somewhat to the left
to make room for the oesophagus. It extends inward, thinning as
it does so, until it terminates about the beginning of the endoplasm.
It is ectoplasmic, but very finely granular as compared with the rest
of the ectoplasm. Mallory’s connective tissue stain ordinarily gives
it a decidedly bluish tinge with slight spots of red only where there
are certain neuromotor fibers. Its action in feeding is very hard to
follow, but its high degree of mobility impresses one when watching
the activities of the organism, and may be demonstrated with fixed
material by its extreme protrusion (pl. 28, fig. 14). In addition to
this, the fact that it is mtimately connected with the neuromotor
apparatus would indicate that it functions in selective feeding. The
oesophagus (ves., fig. I; pl. 27, figs. 4-8) beginning at the cytostome,
passes inwardly, not quite radially but swerving slightly to the right.
It may be followed definitely through the ectoplasm and for a very
short way into the endoplasm where it ends blindly. So far as I
have been able to determine, it is a uniform tube-like opening without
evident enlargements or constrictions. Prowazek (1913), however,
ne . es sehnt sich jedoch
nicht direct trichterformig im die Tiefe, da man von der drei scharfe

gives in part the following description,

Konturen noch nachweisen kann (fig. 1).”’ While studying living
forms stained with neutral red, I have often observed specimens in
the exact position of the one shown by Prowazek (1913, p. 7, text
fic. 1). The lines shown by him were easily recognizable, deeply
stained with the neutral red, but I could interpret them only in the
following way. The most anterior line which he shows seems to be

1922] MeDonald: On Balantidium coli and Balantidium suis 277

clearly the ventral lip of the peristome; the second line was identical
with the deep staining motorium (fig. I) in the forms which I studied,
and the most posterior line seemed identical with the ring of enlarge-
ments on the rootlets of the adoral cilia which he close about the
oesophagus. The margin of the peristome is slightly raised, forming
a ridge or lip. This ridge is most pronounced from the midventral
point, dorsal along the right margin of the cytostome, but as it pro-
ceeds around it becomes rather inconspicuous and wholly disappears
on the left side. Thus the cytostome has the appearance of opening
under a ledge, the ledge formed by the lip of the right side.

The peristome is delimited by an almost complete spiral circlet of
cilia, the adoral ciha (ad. cil., fig. [). The exact point of origin of
the row is hard to determine but it is approximately at the ventral
edge of the peristome, i.e., on the ventral lip of the cytostome. From
here it proceeds in a sort of groove, along the right margin, around
the dorsal margin, and down the left margin of the peristome. A
short distance in advance of the ventral point, the row of adoral cilia
turns into the cytostome and continues in a spiral course down the
oesophagus; entering at the left dorsal side and ending in the ventral
wall about halfway down. Thus these cilia in their entire course make
one complete left-hand spiral, beginning on the ventral lip, passing
around the peristome and down the oesophagus, terminating in its
ventral wall.

Over the lip of the eytostome between the poimt where the adoral
cilia turn into the oesophagus and the midventral point where this
row of cilia has its origin, the longitudinal rows of body cilia turn in.
Each of these rows, of which there are ten or twelve altogether, con-
tinues down the oesophagus until it meets the row of adoral cilia.
Since this latter enters spirally, there is a ciliated patch on the ventral
wall of the funnel-shaped oesophagus which is roughly the shape of a
right triangle, the base of which is the lip of the cytostome. The
hypotenuse is the row of adoral cilia which makes an acute angle with
the longitudinal row of body cilia which enters in the mid line and
which represents the third side of the triangle.

The adoral cilia, except where they lie within the oesophagus, are
completely separate each from the other. This is easily verified by
watching their action especially in a disintegrating animal, or one
cooled to slow up ciliary movement, under which condition codrdina-
tion is frequently interrupted, and a single cilium will be seen acting
independently of its neighbor. Additional evidence is to be found in

278 University of California Publications in Zoology [Vou. 20

the individuality of each basal granule. There is no evidence of fusion
of granules, though adjoining granules are connected by a neuromotor
fiber. However, in the triangular area mentioned above, there is con-
siderable evidence that the cilia are united to form membranelles. It
is very difficult to watch the action of these cilia, and the above con-
clusion was reached largely through a study of fixed material. In
cross-sections (pl. 27, fig. 4) the cilia of the region seem to be very
close together and quite regularly to be connected by, or to form, a
sheet of almost transparent substance. The regularity of this occur-
rence would lead one to believe that it is the normal structure and not
due to entanglement of the cilia in foreign matter. In addition, the
basal granules of this region (pl. 27, figs. 4 and 5) do not stand out
separately, but are so closely packed that they give the appearance
of a single deeply stained mass. I have been unable to distinguish
separate granules in the inner portion of the area and it seems likely
that actual fusion of the granules may have oceurred. The basal
apparatus of this region is so densely packed and takes stain so readily
that in certain views it may easily be mistaken for the motorium. As
a result, it seems plausible to interpret this area as the basal apparatus
of membranelles which lie in a plane transverse to the axis of the
oesophagus. If this region be interpreted as a primitive oral groove
or cytostome, a forerunner of such an elaborate arrangement as occurs
in Huplotes patella (Yocum, 1918) or in Stentor, then comparison is
very significant, for in the latter two the membranelles also run trans-
versely in the eytostome. The only difference, then, between these
organisms and Balantidiwm in this regard would be the difference in
shape and extent of the area.

The adoral cilia (ad. eil., fig. 1) are approximately double the
length of the body cilia, i.e., from 6 to 84 in length or, in the very
large individuals, they may reach a maximum length of 10y. In
fundamental structure they are like the body cilia, but the relative
position of parts is somewhat different. Immediately beneath the
pellicle each cilium bears a basal granule and from this a fiber con-
tinues inward which passes between the adoral plug and the surround-
ing ectoplasm. The sum of all the fibers from adoral cilia marks off
very distinetly the conical eytostomal region from the surrounding
ectoplasm. They seem to constitute the only partition between the
protoplasm of the two areas, for I have not been able to detect any
membrane in this region making a complete separation of the adoral
region from the rest of the apical cone. Where the fibers pass from

1922] McDonald: On Balantidium coli and Balantidium suis 279

the ectoplasm of this cone into the endoplasm, each bears an enlarge-
ment which stains very deeply. This enlargement is undoubtedly
homologous with the inner enlargements of the basal apparatus of
the body cilia for it bears the same relation to the basal granule and
the cilium. It is farther removed from the basal granule but it main-
tains exactly the same relation to the ectoplasm and endoplasm, that
is to say, in either case this enlargement lies in the plane between the
two. From each enlargement a radial fiber extends laterally along
the base of the apical cone to the periphery of the cell. The portion
of the ciliary rootlet which continues inward from this enlargement is
very distinct and may often be traced almost to the posterior end of
the animal (fig. 1). In no instance has it been possible to demonstrate
any attachment or connection of the posterior ends of these rootlets.
They often cross near the center of the organism and then fade out,
becoming indistinguishable in the endoplasm. For a short distance
inward from the oesophagus they are so closely placed dorsally and
laterally that they form a kind of wall, but ventrally they are less
numerous and likely to be much shorter, so that the pseudo-wall is
not complete. It is sufficient, however, to direct the food for some
distance into the endoplasm. There is no such high degree of differ-
entiation here as Sharp (1914) found in the oesophagus of Diplo-
dinium ecaudatum.

The homology of the adoral and body cilia is evidenced by the com-
plete series of gradations from the one to the other which exist in the
cilia of the apical cone (fig. I). The cilia which are proximal to the
adoral cilia are almost identical with them. They are slightly smaller,
but each has the basal granule beneath the pellicle, a fiber connecting
this with an enlargement at the plane of differentiation of ectoplasm
from endoplasm, and the rootlet extending posteriorly. The inner en-
largement is much smaller and the ciliary rootlet shorter than those of
the adoral cilia. These enlargements are connected with the enlarge-
ments of the adoral cilia by the radial fibers mentioned above. Passing
posteriorly the cilia become progressively smaller, the basal granule
and the enlargement closer together (due to the approach of the trans-
verse plane of demarcation between ectoplasm and endoplasm to the
pellicle), and the ciliary rootlets become progressively shorter and less
distinct, until the cilia of the marginal area of the apical cone become
identical with the body cilia. This apical cone then shows us a com-
plete gradation of differentiation between the body cilia and the adoral
cilia, and proves their homology.

280 University of California Publications in Zoology [Vow. 20

Feeding is the chief function of the adoral cilia. They are closely
coordinated in action, as is shown by the fact that in degenerating in-
dividuals they will beat in unison after other cilia beat only erratically
or have ceased entirely. Codrdination is most pronounced in the cilia
of the left lp. The action of the adoral cilia produces a strong eddy
in the surrounding medium with the vortex of the eddy within the
peristome. In the above description it was noted that the right lip
projected shghtly forming a ledge or bank. The current produced
by the cilia strikes against this ledge and solid objects are deflected
into the underlying cytostome. The natural rotation of the animal
on its longitudinal axis as described above aids in this process, for the
right lip thus acts as the blade of an auger. The food particles pass
down the oesophagus and collect at the inner end in a sort of droplet.
After several bits have been collected, the droplet begins its circulation
in the endoplasm as a food vacuole.

The closure of the cytostome in all probability is effected by the
oral plug. The latter is very mobile and contains fibers of the neuro-
motor apparatus. It has frequently been seen to project anteriorly
in a knoblike protrusion (pl. 28, fig. 14). This same phenomenon was
observed by Wising (1871). In view of its situation, its sensitivity,
and its mobility, it seems plausible to interpret it as an oral plug
with essentially the same function as the oral disk of Diplodinium
ecaudatum (Sharp, 1914).

The discharge of indigestible portions of the food takes place at
a constant point at the posterior end of the animal, the eytopyge (cyt.,
fig I). It is an opening, however, only at the time of discharge.
At other times there is only a thinning of the cortical layer which can
be identified with comparative ease in fixed material. In the living
organism the process of defecation was frequently observed. The
undigested particles become segregated at the extreme posterior end
in a sort of vacuole, the rectal vacuole (ret. v., fig. 1; pl. 28, fig. 13).
After this vacuole has become of considerable size (often filling one-
third of the cell in degenerating forms) the pellicle over the cytopyge
ruptures and the contents are discharged. The pellicle quickly forms
again closing the aperture, collection of indigestible particles in the
rectal vacuole continues, and the process is repeated.

The contractile vacuoles (post.c.v., ant.c.v., fig. 1) are two in
number, usually situated on the dorsal side, one anteriorly, well up
toward the apical cone, the other in the posterior one-third of the
organism. There is a great deal of variation in their location in
different animals, though in the individual their situation seems to be

1922] McDonald: On Balantidium coli and Balantidium suis 281

very constant, at least throughout the limit of possible observation.
These vacuoles seem clearly to originate within the’ ectoplasm. When
fully distended they encroach far upon the region of the endoplasm
and it becomes impossible to tell whether or not they are entirely sur-
rounded by ectoplasm; but from their origin such might be suspected
to be the case. Also such is the case in many related forms, for
example, Diplodimum ecaudatum (Sharp, 1914) and Huplotes patella
(Yocom, 1918).

The pulsation of the vacuoles was observed in several instances
for a long period of time. The rate of pulsation varies considerably,
occurring as rapidly as once in every thirty seconds under some
conditions, while under others a complete cycle from discharge to
discharge occupies a period of five minutes. In degeneration the
pulsation is likely to be very much retarded or may cease entirely,
the vacuoles becoming enormously distended and breaking together
thus forming one large vacuole occupying fully one-half of the interior
of the organism. Following this the animal ruptures and disintegrates.
The observation of a considerable number of normal individuals has
shown the usual cycle to be as follows. At two points in the ectoplasm
small droplets of clear liquid appear. These increase in size and
become the vacuoles usually seen. Contributing vacuoles or channels
such as oceur in Paramaecium have never been noted. When they have
reached sufficient size (10u to 152 in diameter in the ordinary indi-
vidual), they change from their spherical shape and begin to bulge,
each on the side toward the other. These bulges elongate until they
meet at a midpoint. At this midpoint a new vacuole arises and into
it, through the channels thus formed, the two vacuoles discharge their
contents. This large middle vacuole almost immediately discharges
to the exterior through the pellicle, and at the same time the other
two vacuoles re-form. However, the discharge of the middle vacuole
may be delayed until the other two are well formed and then the
individual has three vacuoles present. This and other variations are
not uncommon and should be taken into account in the use of the
number of vacuoles as a basis for classification. Leuckart (1861)
deseribed a third contractile vacuole though he did not give its rela-
tive position to the other two and-he did not describe the process of
drop-

cc

contraction. He reported that he had observed the vacuoles
like’’ through the cytoplasm and wandering from place to place.
Solojew (1901) described the two vacuoles and observed a canal con-
necting the two, but he did not explain its function in discharge of
the contents.

282 University of California Publications in Zoology [Vou. 20

ENDOPLASMIC STRUCTURES

Endoplasm.—Within the ectoplasm the body is composed of the
endoplasm (end., pl. 27, figs. 7 and 8; pl. 28, figs. 9 to 12) and the
inclusions therein. The endoplasm is less dense than the ectoplasm
and more coarsely granular. It is quite fluid, having a fairly definite
circulation in the active organism. From the inner end of the
oesophagus the direction of flow is posteriorly along the ventral sur-
face dorsalward just before reaching the posterior end, thence an-
teriorly along the dorsal surface. Just as it reaches the ectoplasm
of the apical cone, it is deflected posteriorly down through the central
portion of the body dorsal to the rootlets of the adoral cilia. This
course can be followed readily by observing the circulation of the food
vacuoles. :

The food vacuoles are customarily globular and may contain starch
granules, bacteria, or indigestible particles, of the food of the host.
The ingestion of bacteria is probably abnormal since it seldom oceurs
except when the number of bacteria in the medium has increased
greatly during incubation. The starch granules may occur in enor-
mous numbers especially when the host has recently been fed on grain.
Red blood cells have not been noted in any of the balantidia observed -
during the work.

That Balantidium may be cannibalistic is the only possible in-
terpretation of several findings. In these instances small individuals
were lying within the endoplasm of extremely large individuals
(100 < 125.) and were in a state of disintegration. The larger indi-
viduals were Balantidiwm coli in every case and the smaller may have
been Balantidiwm swis, for both species were present in the material ;
but disintegration of the latter had progressed so far that specific
identification was uncertain. The possibility of interpreting this
phenomena as sporulation, which was described by Walker (1909),
is precluded by the disintegration of the included organism and the
fact that the normal vegetative phase of the nuclei of the large indi-
vidual is unmodified. Further evidence is to be found in the fact
that never more-than a single individual has been found inside
another, while sporulation would produce several. ‘

Macronucleus.—As is generally the ease in ciliates, balantidia are
binucleate, having a large macronucleus and a small micronucleus.
The macronuclei of Balantidium coli and Balantidium suis are slightly

1922] McDonald: On Balantidium coli and Balantidium suis 283

different with respect to size and proportions as noted above (page
259), but structurally they are so closely alike that one description
will suffice for both. The macronucleus (mac., fig. I, pl. 28, figs. 13
and 14) always lies in the endoplasm but otherwise is not constant
in location. Immediately surrounding it is an area in which the endo-
plasm is less granular and less dense in appearance, due perhaps as
Yocom (1918) has suggested of Euplotes patella to more rapid oxida-
tion in this region. It is elongate and may be straight and rodlike
or it may be sharply bent into a horseshoe shape. In any ease its
diameter increases toward either end, giving it something of a dumb-
bell shape. This constriction in the central region and enlargement
at each end is more marked in Balantidiwm coli than in Balantidium
swis. The nucleus is delimited by a definite nuclear membrane
(nue. m., fig. I) which is especially apparent in material in which
the macronucleus has shrunk due to faulty technique. Within this
membrane are packed rather densely the masses of chromatin. The
chromatin never occurs in equal sized regular granules but rather in
unequal very irregular masses, sometimes of considerable size (1 to 2u
in greatest dimension). Often there is a sort of vacuolated area,
usually near one end of the macronucleus, which is free from chroma-
tin. The significance of this vacuolated area I could not determine.
It does not seem to be due to degeneration and is not related to any
phase of reproduction. It is in no way comparable to the ‘‘reconstruc-
tion band’’ described by Griffin (1910) and by Yocom (1918) in
Euplotes worcesteri and Euplotes patella, respectively. The chromatin
stains black with haematoxylin so that the macronucleus is the most
conspicuous structure in a stained individual. When Mallory’s con-
nective tissue stain is used, the macronucleus takes on an orange hue.

Micronucleus—The micronuclei (mic., fig. I) of both species of
Balantidium parasitic in pigs are exceedingly small, not exceeding
5p in diameter. In the resting or vegetative phase the micronucleus
is subspherical and its flattened side lies close against the nuclear
membrane of the macronucleus. It may even lie in a depression in
the macronucleus in which position it is scarcely distinguishable from
the granules of the latter. It is surrounded by a nuclear membrane
readily recognized when the micronucleus is undergoing mitosis. In
a few instances the chromatin has appeared indistinctly granular but
it is customarily so closely packed that it looks like a single solid mass.

iw)

84 University of California Publications in Zoology [Vou 20

Neuromoror APPARATUS

The term neuromotor apparatus denotes an integrated system of
fibers, with a codrdinating center, which is present in some Protozoa,
and which is credited with the power of conductivity of nervous im-
pulses, and hence functions in the coordination of the motor organelles
of the cell. The term was first used for ciliates by Sharp (1914) in
his account of the structure of Diplodiniwm ecaudatuwm. Since then
it has been employed by Kofoid and Christiansen (1915), Kofoid
(1916) for flagellates, and by Yocom (1918) and by Taylor (1920)
in their studies of the morphology and behavior of Euplotes patella.

The neuromotor apparatus of Balantidium coli ean searcely be
considered apart from the motor organelles of the cell. To insure
clarity in the description and discussion which follows, the motor
organelles, previously described in some detail, will be briefly reviewed.
With the exception of the oral plug which surrounds the eytostome
the organism is thickly beset with cilia. Of these, the adoral cilia are
largest. They are distributed about the margin of the peristome in
a row which ends in the membranellar region in the ventral wall of
the oesophagus. The rootlets of these cilia are exceedingly long, reach-
ing well into the posterior third of the cell, where they end without
any connection or attachment. They have two enlargements, the first
being the basal granule which lies just beneath the pellicle, and the
second being located at the junction of ectoplasm and endoplasm.
The remainder of the cilia are arranged in longitudinal spiral rows.
The most anterior cilia of these rows, 1.e., those nearest the adoral
cilia, are nearly as sturdy as the adoral cilia themselves. But, pro-
gressing posteriorly in the rows, the cilia become continuously smaller
until they reach their minimum size at the base of the apical cone of
ectoplasm. Likewise the ciliary rootlets become shorter, and the dis-
tance between the basal granules and the secondary enlargements of
the rootlets becomes less and less as one approaches the base of the
apical cone. This is shown in figure I. The remainder of the way
posteriorly the cilia are of uniform size, and the secondary enlarge-
ment of the basal apparatus of each cilium is the termination of the
ciliary rootlet and lies in the granular band of ectoplasm near its
plane of junction with the endoplasm.

In Balantidiwn coli five distinct parts constitute the complete
neuromotor apparatus, namely (1) a motorium or codrdinating cen-
ter, embedded in the ectoplasm close by the oesophagus, and from it
fibers pass out to the oral plug and the motor organelles; (2) a cireum-

1922 McDenald: On Balantidium coli and Balantidium suis 285

oesophageal fiber, beginning and ending in the motorium and giving
off branches as it passes through the oral plug; (3) a fiber connecting
the adoral cilia and the cytostomal membranelles with the motorium ;
(4) the adoral ciliary rootlets passing posteriorly from the basal
granules of the adoral cilia, each bearing an enlargement where it
passes from ectoplasm into endoplasm, and finally ending without
attachment in the endoplasm of the cell; (5) the radial fibers taking
origin from the enlargements of the adoral ciliary rootlets and passing
radially to the ectoplasmic layer where they turn posteriorly into the
granular band.

The motorium (mot., figs. I and J) when viewed in ventral aspect
has somewhat the appearance of a reversed letter J. It lies within
the ectoplasm of the apical cone, close to the ventral and right walls
of the oesophagus. The part of the motorium corresponding to the
vertical shaft of the J les along the right wall of the oesophagus, and
its anterior terminus is situated just inside and slightly dorsal from
the point of origin of the right lip of the eytostome. The curved end
of the J is posterior and passes ventrally and anteriorly around the
oesophagus and ends close to the inner termination of the rows of
adoral cilia. In the middle region a sharp slit-like constriction is often
very conspicuous. It is with the part of the motorium anterior to this
constriction, that the cireumoesophageal fiber and the adoral ciliary
fiber have their origin. The portion posterior to this constriction is
quite variable both in size and in stainability. This variation is very
suggestive of the behavior of the parabasal body of certain flagellates
as described by Kofoid and McCulloch (1916), Swezy (1916), Kofoid
and Swezy (1919). These authors have shown that the parabasal body
is not kinetic in function but instead is a reserve or reservoir of
material easily transformed into energy, which reservoir fluctuates
according to the physiological condition of the animal. No direct
evidence can here be advanced establishing such a function for this
posterior portion of the motorium, but the fact of its fluctuation in
volume and its variation in chemical nature (as shown by stains)
would lead one to suspect that such an interpretation is correct.

The circwmoesophageal fiber (cir. oes. f., figs. | and J) takes origin
from the anterior extremity of the motorium and passes into the oral
plug. Its course is very close to the inner, i.e., oesophageal, surface
of the plug. It encircles the oesophagus completely but in the mem-
branellar area it becomes very hard to follow. Certain sections (pl. 27,
fig. 3), however, seem to show that it unites again with the motorium.
It is not a smooth fiber but bears irregular enlargements from which

286 University of California Publications in Zoology [Vou. 20

fibers pass both posteriorly and anteriorly into the ectoplasmic mass
of the oral plug. These branches make no observable connections, but
seem to fade out in the ectoplasm. Morphological evidence would
indicate that this portion of the neuromotor apparatus is concerned
solely with the activities of the very mobile oral plug. The adoral
ciliary fiber (ad. cil. f., figs. I and J) also arises from the motorium.
This fiber attaches to the motorium just anterior to the constriction.
It passes directly to the basal granule of the first cilium of the adoral
row from which it passes on and makes connection with each of the
basal granules of the entire row. It turns inward following the row
of adoral cilia along the dorsal or left-hand margin of the membranelle
area. Here its course is exceedingly hard to determine, but like the
circumoesophageal fiber, some sections seem to show its connection
with the posterior end of the motorium.

The remainder of the neuromotor system is not directly connected
with the motorium. The adoral ciliary rootlets (ad. cil. r., fig. 1) pass
inward from the basal granules of the adoral cilia through the ecto-
plasm of the apical cone into the endoplasm and well into the posterior
third of the organism where they end, not abruptly but by fading out.
There is absolutely no indication of any attachment of their inner
ends. Just posterior to the middle of the cell there is usually a very
distinct crossing of the ciliary rootlets from the opposite sides of the
peristome. Each of these adoral ciliary rootlets bears a decided enlarge-
ment at the point where it passes from the ectoplasm into the endoplasm.
The aggregation of these enlargements gives the appearance in eross-
sections through this region (pl. 27, fig. 6) of a zone of large, deeply
staining granules about the oesophagus. Each of these enlargements
gives rise to a fiber which passes radially outward in a transverse
plane. These have been termed the radial fibers (rad. f., fig. J; pl. 27,
fig. 6). Each of these radial fibers as it passes peripherally connects
with small enlargements of the rootlets of the cilia of the apical cone.
The rootlets of the cilia of the apical cone are like those of the adoral
cilia except that they shorten progressively as they near the periphery,
at which point they seareely extend into the endoplasm at all, becom-
ing identical with those of body cilia. Since the radial fibers and the
enlargements of the ciliary rootlets lie in the plane of contact between
the ectoplasm and the endoplasm they very clearly mark the limit of
the two. At the periphery the radial fibers turn posteriorly and
become lost in the granular band. This last fact is very suggestive,
since the terminal enlargements of the rootlets of the body cilia lie in
these granular bands.

1922] McDonald: On Balantidium coli and Balantidium suis 287

It has not been possible to demonstrate a posterior continuation
of the radial fibers within the granular bands. If they do not continue
posteriorly, making connections with the enlargements of the rootlets
of the body cilia, then the logical alternative seems to be to attribute
the function of conductivity to the granular band of ectoplasm itself.
There must be some means of conduction of stimuli, for the body cilia
are concerted in their action, though not to so high a degree as in
the case of the cilia of the apical cone. Protoplasm is generally con-
ceded to have the power of conducting stimuli. So here in Balan-
tidiwm coli there seems to be a transition from conduction by the
undifferentiated protoplasm which serves as a matrix for the ciliary
apparatus, to a condition in which there is a differentiation of the
protoplasm into fibers or strands to serve the purpose. Moreover, the
increasing degree of differentiation is directly correlated with the
increasingly high degree of codrdination of the motor organelles.

It will be seen from the figures and the above description that
Balantidiwm coli is equipped with a notably integrated system of
fibers bearing such morphological relations as would make the assign-
ing to them of the function of codrdination perfectly logical. The
motorium is connected directly only with the oral plug and the adoral
cilia. But through ciliary rootlets and the radial fibers all of the
cilia of the apical cone are in connection with the adoral cilia. And
if the function of conductivity may be attributed to the granular
bands of the ectoplasm, into which the radial fibers turn and become
lost to view, and in which the enlargements of the ciliary rootlets of
the body cilia lie, then all of the motor organelles of the organism are
reached by the neuromotor apparatus, and by it the codrdination of
these organelles may be explained.

Attributing to the various fibers described above the quality of
conductivity and to the whole neuromotor apparatus the function of
- coordination of all of the locomotor organs in swimming and feeding,
microdissection experiments carried on by Taylor (1920), we may
consider the functions of the various parts from that point of view.
The main role of the motorium is to act as a coordination center. The
cireumoesophageal fiber would serve to correlate the movements of the
oral plug with those of the adoral cilia in feeding and in avoiding-
reactions. The ciliary rootlets and radial fibers make possible the
coordination of all of the locomotor organs in swimming and feeding,
and especially the cilia of the apical cone which are employed in the
boring movement of the organism.

University of California Publications in Zoology [Vou. 20

iw)
lo 2)
(o'6)

DISCUSSION

Up to the present time systems of intracytoplasmic fibers and
accessory neuromotor masses comparable to that found in Balantidiwm
coli have been fully deseribed in several flagellates and a few ciliates.
The very primitive type occurring in Naegleria gruberi (Schardinger)
has been deseribed by Wilson (1916). Kofoid (1916) and Swezy
(1916) have made a eritical comparative study of the motor systems
of those flagellates, in which they have been most carefully studied.
That there is a striking similarity in the neuromotor systems of flagel-
lates and ciliates is clearly pointed out by Yoeom (1918).

Among the ciliates, intracytoplasmic fibers have been known for
some time and there have been several descriptions of them and several
conflicting views as to their function (Engelmann, 1880; Biitsehh,
1889; Schuberg, 1891; Maier, 1903; Prowazek, 1903; Griffin, 1910;
Braune, 1913). Sharp (1914), however, was the first to deseribe fully
a completely integrated fibrillar system with a central neuromotor
mass, to which he applied the term neuromotor apparatus.

Of the neuromotor apparatus of ciliates that of Balantidium coli
is the third to be quite fully worked out. In 1914 Sharp described
the neuromotor apparatus of Diplodimum ecaudatum (Fiorentini).
This apparatus consists of six parts. The central motor mass, or
motorium, lies in the area of thickened ectoplasm at the anterior end
of the animal between the dorsal and adoral membranelle zones. A
fiber connects the motorium with the basal granules of the dorsal mem-
branelles, a branch from which runs along the base of the inner dorsal
lip. Another fiber connects the motorium with the basal granules of
the adoral membranelles. A set of opercular fibers leave the motorium
and pass along underneath the operculum. Lastly, the motorium has
a definite connection by means of a fiber with what Sharp called the
cireumoesophageal ring. There is also a set of fibers in the wall of
the oesophagus, which he termed the oesophageal fibers, and which
he believed took their origin from the cireumoesophageal ring. All of
these structures as well as the micronucleus Sharp found had an
affinity for the acid fuchsin when Mallory’s connective tissue stain
was used.

In Euplotes patella (O. F. Miller) the neuromotor apparatus, as
deseribed by Yocom (1918), is made up of five distinct parts. The

1922] McDonald: On Balantidium coli and Balantidium suis 289

motorium is a somewhat bilobed mass and lies in the ectoplasm at
the anterior end of the organism close to the right anterior corner of
the triangular cytostome. From the left end of the motorium five
main longitudinal fibers pass posteriorly, diverging slightly, and each
joins with one of the five anal cirri. The exact relation of these fibers
to the basal plates of anal cirri has been very carefully determined
by Taylor (1920). Leaving the right end of the motorium a fiber
passes to the membranelles of the adoral zone. Directly connected
with this fiber is the ‘‘sensory structure’’ of the anterior lip. Lastly,
there are dissociated fibers in connection with the frontal, ventral, and
marginal cirri. In Euplotes, as in Diplodinium, all parts of the
neuromotor apparatus as well as the micronucleus stain brilliant red
with acid fuchsin.

For facilitating comparison, it might be well to summarize briefly
the neuromotor apparatus of Balantidiwm coli, as described above.
It consists in this organism of five distinct divisions. The motorium
is a J-shaped mass situated in the thickened ectoplasm of the anterior
end of the animal close to the eytostome and oesophagus. From it
arises the circumoesophageal fiber, which gives off branches to the
oral plug. A second fiber takes its origin from the motorium and
connects with the basal granules of the adoral cilia. The adoral ciliary
rootlets pass inward from the basal granules of the adoral cilia, and
may extend well into the posterior third of the cell. The radial fibers
take their origin from enlargements of the adoral ciliary rootlets,
at the poimt where they enter the endoplasm. They pass outward
radially, making connection with the ciliary rootlets of the cila of
the apical cone, and at the periphery turn posteriorly and cannot
be traced farther in the granular bands of the ectoplasm. As in the
previous forms just summarized, the neuromotor apparatus as well as
the micronucleus of Balantidiwm coli is selective for acid fuchsin.

Of the three examples of neuromotor systems so far fully worked
out and described, each represents a different order of the class
Ciliata: Diplodinium being of the order Oligotricha; Euplotes being
of the order Hypotricha; and Balantidium being of the order Hetero-
tricha. Yet in spite of the diversity of forms there is a remarkable
similarity in the neuromotor system. The presence of a motorium is
common to all three. In each of the three organisms it is located at
the anterior end of the animal and hes wholly within the ectoplasm
near the cytostome. By means of fibers it is connected with a part
or all of the motor organelles of the animal. Another feature of the

290 University of California Publications in Zoology (Vou. 20

fibrillar portion of the apparatus common to all three forms is the
strand connecting the motorium with all of the adoral cilia or mem-
branelles, as the case may be. Finally, the system, in all cases, shows
an affinity for acid fuchsin.

The circumoesophageal ring present in Diplodinium is represented
in Balantidium by the cireumoesophageal fiber running through the
oral plug. The oral plug forms the wall of the greater part of the
oesophagus, and the fiber lies very close to the oesophageal surface.
From this fiber there are given off fibers which pass both anteriorly
and posteriorly in the mass of the oral plug. These fibers are strik-
ingly similar in location to the oesophageal fibers, described by Sharp
(1914) in the wall of the oesophagus of Diplodinium and believed by
him to arise from the circumoesophageal ring. Sharp points out that
these fibers approach very close to the micronucleus though there was
no demonstrable connection with it. In Balantidium this possibility
is precluded by the shortness of the oesophagus which in no case
extends inward to a point anywhere near the micronucleus. At times
when the organism is viewed from the proper angle, the ciliary root-
lets of the adoral cilia may have the appearance of ending in the
proximity of the micronucleus and suggesting a connection with it.
No such connection exists, however, as may be readily demonstrated
in large numbers of whole mounts and still better in sections where
these rootlets may be traced far posterior to the micronucleus, passing
it some considerable distance away. In the vegetative phase, it is
certain that no structural connection exists between any part of the
neuromotor apparatus and the micronucleus.

Of the three neuromotor systems of ciliates here considered, that
of Balantidium is clearly the least centralized, though none the less
a unified structure. In Diplodinium all motor organelles connect
directly with the motorium, and in Huplotes the same is true of all
except the marginal cirri which have no connection whatever, whereas
in Balantidium only the adoral cilia have direct connection with the
motorium while the cilia of the apical cone (and those of the body,
too, 1f they have any connection whatever) are connected with it only
indirectly through the radial fibers and the rootlets of the adoral cilia.
This lesser degree of centralization of the neuromotor apparatus may
perhaps be explained by the lesser degree of specialization of locomotor
organelles. Whereas in Diplodinium the body is devoid of cilia and
the dorsal and adoral zones of membranelles are the sole motor organ-
elles, and in Huplotes the locomotor organelles are restricted to the
cytostomal membranelles and a few cirri on the ventral surface, in

1922] McDonald: On Balantidiwm coli and Balantidium swis 291

the case of Balantidium the entire organism, with the exception of
the oral plug, is covered with cilia. So the slightly lesser degree of
specialization in the neuromotor apparatus would be the logical expec-
tation if modification of intracytoplasmic structures is ‘correlated
with modification of external structures with which they have a direct
connection or of which they form an integral part.

From the point of view of efficiency, also, the arrangement in
Balantidium is readily explainable. The locomotor activities of the
organism may be separated into three main sorts, swimming, feeding,
and boring, this last very likely being used in penetration of the
intestinal wall of the host. Im swimming the codrdination of the entire
locomotor apparatus is necessary. In feeding, and in boring, particu-
larly, the codrdination of the adoral cilia with those of the apical cone
is extremely essential. Such codrdination would be most. effectively
bronght about by a direct connection of the parts concerned, and this
direct. connection is accomplished by the uniting of all of the rootlets
of the cilia of these two regions by means of the radial fibers, without
the interpolation of the motorium.

Throughout the above discussion the assumption of a neural fune-
tion for the neuromotor apparatus, i.e., the power of conductivity of
stimuli resulting in codrdination of parts, has been based on two
general types of evidence, morphological and experimental. The
chemical evidence, that is, the affinity for acid fuchsin, as presented
by Sharp (1914) for Diplodinium and by Yocom (1918) for Huplotes,
seems shghtly less convincing in the case of Balantidium. In the last
named organism not only does the micronucleus, which has no connec-
tion with the neuromotor apparatus, show an affinity for acid fuchsin
but so also do certain cytoplasmic inclusions, which if not food par-
ticles are at least undoubtedly concerned in some way with metabolism
and have no morphological relation to the neuromotor apparatus.
Yocom (1918) states that there is more of the orange G in the micro-
nucleus giving it a different shade from the parts of the neuromotor
apparatus in the case of Huplotes; but in Balantidiwm I have been
unable to detect any such differentiation.

Morphological evidence for attributing neural function to the
neuromotor apparatus has been clearly presented by Yocom (1918)
in his diseussion of the apparatus in Euplotes. The evidence found
in Balantidium is not strikingly different. There is in the latter
organism the same intricate relationship between the neuromotor
apparatus and the motor organelles. The most active cilia, ie., the
adoral cilia, are directly connected by the adoral ciliary fiber with

292 University of California Publications in Zoology [Vou 20

each other and with the motorium. The cilia of the apical cone are
connected with the adoral cilia by the radial fibers and so indirectly
with the motorium. In this case, however, codrdination with the adoral
cilia is most essential and this corresponds with their intimate con-
nection. These morphological interrelations all point to a neural fune-
tion for the neuromotor apparatus.

Very convincing experimental evidence of the neural function is
to be found in the results of Taylor’s (1920) microdissection experi-
ments on Luplotes patella. In Euplotes there is a fiber connecting the
adoral membranelles with the motorium, and also a fiber connecting
each of the five anal cirri with it. In a series of experiments Taylor
severed various ones of these fibers and observed very carefully the
effect on the movements of the animal. He then compared these move-
ments with the normal movements which he had previously carefully
analyzed and classified. Severing of these fibers resulted in lack of
coordination of the parts thus disconnected and resulted in abnormal
movements. Incision made in other parts of the cell, but which did
not sever neuromotor fibers did not so result. In the words of the
author, ‘‘It is apparent, then, that the destruction of the motorium
or the severing of some or all of its attached fibers is alone accountable
for modification in the perfect and efficient codrdination between the
series of membranelles and the anal cirri. We may, therefore, regard
these normal morphological relationships as conditioning the animal’s
usual behavior both in ereeping and in swimming.’’

The general occurrence among Protozoa of protoplasmie modifica-
tion to form organelles for locomotion, feeding, digestion, excretion,
and protection has been known almost as long as have the Protozoa
themselves. The functions of such organelles have not been difficult
to determine. One might equally well expect to find modifications
correlated with the conduction of stimuli, but the establishing of
neural function is not so easy. This difficulty in conjunction with the
traditional idea of the simplicity of the Protozoa has resulted in con-
servatism in crediting any intracellular structures with the function
of conductivity. The recent detailed morphological studies on Proto-
zoa and the experimental work of Taylor (1920), however, leave little
doubt regarding the matter. This account of the neuromotor appa-
ratus of Balantidiwm coli and Balantidium suis presents additional
evidence of the likelihood of a quite general occurrence, in the Pro-
tozoa, of intracytoplasmic specialization resulting in a more or less
integrated system for purposes of codrdination.

1922] McDonald: On Balantidium coli and Balantidium swis 293

SUMMARY

1. Pigs are very generally infected with balantidia as shown by
the findings in previous investigations, many of which were carried
on in foreign countries, and by the finding of 68 per cent infection
among the two hundred pigs examined during the present investi-
gation.

2. There are two species of the genus Balantidium that are para-
sitic in the intestinal tract of pies, namely, Balantidium coli and
Balantidium swis (sp. nov.).

3. Balantidium coli is the species first described by Malmsten
(1857) from man, and later by Leuckart (1861) as a parasite in pigs.

4, Balantidium suis (sp. noy.) has not hitherto been distinguished
from Balantidiwn coli. The former differs from the latter in being
more elongate and being broadest anterior instead of posterior to the
equatorial plane; in having a more slenderly proportioned macro-
nucleus; and in having the mouth displaced ventrally, instead of being
almost terminal, which causes the plane of demarcation between ecto-
plasm and endoplasm in this region to slant posteriorly toward the
ventral surface instead of being perpendicular to the longitudinal
axis, as is the case in Balantidiwm colt.

5. So far as recorded facts will justify conclusions, it seems un-
likely that Balantidium suis occurs as a parasite of man, but instead
that Balantidium coli is the cause of balantidiasis. Whether or not such
is the ease, it is very desirable that the occurrence of the two species
in pigs be taken into account in future work on experimental infection,
for to a failure to distinguish between the two species may be due
the seemingly conflicting results of previous experiments.

6. The cilia of the two species are homologous. Variation occurs
in size and relative position of parts only. The basal apparatus 1s
essentially diplosomic, consisting of a basal granule connected by a
ciliary rootlet to a secondary enlargement. The former is situated just
beneath the pellicle; the latter lies in the plane of demarcation between
ectoplasm and endoplasm. The adoral cilia are largest, the basal granule
and secondary enlargement are farthest removed from one another, as
the ectoplasmic thickening is greatest in this region, and the ciliary
rootlet may extend far into the endoplasm. The cilia of the apical
cone intergrade between the adoral cilia and the body cilia. As one

294 University of California Publications in Zoology [Vou. 20

progresses posteriorly, they become smaller in size, the secondary
enlargement approaches the basal granule as the ectoplasmie layer
becomes thinner, and the extension of the rootlet into the endoplasm
becomes shorter. The body cilia are smallest and their rootlets
terminate in the secondary enlargement. The cilia of the anterior
end are highly concerted in action. They beat in such a way as to
give the animal a remarkable boring motion which probably serves
in the penetration of the mucosa of the intestine.

7. Both species possess a neuromotor system. This is a highly
developed and integrated system consisting of five correlated parts.
The motorium, lying within the ectoplasm near the eytostome, gives
rise to a cireumoesophageal fiber. In addition, there is a heavier fiber
which connects it with the basal granules of the adoral cilia. The root-
lets of the adoral cilia extend far into the endoplasm, usually well into
the posterior one-third of the cell. Where they pass from ectoplasm
into endoplasm each adoral ciliary rootlet bears an enlargement and
from this arises a radial fiber which passes to the periphery, turns
posteriorly, and disappears in the granular band of ectoplasm. In
passing to the periphery these radial fibers connect with enlargements
of the rootlets of the cilia of the apical cone.

Transmitted February 26, 1921.

Zoological Laboratory, University of California,
Berkeley, California.

1922] McDonald: On Balantidium coli and Balantidium swis 295

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BRAUNE, R.
1913. Untersuchungen tiber die im Wiederkaiuermagen vorkommenden Proto-
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1889. ‘‘Ciliata’’ in ‘‘Protozoa’’ (1887-1889) in Bronn, Klass. und Ordn. des
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1909. Researches on the intestinal Protozoa of frogs and toads. Quart. Jour.
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DoFLEIN, F.
1911. Lehrbuch der Protozoenkunde. (Jena, Fischer), ed. 3, xii+ 1043, 951
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EHRENBERG, C. G.
1838. Die Infusionsthierchen als volkommene Organismen. (Leipzig, Voss),
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ENGLEMANN, T. W.
1880. Zur Anatomie und Physiologie der Flimmerzellen. Pfliiger’s Arch. ges.
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GrirFin, L. E.
1910. Euplotes worcesteri sp. nov., I, Structure. Philippine Jour. Sci., 5,
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JENNINGS, H. 8S.
1908. Heredity, variation and evolution in Protozoa. Proc. Am. Phil. Soc.,
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296 University of California Publications in Zoology [Vou. 20

JOHNSON, H. P.
1893. A contribution to the morphology and biology of the stentors. Jour.
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1922] McDonald: On Balantidium coli and Balantidium suis 297

ScHauDINN, F., and Jakosy, M.

1899. Ueber zwei neue Infusorien im Darm des Menschen. Centralbl. f.

Bakt., Abt. I, 25, 487-494, 4 figs. in text.
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1886. Ueber der Bau der Bursaria truncatella mit besonderer Beriicksichti-
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kauermagens. Sitz.-ber. d. phys.-med. Ges. Wurzburg, 1891, 122-137.

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Tuon, K.

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5, 281-321, pls. 12-13, 3 figs. in text.
WALKER, E. L.

1909. Sporulation in the parasitie Ciliata. Ibid., 17, 297-306, 2 pls.

1913. Experimental balantidiasis. Philippine Jour. Sci., 8, (B), 333-349, 7
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WILSON, C. W.

1916. On the life history of a soil amoeba. Univ. Calif. Publ. Zool., 16, 241—

292, pls. 18-23.
WISING, P. J.

1871. Till kannedomen om Balantidiwm coli hos minniskan. Nordiskt Med.

Ark., 3, 1-30, 1 pl.
Yocom, H. B.

1918. The neuromotor apparatus of Euplotes patella. Univ. Calif. Publ.

Zool., 18, 337-396, pls. 14-16, 1 fig. in text.

EXPLANATION OF PLATES
PLATE 27

Camera lucida drawings of a series of transverse sections of Balantidiwm coli
(Malmsten). The series begins at the anterior end and progresses posteriorly
about one-third the length of the organism. X 1000.

Fig. 1. This section shows dorsal portion only of peristome. The adoral cilia
are removed showing their basal granules clearly.

Fig. 2. A portion of the oral plug is shown with the circumoesophageal fiber
and the connected oral plug fibers. The deeply-staining region between proto-
plasm of oral plug and the surrounding ectoplasm, so marked in this individual,
is rarely found.

Fig. 3. The continuation of cilia over the ventral lip and down into the ecyto-
stome are to be noted particularly; the beginning of the motorium on the
(reader’s) left of the eytostome; and the connection of the adoral ciliary fiber
with the motorium.

Fig. 4. In this figure the cilia of the cytostome give the appearance of mem-
branelles; the circumoesophageal fiber makes connection with the motorium.

Fig. 5. This shows the beginnings of the enlargements of the adoral ciliary
rootlets.

Fig. 6. Here the oesophagus is completely surrounded by the enlargements
and radial fibers may be seen arising from some of them.

Fig. 7. The extreme posterior tip of the motorium appears within the circle
of enlargements; the dorsal half of this section is through the endoplasm.

Fig. 8. This section is posterior to the adoral apparatus with the exception of
the rootlets of the adoral cilia which in cross-section can not be distinguished from
the granules of the endoplasm.

[298]

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PLATE 28

Figs. 9-12. Series of longitudinal sections through the anterior end of Balan-
tidium coli (Malmsten). X 1000. These sections are oblique, being more nearly
frontal than sagittal, however. The section shown in figure 9 is tangential to the
margin of the peristome some distance to the right of the most dorsal point of the
latter (see fig. J in text). The remainder progress toward the left ventral lip.
The oral plug fibers, the radial fibers, and the adoral ciliary rootlets are shown
clearly in these sections.

Fig. 13. A sagittal section through Balantidium suis (sp. nov.). X 1000.
The specific characters, viz., elongate body and macronucleus, and oblique plane
delimiting apical cone of ectoplasm, are shown in this section. The eytopyge is
distinct and open to the exterior. A paramylum body of considerable size is
present in the endoplasm.

Fig. 14. An oblique longitudinal section through Balantidiwm coli (Malmsten)
showing the protrusion of the oral plug. X 750.

[300]

gq

PLATE 2%

[ MCDONALD ]}

)

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Grinnell. Pp. 431-433. os
Nos. 14 and-15 in one cover. April, 1918 -.220. earnest

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IN

ZOOLOGY

Vol. 20, No. 11, pp. 301-307, 7 figures in text April 21, 1922

MITOSIS IN ENDAMOEBA DYSENTERIAE IN
THE BONE MARROW IN ARTHRITIS
DEFORMANS

BY
CHARLES ATWOOD KOFOID anp OLIVE SWEZY

(Contribution from the Zoological Laboratory, University of California, and from the Division of
Parasitology, Bureau of Communicable Diseases, California State Board of Health)

The coincidence of the presence of Endamoeba dysenteriae in the
human bowel and a diagnosis of arthritis deformans has been a matter
of observation and record in our laboratory for several years. We
have previously called attention to this relation to rheumatism of the
joints (Kofoid, 1920). We later (1922) identified amoebae in the bone
marrow in material submitted by Dr. Ely in Ely’s (1920) second
type of arthritis deformans, and our discovery was announced coinci-
dently by Ely, Reed, and Wyckoff (1922) with ourselves. This note
is a preliminary statement of the proof that the cells interpreted by
us as amoebae are in reality parasitic rhizopods and not amoeboid
human cells.

The proof rests upon the fact that mitosis in the Rhizopoda and
in the Metazoa, including that in human cells, is of two distinet types,
clearly distinguishable by well-known eytological criterions of such
definiteness that there can be no possibility of any trained cytologist
or protozoologist confusing them. If the amoeboid cells found by
us in the bone marrow of the lesions in the excised head of the femur
are really amoebae, they will exhibit, when they divide, the character-
istic behavior and resulting morphology in fixed and stained material
which oceurs in the rhizopods. If they are only wandering amoeboid
human ¢ells, they will exhibit the human type of mitosis, or, if the
tissue is degenerating, some modification of it. It is hardly probable
or possible in the light of known degenerating human cells that degen-

302 University of California Publications in Zoology [ Vou. 20

eration should reproduce in each instance which we have detected of
division in the supposed amoebae, the rhizopod type of mitosis.
Through the courtesy of Dr. C. L. Ely of Stanford University
Medical School, unstained sections and undeealcified portions of the
excised head of the femur of his case 187 of the second type (non-
baeterial) of arthritis deformans have been placed in our hands for
investigation. This excised bone had been fixed promptly in formalin

Fig. 1 Fig. 2

Fig. 1. Lateral view of a small epithelioid (?) cell in the metaphase of
mitosis from a hypertrophied ‘cervical gland of man in Hodgkin’s disease. Note
the absence of nuclear membrane, and the spindle fibers running from the centro-
somes at the apices to the massed, deeply stained chromosomes in the equatorial
plate. From a section stained in iron haematoxylin. X 2500.

Fig. 2. Lateral view of the anaphase of mitosis in a similar cell from the same
source. X 2500. Note the elongated divided chromosomes retreating towards
the poles of the spindle, and the lateral constriction, indicating approaching sep-
aration of the two daughter cells. Only part of the chromosomes (about 16) of
the cell have been drawn, because of the superposition of the deeply stained rods
which form the chromosomes. We are indebted to Dr. Doxey Wilson and Dr. East-
man of San José for the material from which these sections were made.

and was well preserved, though difficult to stam, and somewhat difficult
to deeolorize normally. The cells are splendidly preserved and the
tissues came through decalcification with little if any disturbance.
Prolonged and assiduous search among the amoeboid cells which
we interpreted as amoebae has brought to ght several imstances of
stages of mitosis and several binucleate amoebae, indicating that
asexual binary fission was in progress among the amoebae in the mar-

coins of the necrotic areas in the head of the femur.

—

1922] Kofoid-Swezy: Mitosis in Endamoeba dysenteriae 303

We have also found dividing human cells in the hypertrophied
cervical gland excised from a case of Hodgkin’s disease. We have
not found human cells of any kind dividing in the lesions of the bone.
A comparison of the two types of division clearly indicates that the

Fig. 6

Fig. 3. Endamoeba dysenteriae from lesions in bone marrow in the head of
the femur in Ely’s case 187 of the second, non-bacillary type of arthritis. Note
spherical vesicular nucleus with peripheral chromatin, central karyosome, and
spoke radiations.

Fig. 4. Same with nucleus in prophase with peripheral chromatin in strands
(chromosomes?), some of them split.

Fig. 5. Binucleate amoeba resulting from mitosis. Halo around central
karyosome.

Fig. 6. Endamoeba dysenteriae in early anaphase. Note small number of
chromosomes, probably six, intact nuclear membrane and intradesmose joining
centrosomes at poles.

Fig. 7. Late anaphase with distinct intradesmose. All figures 2500
diameters, from sections of the excised head of the femur in Ely’s case 187.

304 University of California Publications in Zoology [ Vor. 20

two types are quite distinct and that the type in the cells under sus-
picion is distinetly that of the rhizopod rather than that of the mam-
mahan or human eell.

Two human cells from the lymph gland are shown in the metaphase
and anaphase of mitosis in figures 1 and 2.

The human eell at mitosis is characterized by three fundamental
structural features which are common to mammalian, if not to meta-
zoan cells, at mitosis. These are, first, the entire disappearance of
all traces of the nuclear membrane. This leaves the spindle of the
dividing cell with its equatorial (fig. 1) or divided (fig. 2) group of
chromosomes exposed free in the enveloping cytoplasm. In the com-
parable stages in the mitosis of amoeba (figs. 6 and 7), the whole
spindle is contained within the persistent nuclear membrane. This
membrane can be distinetly followed in focussing as a slightly stained
wall surrounding the spindle. The spindle lies within it, its spindle
fibers being few (fig. 6) in comparison with the many fibers of the
human spindle (figs. 1 and 2) and lightly stained. The centrosomes
are minute dots at the poles (fig. 6) within the nuclear membrane
of the dividing nucleus of amoeba.

A second point of difference is the absence in the human spindle
during the period of late prophase to the close of the anaphase of a
deeply staining strand joining the centrosomes which we (1921) have
called the intradesmose. The intradesmose is formed from the
parental centrosome and as this divides and the resulting daughter
centrosomes move to the poles of the spindle, a deeply staining sub-
stance similar, in iron haematoxylin, to that in the polar masses which
constitute or contain the centrosomes, is spun out in a meridional
thread often applied to the inner face of the nuclear membrane. If
heavily decolorized, this is destained. If laterally located, it may be
difficult to detect. In favorable material (fig. 7) it is clearly seen.
This structure has been found by us in dividing nuclei in the intestinal
eysts of Endamoeba dysenteriae (not yet published), in EL. coli (Swezy,
1922, in press) and in Councilmania lafleuri (Kofoid and Swezy,
1921). It corresponds to the extranuclear paradesmose of the flagel-
lates, and to the axial strand of linin fibers in the mitotie spindle of
the dividing metazoan cell. In the dividing human eells, from the
lymph glands of Hodgkin’s disease, no structure such as the intrades-
mose can be found. In fact, none is known in cells of mammals, nor,
in so far as we know, in any metazoan cells. In the Protozoa, it
occurs in the Rhizopoda.

1922) Kofoid—Swezy: Mitosis in Endamoeba dysenteriae 305

A third criterion exists in the number of chromosomes. The
number of chromosomes in man appears to be either 24 or 48, or pos-
sibly both, the former being the diploid and the latter, the tetraploid
condition. Guyer (1910, 1914) reported 22, but Montgomery (1912)
and Wieman (1917) raised the number to 24. However, double this
number have been reported by Evans (1918, 1921) and Painter
(1921) who suggest 45 to 48. The finding of 47 chromosomes by
Winiwarter (1917) in the testis suggests the possibility that there
may be 47 in the male and 48 in the female in the tetraploid phase,
and, by inference, in the diploid phase 23 and 24.

We have found (Kofoid and Swezy, 1921) 8 chromosomes in
Councilmania laflewri, and 6 (see also Swezy, 1922, in press) in Enda-
moeba coli, and probably 6 (not less than 5 nor more than 7) in
Endamoeba dysenteriae. The number of chromosomes in the dividing
cells which we interpret as dividing amoebae is not 24, much less 48.
It is at the most a small number (fig. 6), probably 6. The number
in the dividing human eells observed by us in lymph glands of
Hodgkin’s disease (figs. 1 and 2) is many more than 6. The chromo-
somes are so crowded in the equatorial plate (fig. 1) or so overlie
each other, especially at the margins of the divided equatorial plate
in lateral view, that the exact number can not be ascertained with
certainty. We have not yet found a polar view in which the prac-
tieability of counting will be enhanced. The individual chromosomes
are indicated by the projecting lobes representing their free ends.
The number of these free ends on either side (fig. 2) or of elbows on
the polar side of each group may be estimated in the most favorable
region, namely, a sector of 90° on one surface. The number in our
sections appears to be not less than 24, and not the tetraploid (?) 48.
It is certainly not 6. In view of the general acceptance by cytologists
of the view that the somatic chromosome number is both constant and
characteristic, we are constrained to believe that this criterion is valid
and critical to determine the non-human and the amoebie identity of
these cells.

The chromosomes of the human cells are also larger and propor-
tionately longer than those of the amoebae. They are consistently so
in all the mitotic figures at the metaphase or near it of the amoebae
which we have been able to detect, and we find no evidence in our
material which connects these types of mitosis with any observed form
of degenerating human cells. They appear to be normal parasitic
amoeba in the normal phases of mitosis.

306 University of California Publications in Zoology [ Vo. 20

The presence of the amoebae in the lesions of Ely’s second or non-
bacterial type of arthritis deformans supplies the missing etiological
factor in this disease. The term, amoebiasis of the bones, would
therefore more accurately link the disease with its causative organism,
in the event that an etiological relationship is confirmed by future
work.

The question as to the exact species of amoeba found in the bone
lesions is perhaps still an open one. It ean not be Councilmania
lafleuri, which has eight chromosomes and a granular karyosome. The
only amoebae definitely known to be tissue invaders are Endamocba
dysenteriae and E. gingivalis. Mitosis and chromosome number in
the latter are unknown. In our material and in published accounts
of this species, the morphology of the nucleus differs in one minor
particular, the absence of spoke-lke radiations. In view of this fact,
we tentatively inclme to the view that the amoeba in the bone lesions
is EZ. dysenteriae rather than EF. gingivalis, on the assumption and
perhaps rather widely accepted view that the two are distinet species.
There is no satisfactory evidence that they are identical, though evi-
dently rather more closely related in morphology to each other than
is either to any of the other amoebae of man.

SUMMARY

Active amoeboid cells with the type of nucleus found in the cysts
and active stages of Hndamoeba dysenteriae in the bowel and in
active amoebae in the lesions of amoebiasis are found in the lesions
of the bones in arthritis deformans of Ely’s second or non-bacterial
type. These amoebae at mitosis have an intact persistent nuclear
membrane, a meridional intradesmose, and a small number of chromo-
somes, probably six. This type of mitosis is that of the Rhizopoda,
not of the Metazoa, Mammalia or man. It is in these details exactly
simiar to that found in F. dysenteriae of the bowel. Human eells at
somatic mitosis have, excluding sex differences, 24 (diploid) or 48
(tetraploid?) chromosomes, no intradesmose, and the nuclear mem-
brane disappears. The mitoses in the amoebae appear to be normal,
and no intergrades with degenerating human eells have been found.
The amoeba of the bone lesions appears to be H. dysenteriae rather
than EF. gingivalis of the gingival abscesses and tonsils. The morpho-
logical evidence supports the conclusion that EH. dysenteriae is the

1922 | Kofoid-Swezy: Mitosis in Endamoeba dysenteriae - 307

parasitic etiological factor in Ely’s second type of arthritis deformans.
This disease might therefore be designated as amoebiasis of the bones
provided the etiological relationship of the parasite to the disease ean
be established.

Literature cited will be found at the end of the following article.

ZOOLOGICAL LABORATORY,
UNIVERSITY OF CALIFORNIA.

Transmitted April 13, 1922.

ri

UNIVERSITY OF CALIFORNIA PUBLICATIONS

IN
ZOOLOGY
Vol. 20, No. 12, pp. 309-312, 4 figures in text April 21, 1922

ENDAMOEBA DYSENTERIAE IN THE LYMPH
GLANDS OF MAN IN HODGKIN’S DISEASE

BY
CHARLES A. KOFOID, LUTHER M. BOYERS, M.D., anp OLIVE SWEZY

(Contribution from the Zoological Laboratory, University of California, and the Division of Parasitology,
Bureau of Communicable Diseases, California State Board of Health)

Hodgkin’s disease has come to be known since the investigations of
Reed (1902) and others as a clinical and pathological entity with a
specific histological picture, not a simple hyperplasia, but with changes
suggesting a chronic inflammatory process. The pathological agent,
however, is unknown. It is the purpose of this preliminary note to
make an announcement of the discovery of Endamoeba dysenteriae
in the lesions of the lymph glands in this disease. In a previous
communication (1922), we announced the coexistence of intestinal
amoebiasis with the cysts of Endamoeba dysenteriae in the stools, and
a diagnosis of Hogdkin’s disease in the first two cases we examined,
with a probability of the coexistence in a third (incoln’s case). The
mathematical probability of such a coincidence in the first three cases
on the basis of an incidence of amoebiasis at large of 4 per cent is
1 in 15,625. On the basis of 1 per cent incidence, it is 1 in 1,000,000.
Since then a fourth case has been examined with an added instance
of coincidence, thus increasing the probability of an etiological con-
nection between this parasite and this disease of hitherto unknown
cause.

This last case was an inguinal gland apparently but recently
attacked by the disease. Sections of this gland have been stained in
iron haematoxylin and systematically searched for evidences of amoebic
infection. In the territory of the gland most involved in the progress
of the pathological modifications due to the disease we have found
amoeboid cells with vesicular nuclei which we interpret as amoebae

(figs. 1, 2, and 3).

310 University of California Publications in Zoology [ Von. 20

As has been stated in the previous article, more evidence is required
to establish a critical determination of these suspected cells as parasitic
amoebae, since they may be only wandering amoeboid cells with an
unusual type of nucleus for a human cell, a product of the pathogenic
process.

Figs. 1-8. Endamoeba dysenteriae in the active state with pseudopodia,
spherical vesicular nucleus with peripheral chromatin, central karyosome, and
spoke radiations. X 2500.

Fig. 4. An amoeba with nucleus in the telophase of cell division, with
intact nuclear membrane, a few (probably not over six) chromosomes at either
pole. The intradesmose is the black line connecting the two masses of chromo-
somes. It presumably connects the centrosomes at the apices of the nucleus.
2500. All drawings from amoebae in sections of inguinal gland stained in
iron haematoxylin. We are indebted to Dr. L. A. Walker of Helena, Montana,
for this gland.

We have, however, the same criterion to apply here as in the
parasitic amoebae of the bone marrow, namely, the morphology of the
nucleus at mitosis and the number of chromosomes. We have accord-
ingly made an intensive search of these sections, and have found
among the supposed amoebae more than ten instances of some phase
of the mitotie process.

1922] Wofoid—Boyers—Swezy: Endamoeba in Hodgkin’s Disease 311

The amoebae are often distinguished by slight differences in the
stainability of the cytoplasm. In some instances, there is a suggestion
of deterioration, but not in all. A normal amoeba with vacuolated
protoplasm, pseudopodia, and small spherical nucleus with central
spherical karyosome and heavy peripheral rim of chromatin is
shown in figures 1 and 2. A deeply stained, clouded nucleus closely
resembling those often found in amoebae in sections of intestinal wall
stained in iron haematoxylin is shown in figure 3.

The appearance of such amoeboid cells at mitosis is shown in
figure 4. The nucleus of this amoeba is in the late anaphase, the
chromosomes have reached the ends of the elongated nucleus, where
their individuality is somewhat masked by overlapping and fusion.

If we apply to this nucleus the same criterions which were applied
in the preceding article to dividing amoebae in the bone marrow, we
find that this nucleus meets these tests. It has, in the first place, an
intact nuclear membrane which has persisted to a late phase of mitosis.
In the second place, there is a meridional intradesmose on the inner
face of the nuclear membrane connecting the two poles of the nucleus.
In the third place, the number of chromosomes is neither 24 nor 48,
the numbers reported for human cells. It is apparently not over 6.
It is impossible to be certain of the full number beeause of the over-
lapping of these small bodies.

We therefore conclude that these amoeboid cells are not human
cells, and that they are parasitic amoebae. In view of their similarity
in the active state and in mitosis to Endamocha dysenteriae and also
because of the coexistence of the intestinal infection by this amoeba
in this specific case, we infer that it is this same infection that has in
this instance reached the inguinal gland. This conclusion opens the
possibility that Hodgkin’s disease may be amocbiasis of the lymphatie
system and calls for investigation along other than morphological lines.

—

312 University of California Publications in Zoology [ VoL. 20

LITERATURE CITED IN THE TWO ARTICLES

A laboratory and clinical

Ey, L. W.
1920. The second great type of chronic arthritis.
study. Arch. of Surgery, 7, 158-183, 21 figs. in text. .

Ey, L. W., REED, A. C., AND Wyckorr, H. A.
1922. The amoeba as the cause of the second great type of chronic arthritis.
California State Jour. Med., 20, 59.

Evans, H. M.
1918. Unpublished work cited by Babeock, E. B., and Clausen, R. E., im
“*Genetics in Relation to Agriculture’’ (McGraw-Hill Book Co.,

New York and London), p. 538.
Unpublished work cited by Sharp, L. W., in ‘‘An Introduction to

1921.
Cytology’’ (MeGraw-Hill Book Co., New York), p. 362.

Guyer, M. F.
Accessory chromosomes in man. Biol. Bull., 19, 219-234, pl. 1.
Science, n.s., 39, 721-722; 941-942.

1910.

1914. Aecessory chromosomes in man.
Koro, C, A.

1920. Hookworm and amoebiasis in California. California State Jour. Med.,

18, 329-332.
Kororp, C. A., AND SweEzy, O.
1921. On the free, encysted, and budding stages of Councilmania lafleuri, a
parasitic amoeba of the human intestine. Univ. Calif. Publ. Zool.,
20, 169-198, pls. 18-22, 3 figs. in text.
On the occurrence of Hndamoeba dysenteriae in the bone lesions in
California State Jour. Med., 20, 59.

1922.
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Koro, C. A., Swezy, O., AND Boyrers, L. M.

1922. On the coexistence of Hodgkin’s disease and amoebiasis. Jour. Am.

Med. Assoe., 78, 532.
Montcomery, T. H.
1912. Human spermatogenesis, spermatocytes and spermiogenesis; a study
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PAINTER, T. S.
1921. The Y-chromosome in mammals. Science, n.s., 53, 503-504.
REED, DorotHy M.
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SweEzy, O.
1922. On mitosis in Endamoeba coli. Univ. Calif. Publ. Zool., 20 (in press).

Amer. Jour. Anat., 21,

WIEMAN, H. L.
c 1917. The chromosomes of human spermatocytes.
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WINIWARTER, H. VON.
Etudes sur la spermatogenese humaine. I. Cellule de Sertoli. II.
Arch. ad.

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Biol., 27, 91-189, pls. 6-7.

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by Charles Atwood Kofoid and Olive Sweny: Pp, “21-40, plates 3-4, 2.
firnres 311 Cex hs Py LTO ate ee eo
3. Studies on the Parasites of the Termites. IIT. On Trichonympha campanula—
sp. nov., by Charles Atwood Kofoid and Olive Swezy.. Pp. 41-98, plates
§-12,-4 figures Ie text DAY, 1919 na as
4, Studies on the Parasites of the Termites, IV. On Leidyopsis sphacrica gen.
nov., sp. nov., by Charles Atwood Kofoid- and Olive Swezy. Pp. 99-116, =,
plates 13-14, i figure in text. July, 1919 .....
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Flagellate of the Human Intestine, by Charles A, Kofoid and Olive Swezi
Pp: 117-144, plates 15-17, 2 figures in text, April, 1920 2... ,
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Pp. 145-168, 9 figures in text. June; 1920 2 nw 2 cn eg ene
7. On the Free, Encysted, and Budding Stages of Councilmania Tajienré: a Para-
sitie Amoeba of the Human Intestine. by Charles Atwood Kofoid and”
Olive Swezy. Pp. 169-198, plates 18-22, 3 figures in text. June, 1921- Sites
8. Mitosis and Fission in the Active and Encysted Phases of Giardia enterica.
(Grassi) of Man, with a Discussion of the Method of Origin of Bilateral
Symmetry in the Pelymastigote Flagellates, by Charles A. Kofoid and
Clive Swezy. - Pp. 199-234, plates 23-26, 11 figures in-text. March:

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April, 1922
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; “Pp. 248-300; plates 27, 28, 15 figures "im text <2. ee tees
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IN

ZOOLOGY
Vol. 20, No. 13, pp. 313-332, plates 29-31 May 20, 1922

MITOSIS IN THE ENCYSTED STAGES OF
ENDAMOEBA COLI (LOESCH)

BY

OLIVE SWEZY

CONTENTS

TERUTROITIOTIOT, pageee naSetctee cate teense Se a ae nae eer SESE 313
Acknowledgments ....

TMICGTA OTTO a oe ee Re Rc ee Se ESE Gee speres 05)
STORET TS E57 Se ec wena wee ; 317
TROVE) ops nee ee ae erect reer aC eon Soes bape eos cas Cnceteatecaenenss-e= 317
INAGIIDFSTOSO cecsectcece pena eacteenepe tr nen eee Ee Eo ce ere ey WI)
ANDER UNAS aos orcas corer ce ere Serer eer 320

MEN Cay oN See see ere ee eae SE cerita sre be emegnccscocseaeeaeoee 320
Discussion ..... + pt eee oak
Summary RV a ee 325
TRONS UUTC 5 CLUC Case acces canned enna set) AS pe iO eae

XY,

ie “o
aha +9\)
INTRODUCTION.222! Mu

%

A complete study of the various phases of the life history of the
intestinal amoebae of man is confronted by several difficulties which,
thus far, have proved almost insurmountable. One of these is the
difficulty of culturing these parasites outside of the human body. It
is true that cultures have been, made which yielded an abundance of
amoebae (for example, Yoshida, 1920), but, in most cases at least,
either the amoebae have not been the true intestinal species, or they
have shown undoubted evidences of degeneration which render them
unsuitable for further investigation, either cytological or develop-
mental. In the latter case, the amoebae are possibly only those which
survived from the original inoculation in a more or less degenerate

condition.

314 University of California Publications in Zoology [Vou. 20

Another difficulty is correlated with the change in habitat of the
cysts of these organisms on discharge in the stool. If the seat of
infection is the upper part of the colon where the conditions differ
somewhat from those prevailing in the rectum and stool, such differ-
ences undoubtedly react on these delicate organisms so that only those
protected by a fully developed cyst may pass out of the body without
showing some of the deleterious effects of this great environmental
change. Such changes as those incident to passing from the region
of the mucosa to the lumen and thence downward in the stool appear
to be associated with the process of encystment. It is also possible
that changes in the temperature of the environment or in available
oxygen may temporarily suspend or accelerate developmental changes
within the organism, such as mitosis, until a more favorable location
is reached, when normal development may again proceed.

It is evident to any one who works over amoebic cysts that a vary-
ing number of the cysts ordinarily met with in stool examinations
show the results of degenerative changes, particularly if the specimen
has remained in the laboratory some time before examination. In
any consideration of the various stages of the life history, these forms
must be eliminated or the results will be disastrous from a cytological
standpoint. It is also doubtful how much value can be placed on the
cytological study of the amoebae which have been inoculated into
kittens, since an alien host. may tend to produce some morphological
changes.

Endamoeba coli is a common infection of man but is rarely present
in great numbers in human stools. The different stages of mitosis are
seen only occasionally and are rare in the cysts in most stools. It is
therefore difficult to make a sufficient number of observations of
mitosis in the cysts of this human intestinal amoeba to use as a basis
for the investigation of this process.

The material on which this study was made, and from which the
figures illustrating it were mainly drawn, was found in a single stool
from an overseas ex-soldier in the University Infirmary. It occurred
among more than one thousand stools containing infections of LZ. coli
which were examined among more than six thousand routine examina-
tions with a total of four thousand two hundred persons. <A fresh
stool was available and many of the cysts were found with mitosis
in progress. An attempt was made to secure further development
by keeping the stool in a warm oven and making slides at intervals
of a few hours. In the first set made, the slides showed two and four

1922] Swezy: Mitosis in Endamoeba Coli 315

nuclei in the majority of the cysts; those made later contained prac-
tically no cysts with dividing nuclei but a relatively greater number
of eight-nucleated cysts. This would seem to indicate that division
proceeded normally in this case. The only good mitotic figures were
found on the first set of slides, with the exception of that shown in
figure 23, plate 31, which was taken from a set of slides made after
the stool had been standing in the laboratory for twenty-four hours.
The fact, however, that this single stool was the only one among
several thousands which gave an abundance of division forms indicates
the rarity of the occurrence of mitosis in cysts in stools examined in
the ordinary manner. The fortunate coincidence of discharge during
a phase of normal nuclear multiplication and prompt access to the
material and its early fixation has made this study possible.

ACKNOWLEDGMENTS

The work which forms the basis of the following study was done
while the author held the Sarah Berliner Research Fellowship, and
it is with pleasure that the indebtedness of the author to this founda-
tion is acknowledged. The author is also greatly indebted to Professor
Charles Atwood Kofoid for the abundant resources of his laboratory
at the University of California which have been placed at her disposal.

MORPHOLOGY

The active amoeboid stages of Hndamocba coli may be readily dis-
tinguished from Coxuncilmania laflewi, in both fresh and stained
material, by granular pseudopods and lack of clearly marked ecto-
plasm, as well as by more sluggish movements in the living forms.
The eysts are distinguished on careful examination of the karyosome
when unstained or in iodine-eosin, and may be more readily differ-
entiated in fixed and stained preparations. These differences have
been summed up in a recent paper (Kofoid and Swezy, 1921) and
need not be repeated here, except to point out the fact that they may,
in some cysts, be almost entirely obscured during certain stages of
the process of mitosis, such as the early prophase and late telophase,
when chromosomes cannot be counted in the eneysted amoebae. The
same fact will probably hold true of division in the active forms of
these two species, but, thus far, division in the active amoebae has not
been found in our material. More data are needed on this point.

316 University of California Publications in Zoology (Vou. 20

In the following paragraphs a brief description of the eneysted
forms only of Endamoeba coli will be given, since this study is con-
fined to the process of mitosis in that phase only of its life history.

The cysts of HEndamoeba coli vary from 10 to 25 in diameter.
Larger cysts than these have been reported in the literature but they
may have been the cysts of Councilmamea lafleuri, and not of Enda-
moeba cola.

In unstained material in fresh physiological salt solution, the
cysts appear homogenous, the highly refractive eyst wall standing out
sharply against the material of the stool. In cysts that are moribund
the nuclei become visible, sometimes appearing with great clearness
even under low powers of the microscope. In preparations stained
with iodine-eosin the contained structures may usually be readily
seen, including the nuclei, the chromatoidal bodies, and the glycogen
mass, if the latter be present. The nuclei show the typical excentric
karyosome, and chromatin granules on the membrane. In permanent
preparations stained with iron-haematoxylin or other stains, a few
scattered granules may be observed on the linin reticulum whieh fills
the nuclear spaces.

Uninucleate cysts have been exceedingly rare in the material under
observation, and in nearly every case have presented a prophase stage
of mitosis (pl. 29, fig. 1). In the binucleate and quadrinucleate
cysts the majority, in the stool above referred to, showed some phase
of the mitotic process, but occasional individuals have been found
with the nuclei in the ordinary resting condition (pl. 29, fig. 2). These
show the typical excentric karyosome, chromatin granules on the
nuclear membrane, and a few seattered granules on the linin reticu-
lum. The greater part of the cyst in the binucleate phase is usually
occupied by a single, large, centrally located glycogen vacuole or by
several smaller vacuoles, forcing the two nuelei out to the periphery.
The remaining space is often closely packed with acicular, splinter-
like, chromatoidal bodies (pl. 29, fig. 2). These vary in quantity in
different cysts but are almost always present in the binucleate and
quadrinucleate ones, sometimes in such abundance that detection of
the nuclei becomes an impossibility, particularly in the various mitotie
phases. In later stages the chromatoidal material generally becomes
ereatly reduced in quantity, either beimg massed in a few large clumps
(pl. 31, fig. 25) or appearing as slender splinters (pl. 31, fig. 28).
Oceasionally a few black, rounded-up granules are the only vestiges
of chromatoidal material remaining in the eyst (pl. 31, fig. 26).

1922] Swezy: Mitosis in Endamoeba Coli 317

At the beginning of encystment the cytoplasm generally contains
a large amount of glycogen. This may be so great in extent as to
give a dense brownish color to the entire cyst in iodine-eosin stain,
or it may be localized in’small spheres with outlines more or less
sharply defined. With the process of fixation the glycogen forms a
single mass or several large masses whose locations, in stained prep-
arations, appear as large, clearly defined vacuoles (pl. 29, figs. 3-7),
the glycogen itself having been dissolved in the aqueous solutions in
the process of staining.

MITOSIS

Binary fission of Endamoeba coli in its active form has not thus
far been described by any investigator. It is probable that it occurs
. mainly in the upper part of the colon and occupies such a short time
that individuals undergoing this process do not reach the exterior
before its completion. Binucleate free individuals may sometimes be
present (Dobell, 1919), but no clue to their further history has been
found.

In the following paragraphs, an outline of the process of mitosis in
the eneysted form of Endomocba coli will be given. Some minor gaps
in this process have yet to be filled, but a sufficient number of stages
are here presented to indicate the line of development throughout.

PROPHASE

The prophase of mitosis is marked by a considerable increase in
the amount of chromatin contained within the nucleus. This is rela-
tively small in the normal trophozoite and is distributed in typical
fashion, with chromatin granules attached to the nuclear membrane,
and a small, spheroidal excentric karyosome (pl. 29, fig. 2). A few
chromatin granules may also be found on the linin reticulum which
fills the nucleus. At the approach of the prophase the linin network
disappears and the karyosome becomes lost in the mass of chromatin
found in the center of the nucleus (pl. 29, fig. 1), usually distributed
in irregular masses and granules. At the same time the nucleus
becomes elongated and the larger masses of chromatin become dis-
tributed as numerous small granules filling more or less of the interior
(fig. 3). This is the stage most abundant in amoebae containing two
nuelei. In fact, most individuals of the two- and four-cell stages in
Endamoeba coli in our material seem to be in the early prophase.

318 University of California Publications in Zoology |Vou. 20

In the one- and two-cell stages, when many chromatoidal bodies,
as well as the nuclei, fill the narrow space between the glycogen mass
and the periphery, the nuclei are exceedingly difficult to differentiate,
particularly if the chromatin granules have left their position on the
nuclear membrane. In a few cases only have the cysts been devoid,
or nearly so, of chromatoidal material (pl. 29, figs. 6, 7). The nuclei
are often obscured also by the change in the staining reactions of the
chromatin that may be noticed at this stage, in its failure to take
a dark stain with iron-haematoxylin or in its ease in destaining.
Frequently, as the chromatin gathers at the center of the nucleus, it
stains less intensely with iron haematoxylin, appearing as a grey mass
with a few darker staining granules within it (pl. 29, fig. 3). Later
the central mass takes an intensely dark stain and at the same time
shows a considerable increase in volume (pl. 29, fig. 4).

It is impossible to say whether any chromatin substance is extruded
from the nucleus, owing to the amount of darkly staining material
found in the extranuclear regions. There is no evidence of an
exchange of material in either direction, though it may well take
place. It is equally impossible, however, to consider the abundant
chromatoidal bodies as formed by nuclear extrusions alone, as they
are often present in great quantity and size before division begins
(pl. 29, fig. 1). Their abundance in the earlier stages of mitosis and
their gradually lessening quantity as mitosis proceeds, as is the case
also with the glycogen, would seem to indicate that both these sub-
stances serve as reserve food materials that are gradually used up
in the great metabolic activity required by the repeated processes of
mitotic division. The glycogen is formed before the chromatoidal
bodies appear, and it likewise is absorbed before the latter are. Cysts
containing more than four nuclei very rarely show any traces of
glycogen, while the presence of chromatoidal bodies is frequently
noted in such eysts.

About the time the chromatin granules have all disappeared from
the nuclear membrane, the chromatin in the central mass has assumed
the appearance of small, oblong granules, or rods, without definite
number or arrangement, but scattered through the major portion of
the interior of the nucleus (pl. 29, figs. 4, 5). A definite spireme or
skein was not observed, nor could the formation of the chromosomes be
followed in detail. The mass of granules becomes less in amount,
either by condensation or other metabolic change, and the double
chromosomes appear (pl. 29, fig. 7, lower nucleus).

1922] Swezy: Mitosis in Endamoeba Coli 319

The polar masses or daughter centrosomes are probably formed
from a part of the original karyosome but it has not been possible
to trace this throughout the changes in general massing of the
chromatin in the central part of the nucleus. As the polar masses
divide and move to opposite poles, a slender thread, the intradesmose,
is drawn out between the two portions and lies at all times within the
nuclear membrane. This is the same intranuclear structure as that
found in Councilmania laflewri which we called intradesmose (Kofoid
and Swezy, 1921) to distinguish it from the paradesmose of the flagel-
lates, the latter structure being outside of the nuclear membrane
(Kofoid and Swezy, 1915).

Along with the formation of the chromosomes, the nucleus becomes
elongated (pl. 29, figs. 4-7). The spindle is formed between the polar
masses within the nuclear membrane which persists throughout the
entire process of division. In the equatorial plate stage, the spindle
fibers, to which the chromosomes are attached, are found to le in the
outer surface of a double cone (pl. 30, fig. 13). The intradesmose is
found to lie close to the spindle fibers (pl. 29, figs. 8, 9; pl. 30, fig. 15),
and, as it soon loses its intense staining reactions, it becomes diffieult
to differentiate from the spindle fibers. It is probably retracted in
the late telophase along with the drawn-out nuclear membrane.

The first division of the nucleus was not found in the material
under observation, but it is probable that the mode of procedure is
the same in this as in the later divisions. In eysts containing more
than one nucleus the time of division of each is not always synchronous
for the different nuclei (pl. 29, fig. 7; pl. 31, figs. 19-23), which
accounts for the frequent appearance of individuals of Endamoeba
coli with five, six or seven nuclei, or any number between eight and
sixteen.

METAPHASE

There is some suggestion that the chromosomes divide early in the
prophase, as they appear double before the spindle is formed (pl. 29,
fig. 7). No conclusions regarding the relative positions of the chromo-
somes at this stage, or as to how they become attached to the spindle
fibers, could be drawn from observation on the material studied.
When the metaphase is reached the appearance of division in the
chromosomes is lost, and they are attached to the middle of the spindle
as twelve slightly irregular, compact, rod-like granules. In end view
these form a ring (pl. 30, fig. 13) in the middle plane of the nucleus
with a clear area in the center of the spindle.

320 University of California Publications in Zoology [Vou. 20

ANAPHASE

The anaphase is probably of longer duration than is the metaphase,
judging by inference from the relative number of individuals of both
stages observed. In fact, the majority of dividing nuclei found in the
material were in this stage, the next in abundance being the early
prophase.

In most eases, there seems to be little difference in the size and
shape of the chromosomes during the anaphase, although occasionally
one or two chromosomes may be somewhat smaller in size than the
others (pl. 30, figs. 10, 18; pl. 31, fig. 28). The chromosomes range
in size from .dp to 1.5p.

As the division of the chromosomes seems to occur before the
spindle is formed (lower nucleus in figure 7, plate 29), the separation
in the anaphase probably follows the original split, as in Giardia
(Boeck, 1919), and Trichonympha (Kofoid and Swezy, 1919). The
separation of the two moieties is not synchronous for the six chromo-
somes, but considerable variation is found between them in almost
every case examined. In many nuclei the variations in this respect
are striking, some of the chromosomes being well on their way toward
the poles before the final parting of the last daughter chromosomes is
completed (pl. 30, figs. 10, 11, 15; pl. 31, fig. 19).

As the chromosomes divide they move toward the poles, as the
spindle fibers shorten (pl. 30, fig. 15). Along with this change the
nucleus begins to elongate somewhat, becoming less rotund through
its middle and gradually assuming a dumb-bell shape (pl. 30, fig. 16).

TELOPHASE

With the migration of the chromosomes to the poles they soon lose
their identity and become massed together (pl. 30, fig. 16; pls. 31,
figs. 19-21). This process is accompanied by an elongation of the
nucleus and its constriction near the middle. The latter change
proceeds until the nucleus has become a long, slender dumb-bell (pl.
31, fig. 21), which may be drawn out until the sister nuclei come to
lie on opposite sides of the eyst. The nucleus parts in the middle,
the drawn-out points are retracted and two new spherical nuclei are
formed (pl. 30, fig. 17).

The complete reconstruction of the nucleus evidently takes place
before the prophase of the next division is entered upon (pl. 30, fig.
18). Corresponding steps in the process of mitosis of the nuclei seem

1922] Swezy: Mitosis in Endamoeba Coli 321

to be closely similar, if not identical, in the two-, four-, and eight-cell
stages. There is considerable decrease in actual size of the nuclei
with each successive division (pl. 31, fig. 23), giving to the eysts with
eight or more nuclei a greater disproportion between the size of the
eyst and that of the nuclei (pl. 31, fig. 22) than may be found in the
earlier stages, where the nuclei are relatively large (pl. 29, fig. 2).
Thus in the one- and two-cell stage the diameter of the nucleus may
be equal to one-third that of the cyst, while in the eight-cell stage the
diameter of the nucleus may be one-sixth, or even less than that, of
the cyst.

Division of the protoplasmic body has not been observed in Enda-
moeba coli, either in the active or in the eneysted amoeba. In the
latter case it may well be that it is delayed until after leaving the
body of the host and entering a new one. Figure 25, plate 31, would
suggest that the multinucleate somatella is released from the cyst
before dividing into the individual amoebas, though this may be the
result of repeated divisions in the active amoeba without plasmotomy
intervening, as individuals with two or more nuclei may occasionally
be observed.

DISCUSSION

The literature on the subject of division in the parasitic amoebae
is filled with confusion and misinterpretations, the result, largely, of
generalizations on too scanty data. The earlier conceptions of this

”?

process as a ‘‘simple amitosis’’ were, perhaps, unavoidable, but with
the constantly growing knowledge of the types of mitosis that may be
found in the Protozoa at the present time, modifications of these
earlier conclusions are to be expected as the result of further investi-
gations along this line. It seems very probable that simple amitosis
is as rare in the higher Protozoa as in the Metazoa.

In Dobell’s recent discussion (1919) of the nuclear behavior of
the human intestinal amoebae, he gives a number of division figures
of Endamoeba dysenteriae, taken from active amoebae from intestinal
ulcers of cats experimentally infected with human amoebae. The
type of division shown in these figures differs somewhat from that
shown herewith for Endamoeba coli, and it may be that the alien
environment which the human amoebae find in the cat has produced
some modifications which are not present when the amoebae occupy
their normal habitat, or that division in the active form differs from

322 University of California Publications in Zoology (Vou. 20

that in the cyst. In Hndamoeba coli Dobell found a typical spindle
in the first nuclear division of the eneysted amoeba, but he also states
that ‘‘the subsequent nuclear divisions are all similar, and resemble
those of Endamoeba histolytica.’’ Of the latter amoeba he says, ‘‘I
do not consider the nuclear division of Endamoeba histolytica to be a
regular mitosis. On the other hand, I cannot eall it an amitosis. It

,

seems rather to belong to an intermediate category.’’ Certain resem-
blances may be found in his figure 64, plate 4, to some which are
presented in this paper.

In Councilmania lafleuri (Kofoid and Swezy, 1921) the type of
mitosis is closely similar to that shown herewith for Endamoeba coli,
both having polar masses or centrosomes and an intradesmose. Couwn-
cilmama laflewri has, however, eight chromosomes, differing in this
respect from Hndamoeba coli with its six chromosomes.

The nuclear division of the active amoeboid phase of Hndamoeba
coli has not as yet been observed by any investigator. Free amoebae
witb two nuclei are occasionally met with and in one ease (pl. 31,
fig. 25) one with eight nuclei was seen. These are not common, how-
ever, and it is probable that binary fission is the general rule among
the active amoebae, and that it occurs before the organisms leave the
upper part of the large intestine. The individual shown in figure 25,
plate 31, may be one that has escaped from its cyst without dividing,
but it is also possible that such somatellas are sometimes formed with-
out eneystment as well as in the cysts, as in the ease of the parasitic
flagellates, Giardia (Kofoid and Christiansen, 1915) and Trichomonas
(Kofoid and Swezy, 1915).

The question of a division center or centrosome in the parasitic
amoebae is one about which conflicting opinions may be found in the
literature on the subject. Dobell (1919) found no structures which
he could regard as centrosomes in Endamoeba dysenteriae. In a brief
description of the division of Endamoeba dysenteriac, Mathis and
Mercier (1916) find, however, that such structures are present. In
1917 the same authors figure division in Hndamoeba legeri from
Macacus as a mitosis with centrosome, spindle, and centrodesmose
(= intradesmose). The centrosome originates in the karyosome and,
as it divides, the two daughter centrosomes draw out between them
a slender, darkly staining thread, which we have ealled the intra-
desmose. The spindle fibers are formed between the centrosomes
within the nuclear membrane (their figs. II, b-d, h). This method of
procedure is closely similar to that outlined above for Hndamoeba

1922] Swezy: Mitosis in Endamoeba Coli 323

coli. The prophase of the two amoebae seems also to be similar, as
their figure Ill-e differs from the same stage in Endamoeba coli only
in the lack of characteristic chromatoidal bodies.

The abundance of chromatoidal substances present in the early
prophase of Hndamocba coli obscures much of the development in
that period, the individuals from which the present studies were made
being especially rich in that substance. The lack of this substance in
Endamoeba legeri makes that species a more favorable object for
study of these early stages.

The polar masses or centrosomes in Hndamoeba coli are rather
large (spread out) and often diffuse (pl. 29, fig. 8, 9), or may be
small (pl. 30, fig. 13). In the telophase they are incorporated with
the chromosomes (pl. 30, figs. 15, 16) but their further history could
not be traced with certainty. It is probable that the centrosome forms
the karyosome in the new nucleus, while the chromatin of the chromo-
somes becomes wholly, or in large part, distributed over the linin
reticulum and nuclear membrane.

It is possible that a definitive centrosome may be present within
the larger structure which forms the polar mass, a suggestion of which
is found in the fact that this may be very small in some individuals.

The intradesmose is formed when the centrosome or karyosome
divides, and connects them until the final parting in the telophase.
The origin of this structure is shown with greater clearness in the
figures of Endamoeba legeri by Mathis and Mercier (1917), referred
to above, and is called by them the centrodesmose. It is often difficult
to differentiate the intradesmose from the spindle fibers, hence its
absence in some of our figures.

The differences to be found between the type of division described
above for Endamoeba coli and those described by other workers for the
various species of non-parasitic amoeba consist largely in the structure
of the spindle, centrosome, and intradesmose and in the number and
size of the chromosomes. The differences, however, are more apparent
than real, as a closer examination will show. The residual mass of
chromatin derived from the karyosome and forming the polar caps
and central spindle (intradesmose) is greater in amount in Naegleria
gruberi (Wilson, 1916) and Amoeba tachypodia (Glaser, 1912), than
in Endamoeba coli or Endamoeba legeri, but the behavior of the
structures is essentially the same in both groups. The division of
Amoeba hyalina, as described by Hartmann and Chagas (1910),
repeats the same process in its essential details, although the amount
of chromatin involved in the centrosomes is here quite minute.

324 University of California Publications in Zoology [Vou. 20

The division of these amoebae differs from that of the parasitic
flagellates in that the centrosomes and intradesmose are found within
the nuclear membrane, the homologue of the latter structure, the
paradesmose, and the centrosome of the flagellates always being out-
side of the nuclear membrane, the relation of the centrosome to the
flagella in the flagellate making the extranuclear position an essential
one for these structures in that group.

In a recently published paper Yoshida (1920) describes a process
of autogamy in Endamoeba coli in which two, four, or even six, nuclei

”?

may fuse to form a ‘‘synearyon: On the basis of his experiments,
he claims that ‘“‘the daughter-nuclei in the cyst stage correspond
exactly to the gamete nuclei of other Protozoa,’’ but gives no eyto-
logical evidence of gamete formation. As a further step in the
process of autogamy he finds that the amoebae develop numerous
vacuoles and their nuclei shrink and degenerate until they finally
burst. The phenomenon he has described is one occurring occasionally
in parasitic amoebae when undergoing degeneration outside of their
normal host, and has no known relation to sexual or ordinary develop-
mental processes. The process of ‘‘autogamy’’ was first described for
Endamoeba coli by Schaudinn (1903) and later figured by Hartmann
(1909) from descriptions in the earlier paper. Some of these figures
are similar to those shown on plate 29, of the one- and two-nucleate
amoebae with the large glycogen vacuole filling the center. However,
instead of adding the third stage, a four-nucleate amoeba, the first
stage, or a Blastocystis was again arbitrarily used as that resulting

7

from ‘‘autogamy’’ in the two-nucleate cyst. Along with these stages
Schaudinn added other phases of the yeast-like Blastocystis frequently
found in human faeces (Kofoid, 1921). This process has since been
figured in textbooks as the normal sexual process in amoebae. It has
been repeated in textbooks again and again for amoebae but thus far
no satisfactory cytological evidence has been brought forward to sup-
port such an interpretation for the ordinary steps in nuclear division
(pl. 29, figs. 1-7), or for the degeneration phases of amoebae that
have been placed in an alien environment.

1922] Swezy: Mitosis in Endamoeba Coli 325
SUMMARY

Nuclear division in Endamoeba coli is a simple mitosis which takes
place within the nuclear membrane.

The chromosomes are probably six in number and divide before the
spindle is formed.

Polar masses or centrosomes are formed by division of a chromatin
body probably derived from the karyosome. An intradesmose con-
nects these bodies as they separate and continues to unite them until
the nuclei part in the telophase.

The centrosomes, spindle, and intradesmose are contained within
the nuclear membrane, which is present throughout the entire process
of mitosis.

Three successive mitotic divisions take place, resulting in the pro-
duetion of eight nucleated cysts. A fourth division may also occur.

LITERATURE CITED
Borck, W. C.

1919. Studies on Giardia microti. Uniy. Calif. Publ. Zool., 19, 85-136, pl. 1,

19 figs. in text.
DOBELL, C.

1914. Cytological studies on three species of amoebae—4A. lacertae Hartmann,
A, glebae n.sp., A. fluvialis n.sp. Arch. f. Prot., 34, 139-189, pls.
(uke

1919. The amoebae living in man (London, John Bale, Sons & Danielsson).
Pp. vi+ 155, 5 pls.

GLASER, H.

1912. Untersuchungen iiber die Teilung einiger Amoében, zugleich ein Beitrag
zur Phylogenie des Centrosoms. Arch. f. Prot., 25, 27-152, pls. 3-8,
5 figs. in text.

HarTMANN, M.

1909. Autogamie bei Protisten und ihre Bedeutung fiir des Befruchtungs-
problem. Ibid., 14, 264-334, 27 figs. in text.

HARTMANN, M. and Cuaaas, C. ;

1910. Ueber die Kernteilung von Amoeba hyalina Dang. Mem. Inst. Oswaldo
Cruz, 2, 64-126, pls. 4-9, 3 figs. in text.

Koro, C. A.

1921. A eritical discussion of the chromidial formation of nuclei, of autogamy,
and of the multiple division of polyenergid nuclei, with special
reference to the unity of the mechanism of heredity in the Protozoa
and Metazoa. Anat. Rec., 20, 223-225.

326 University of California Publications in Zoology (Vou. 20

Koro, ©. A., and CHRISTIANSEN, E. B.
1915. (Qn binary and multiple fission in Giardia muris (Grassi). Univ. Calif.
Publ. Zool., 16, 30-54, pls. 5-8, 1 fig. in text.

Korop, ©. A., and Swexzy, O.

1915. Mitosis and multiple fission in trichomonad flagellates. Proe. Amer.
Acad. Arts Sci., 51, 289-278, pls. 1-8, 7 figs. in text.

1919. Studies on the parasites of the termites. III. On Trichonympha cam-
panula sp. nov. Univ. Calif. Publ. Zool., 20, 41-98, pls. 5-12, 4 figs.
in text.

1921. On the free, eneysted, and budding stages of Councilmania lafleuri, a
parasitic amoeba of the human intestine. Ibid., 20, 169-198, pls.
18-22, 3 figs. in text.

Marais, C., and Mercier, L.
1916. La division simple chez Entamoeba dysenteriae. C. R. Soc. Biol., Paris,
79, 982-984.
1917. Affinités d’Hntamocba legeri Mathis et d’E. coli. Arch. Zool. Exp. et
Gen., 56, 63-72, 4 figs. in text.

ScHaupinn, F.
1903. Untersuchungen iiber die Fortpflanzung einiger Rhizopoden. Arb. Kais.
Gesundh., 19, 547-576.

Witson, C. W.
1916. On the life-history of a soil amoeba. Univ. Calif. Publ. Zool., 16,
241-292, pls. 18-23.
Yosurpa, K.
1920. The encystment of dysentery amoebae in vitro. Jour, Exper. Med., 28,
387-413, pls. 30-43.

EXPLANATION OF PLATES

All drawings are of Endamoeba coli and were made with camera lucida from
preparations fixed in Schaudinn’s fluid and stained with iron haematoxylin. Mag-
nification, 2500.

PLATE 29

Fig. 1. Mononucleate cyst with nucleus in the prophase and the cytoplasm
filled with typical chromatoidal bodies.

Fig. 2. Binucleate cyst with nuclei in the resting phase.

Fig. 3. Binucleate cyst with nuclei in early prophase.

Fig. 4. A later prophase with the chromatin leaving the nuclear membrane.
Fig. 5. Prophase with the nuclear membrane entirely free from chromatin.
Fig. 6. Prophase.

Fig. 7. Late prophase with the spindle formed in one nucleus and not yet

begun in the other. Note evidence for splitting of the chromosomes in lower
nucleus.

Fig. 8. Anaphase. Note abundance of chromidial bodies in cytoplasm.
Fig. 9. Same stage as that of figure 8.

[328]

NWAOIE: H2X0)

UNIV, CALIF, PUBL. ZOOL

PLATE 30

Fig. 10. Early anaphase or late metaphase. Some chromosomes in each
nucleus not yet divided.

Fig. 11. Anaphase with one chromosome not yet divided.
Fig. 12. Anaphase.

Fig. 13. One nucleus in metaphase—seen from one pole—and the other in
early anaphase. Large glycogen vacuole absent.

Fig. 14. Anaphase.

Fig. 15. Late anaphase.

Fig. 16. Telophase. Chromosomes have begun to lose their individuality.
Fig. 17. Telophase. Note lack of synchronism in division of nuclei.
Fig. 18. Reorganization nearly completed in the four nuclei.

[330]

UNIV. CALIF. PUBL, ZOOL. VOL. 20

PLATE 31

Fig. 19. Third division of nuclei. Each of the four nuclei shows a different
phase. ;

Fig. 20. Telophase. Beginning of constriction of the nuclei.

Fig. 21. Later telophase. Glycogen vacuole absent in these stages.
Fig. 22. Completion of third division.

Fig. 23. Early prophase of third division.

Fig. 24. Fourth division on its way.

Fig. 25. Active amoeba with eight nuclei.

Fig. 26. Active binucleate amoeba with huge glycogen vacuole.

Fig. 27. Eight-nucleate eyst with chromatoidal substance almost absorbed.
Fig. 28. Hight-nucleate cyst with typical, splinter-like chromatoidal bodies.

[332]

UNIV, CALIF. PUBL. ZOOL, VOL, 2G [SWEZY ] PLATE

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UNIVERSITY OF CALIFORNIA PUBLICATIONS

IN
ZOOLOGY
Vol. 20, No. 14, pp. 333-364, plates 32-36, 5 figuresintext November9, 1922

THE NEUROMOTOR APPARATUS OF
PARAMAECIUM

BY

CHARLES WILLIAM REES

CONTENTS oren
Introduction................ Perens be 334
Acknowledgments..... wa 334
Review of literature .... ie 334
Micro-injection.................. ne oa 336
Technique and Methods...................... : 336
@uilture me se : pesake ate 336
SNE CHIN GUC eee retececs eoce-cs. ws 336
IVIOT NOLOGY fac seccsceecseeeestacess: ae 338
Brief description of Paramaeciwm 338
Neuromotor apparatus..... Afi. 338
Neuromotor center ........ 340
Peripheral fibers.......... ; 341
Fibers of the cytostome and cytopharynx. : 342
The neuromotor system during division.. 344
The neuromotor apparatus of Paramaecium compared with that of the
flagellates and with other ciliates........... Le ALS eet Ont 345
FF RTS OUT ET ell Meee Ee cc keecr Suna cavenivers aa ee 346
Micro-injection of vital dyes...... it ; . 846
Intravitam staining........ 2 Ooh A SPR ORT fs 346
PNsxi abe reiclemits tee ee este case cacstncsceseenss 347
Micro-dissection «00.00.00... ee tee eee : é 349
NDISCLISS] OL eee 8 ee ne eters sass : es : : Jee 350
\SUHTODE SFI a s-eeconeoceupeqeeeRBee ieee c: ee : ee eo
intberatbure Cubed: esescassvctersts-ces-osee+-0e-> eee pee cccyae ts srenset eevaresutesetersvastatecuetvese: 353

Piscp lent OMMO IGP ALES rere teceeeyrecerecasssacecsssssseccaesesecsscececsesneaseerncee peers Re O00

334 University of California Publications in Zoology [Vou. 20

INTRODUCTION

Paramaecium, because of its universal occurrence in aquaria and
the ease with which it may be cultured, has long been a favorite
organism for use in class instruction and laboratory investigation.
It is well adapted for this study because it is a generalized rather
than a highly specialized type. For the same reason a description of
the system of neuromotor fibers in Paramaecium, a system equalling
in complexity the nervous systems of many of the lower Metazoa, will
be of general interest.

ACKNOWLEDGMENTS

The present work was conducted during the past two years at the
University of California under the direction of Dr. Charles A.
Kofoid, and to his profound insight most of the discoveries were due.
Dr. Charles VY. Taylor rendered invaluable assistance in explaining
and demonstrating micro-injection and micro-dissection equipment.
The fixing, staining, imbedding, and sectioning were directed by Dr.
Olive Swezy, whose expert advice in the use of Heidenhain’s iron
haematoxylin made possible the wonderfully clear differentiation of
the neuromotor fibrils. Professor A. B. Domonoske, of the Depart-
ment of Mechanical Engineering, Mr. H. N. Cooper, Curator of the
Department of Physical Chemistry, and Mr. W. J. Cummings also
gave much appreciated assistance on technical matters of apparatus.

REVIEW OF THE LITERATURE

A review of most of the literature on conduetile fibers in the
Protozoa was written by Dr. Taylor (1920). The only addition made
here is a discussion of some of the papers pertaining particularly to
Paramaecium.

In 1902 Neresheimer made attempts to stain nerve elements in
Paramaecium, Spirostomum, and Stentor coeruleus with Mallory’s
triple stain. He found certain myonemes in Stentor and Spirostomum
but failed to find any in Paramaecitum. He concluded, therefore,
that none were present and set about to prove this by the following
method: He treated both Paramaecium and Stentor with equally

1922] Rees: The Neuromotor Apparatus of Paramaecium 335

dilute solutions of narcotics, e.g., morphine, strychnine, caffein,
curare, and nicotine, and found Stentor to be easily narcotized, while
Paramaecium remained apparently normal.

Khainsky (1910) made a contribution to the morphology of
Paramaecium, the importance of which he did not realize. He fixed
the animals with concentrated bromine water and found that none
of the trichocysts was discharged, whereas they are frequently dis-
charged in other fixatives. In his plate 1, figures 6 to 8, he shows
distinet fibers connected to the basal granules of the cilia. He
comments as follows: ‘‘Die Gleichartigkeit der Fibrillen mit dem
tibrigen Cilienteile begeist dass ihre Hauptrolle in der Ernahrung
und dem Wachstum der Cilie besteht.’’

Most of the recent work on contractile and conductile elements of
protozoans has been done by the University of California Zoological
Laboratory. The name neuromotor apparatus was given by Kofoid
(1915) to the integrating fibrillar complex in Giardia associated with
the blepharoplasts, parabasal bodies, and other active organelles of
locomotion. The literature is already extensive, comprising 473
printed pages and 42 plates in fifteen special research papers.

A neuromotor apparatus was described by Sharp (1915) in
Diplodinium ecaudatum, a very complex member of the order Oligo-
tricha. Parts of this system had been previously deseribed (Braun,
1914), but either a supporting or contractile function had been
ascribed to it. Yocom (1918) described a similar system in the
hypotrichan Euplotes. These two ciliates, the one a parasite, the
other a free-living form, possess neuromotor systems easily com-
parable. In each the system consists of a motorium from which
fibers extend to the motor organelles and also to the membranelles
surrounding the cytostome. The motorium is in no way connected
with the nucleus. Taylor (1920), by the method of micro-dissection,
demonstrated that the system in Euplotes possesses conductile fune-
tions. His conclusions are based on three hundred and fifteen experi-
ments on which extensive notes were taken. His conclusions were as
follows:

These experimental evidences do not support the assumption that the fibrillar
system in Euplotes is either contractile or supporting in function, but they
indicate that this complex system of fibers does possess conductive properties

functioning in the codrdination of the movements of the locomotor organelles
with which it is intimately associated (Taylor, 1920).

336 University of California Publications in Zoology [ Vou. 20

MICRO-IN JECTION

The successful search for a neuromotor apparatus in Paramaecium
was the outgrowth of micro-injection studies (Rees, 1922). To
accomplish micro-injection, it was found necessary to isolate the
organisms in rounded drops, so that they would not be disturbed by
the forces of surface tension (Rees, 1922). This led to a search for
a method of making stained preparations of undistorted animals,
which was accomplished by fixing them in centrifuge tubes instead
of applying the fixative after they had been attached to the slide by
means of egg albumen.

TECHNIQUE AND METHODS

CULTURE

The animals for this work were obtained from wild stock that had ~

been brought in with débris from the creek bed and cultured in
covered battery jars. After being injected or otherwise operated
upon, they usually thrived if given plenty of food. The importance
of food was demonstrated only after the death of many organisms
within three or four hours after the operation. The practice had
been to place them in small drops of culture fluid from which other
individuals had been removed by coarse filter paper. Even the
controls did not survive twenty-four hours under such conditions.

A careful search was first made for some toxic substance. It was
thought that volatile poisons were present in the vaseline used to seal
down the covers or that sufficient of the salts of mercury were formed
in the injecting and isolating instruments to prove fatal. After
weeks of investigation, a rich autoclaved and fully ripened hay
infusion was substituted for the filtered culture fluid. The result was
astonishing. The animal survived after the operation and grew
rapidly, often dividing twice within twenty-four hours.

TECHNIQUE

Contrary to the statement of Neresheimer (1902), a fibrillar
system may be differentiated in Paramaecium with Mallory’s triple
connective tissue stain, even when the animals are attached to the
slide coated with egg albumen. They were fixed in Zenker’s fluid

1922] Rees: The Neuromotor Apparatus of Paramaecium 337

which was dropped on the slide while hot. The same processes of
staining were employed as were used by Sharp (1917) and Yocom
(1918). The system appears as a network of fine anastomosing
fibrils in the ectoplasm.

Another method of staining with Mallory’s was as follows: The
organisms were killed and fixed either in Zenker’s fluid or picro-
mereuric (Yocom, 1918) solution in centrifuge tubes. They were
placed by means of a pipette in a small piece of folded silk bolting
cloth. In this they were run through the stains and alcohols to
xylol, then transferred to a Lefevre watch glass and concentrated in
the groove by means of a camel’s hair brush. A drop of balsam was
added and they were placed on the slide by means of a pipette. The
surface fibrils did not show in animals so stained, but clear views of
the eytostomal and cytopharyngeal systems were obtained.

Tron alum haematoxylin was by far the best stain. Schaudinn’s
solution used hot was the best fixer. The animals nearly always dis-
charged their trichocysts. Picro-mercuric used hot sometimes fixed
the animals with trichoeysts intact, but they did not stain so well as
when Schaudinn’s fluid was used. The fact that trichocysts are dis-
charged in Schaudinn’s is fortunate for whole mounts, as otherwise
the dorsal and ventral whorls of fibrils would not be made visible. In
only four of over twenty trials was success attained with whole
mounts, the difficult part of the process being the destaining, which
required from two to four hours with cold 2 per cent iron alum.
Sometimes the animals turned yellow or became opaque and had to
be discarded. When destaining goes on as is desired, the organisms
change color from black to blue and then to bluish gray, and the
protoplasm is always quite clear. The end of the destaining process
is reached when they are quite gray with the nucleus still quite dark.
Only faint outlines of the neuromotor system can be seen at, this
time even with oil immersion. The whorls of ramifying fibrils become
clearly differentiated only after clearing in xylol and balsam.

Infiltration and imbedding were carried out at first in gelatine
capsules as described by Metealf (1908), but later it was found that
glass capsules made of tubing by sealing one end in the flame were
more satisfactory.

As will be shown by an examination of the plates, the thick sec-
tions (15) were very valuable because they gave more extensive
views of the cytopharyngeal fibers than did the thin ones. The lines
of basal granules and trichocysts were most clearly seen in unstained

338 University of California Publications in Zoology [Vou. 20

living animals isolated in small drops, but after having been dis-
covered there they were found in whole mounts stained with both
Mallory’s and iron haematoxylin. The lines of basal granules were
also brought out when the animals were killed in 10 per cent formalin
and fixed twenty-four hours in the dark in Von Rath’s osmic acid
picro-mereurie fixative.

When animals were killed in 1 per cent strychnine solution, the
cytoplasm, with the included trichocysts, contracted to a small sphere,
leaving the transparent pellicle in its original position. The tricho-
eysts were then discharged and looked like astral rays protruding
from the cytoplasm. The clearest views of the ciliary lines in the
pellicle were then obtained. Wonderfully clear views of trichocysts
and basal granules were also obtained when animals killed in con-
centrated bromine water were placed on a slide and a little aniline
blue solution run under the cover.

MORPHOLOGY
BrigF DESCRIPTION OF PARAMAECIUM

In deseribing Paramaecium, the terms oral and aboral will be used
instead of dorsal and ventral. Otherwise, the terminology is the
some as that used for bilaterally symmetrical animals.

Paramaecium was well described by Ellis (1769) as ‘‘an animal
with a longish body and a groove in one end like a gimlet.’’ The
eytostome, a slit-like aperture, extends from the caudal end of this
groove and lies in the posterior two-thirds of the body. It is con-
tinued as the eytopharynx which runs obliquely caudad to the
vicinity of the posterior contractile vacuole. There are two contractile
vacuoles, one anterior and one posterior, which pulsate rhythmically
about twenty times per minute. The macronucleus is oblong, deeply ~
staining about one-third as long as the body, and its posterior end
lies near the anterior end of the ecytostome. The micronucleus com-
monly lies in a little groove in the macronucleus. The cytoplasm

‘

contains numerous food vacuoles in continual cyclosis.

Neuromotor APPARATUS

In this work, studies have been made of the outer endoplasm and
the ectoplasm and pellicle. The ectoplasmic structures include the
eytostome and ecytopharynx, also longitudinal rows of cilia, and
trichocysts which we have found to be arranged in whorls. The

1922] Rees: The Neuromotor Apparatus of Paramaecium 339

membranelles of the cytopharynx and the motor and defensive
organelles covering the surface of the body have been found to be
connected by an integrated fibrillar system which has its center in
the outer endoplasm at the anterior end of the eytostome.

Maupas (1883), Biitschli (1887-89), Kélsch (1902), Maier (1903),
Schuberg (1907), and Khainsky (1910) have described the pellicle
of Paramaecium. All are agreed that the cilia arise from depressions
in the surface, while the Indian club-shaped trichocysts pierce
deeply into the ectoplasm and reach the surface of papillae arranged
in a definite pattern. The fact that the ciliary and trichocyst lines
are not parallel was also discovered. They were described by
Biitschli (1889) as ‘‘rechtwinklig gekreuzt.’’ Maupas describes the
pellicle as a pattern of rhombohedral plates, the cilia being in the
center, the trichocysts in the corners of these plates. Biitschli,
Kolsch, and Maier thought the plates were hexagonal. Schuberg
found rhombohedrons, hexagons, and parallelograms. Khainsky’s
observations confirm those of Schuberg.

But it is only when one makes maps of the entire pellicle that the
true relationships of these various patterns are revealed and the
idea of grooves and ridges rather than depressions and papillae is
obtained. The terminology employed by Kofoid and Swezy (1919)
to deseribe Trichonympha campanula is here employed to describe
Paramaecium. In Trichonympha the cilia arise from the ridges, in
Paramaecium they arise from the grooves, but in both animals they
are arranged in longitudinal rows. On the aboral surface of the latter
they are almost parallel; on the oral side, slightly oblique and the
rows from opposite sides meet in a series of V’s. The apices of these
V’s lie in a line that runs longitudinally through the cytostome and
hence obliquely through the oral surface from the anterior to the
posterior end. This is the ciliary suture (fig. A, ¢.s., also pl. 35, fig. 26,
and pl. 36, fig. 30).

The ridges differ from those of Trichonympha in that they are not
parallel, but are arranged with reference to the neuromotor system
in whorls (figs. A and B, also pl. 35, figs. 27, 28, and pl. 36, figs. 31,
33). For this reason, they are cut across at regular intervals by the
grooves so that they appear as papillae which constitute interrupted
ridges. It is the intercrossing of these whorls of trichocysts and rows
of cilia that gives the various rhombohedral, hexagonal, and paral-
lelogrammic patterns previously described. The courses of the whorls
being the same as those of the neuromotor fibers, it will not be
necessary to describe them separately.

340 University of California Publications in Zoology —[ Vou. 20

NEvROMOTOR CENTER

All the fibers of the neuromotor system ramify out from the
neuromotor center (fig. A, .c.) which is just anterior to the eyto-
stome near the oral surface. In plate 35, figure 22, are seen two
bodies to which fibers converge. This figure was drawn from a

MU ned,

{ry

mace~ -+-
- ---memb.

----cyt.

ab. wh,--+-=
-- cyp.

tre

~.- ---p.fibe-+—- - AEE

Fig. A. Semi-diagrammatie sketch of Paramaecium caudatum, oral view,
showing oral whorl of peripheral fibers and the ciliary lines, ciliary suture, and
trichocyst ridges. :

Fig. B. Aboral view of Paramaecium caudatum, showing aboral whorl,
ciliary lines, and trichocyst ridges. Abbreviations: c.v., contractile vacuole;
c.s., ciliary suture; o.gr., oral groove; mac., macronucleus; c.g7r., ciliary
grooves; n. c., neuromotor center; or. wh., oral whorl; mic., micronucleus; memb.,
cytopharynx membranelle; cyt., cytostome; cyp., cytopharynx; t.r., trichocyst
ridges; p. fib., peripheral fibers.

Figures A and B are reprinted by permission of the editor of the American
Naturalist from Rees (1921).

1922] Rees: The Neuromotor Apparatus of Paramaecium 341

section fifteen microns thick which was fixed in picro-mercurie,
stained twelve hours in haematoxylin, and destained until almost
white. A small body to which the cytostomal fibers converge may
also be seen in well destained whole mounts. The fact that all the
fibers of the system converge toward a point in this region suggests
a motorium: a body that functions as a codrdinating center or in some
way provides a contact of fibers effecting a basis for such inter-
connection and coordination.

PERIPHERAL FIBERS

For convenience the neuromotor system will be deseribed under
the two headings, peripheral fibers, and eytopharyngeal fibers. The
peripheral fibers are arranged in two whorls, one on the oral, the
other on the aboral side (figs. A and B, or. wh., ab. wh.; pl. 32, figs.
1-6, pl. 34, figs. 18-19).

The oral whorl is the more extensive, covering the entire oral side
and extending, on the left, over part of the aboral side. The fibers
lie very near the surface, impinging upon the endoplasm as they
approach the neuromotor center. They are gray when stained with
iron haematoxylin, and red when stained with Mallory’s. The thick-
ness of the individual fibers has not been determined but they appear
very fine when seen under a magnification of 1330 diameters. In the
oral groove when followed from their diverging ends in the ectoplasm
toward the neuromotor center, their course is obliquely caudad to
the margin of the ecytostome, where they turn and converge obliquely
cephalad. Over the remainder of the surface, they run in gracefully
curved lines directly toward the neuromotor center. The anastomos-
ing ends in the ectoplasm are very numerous and profusely branched,
forming a thick fibrillar complex (figs. A and B; pl. 32, figs. 1, 3, 4;
pl. 34, figs. 18-19). z

The fibers of the aboral whorl converge, forming a large V-shaped
figure with the apex on the left, opposite but slightly posterior to the
eytostome (fig. B; pl. 32, figs. 2, 3, 4). Here they appear to dip
into the endoplasm in their course toward the neuromotor center.
The ramifications of the fiber ends of this whorl resemble, in every
respect, those of the aboral whorl.

The meeting places of the fibers of the oral and aboral whorls in
the ectoplasm cannot be made out very distinctly, because, in order
to see them, the animals must lie either on their right or their left

342 University of California Publications in Zoology [Vou. 20

sides. Being thicker through, in this direction, too much of the light
is shut out. It will be noted that the oral whorl converges from both
sides toward the ‘‘center,’’ while the aboral whorl converges from
only the right side. For this reason, the meeting place on the right
side of the animal is formed of diverging fiber ends (pl. 32, fig. 4).
On the left the fibers of the oral whorl meet the converging apex of
the aboral whorl (pl. 32, fig. 2). Thus the entire ectoplasm is supplied
with a thick complex of diverging fibers.

2?

Actual connections of fibers to the basal granules are figured in
plate 33, figures 7-12, and plate 35, figures 21. 25. Kahinsky (1910)
confirms the presence of these fibers, but calls them ciliary rootlets.
They can be made out clearly only in thin sections (not over
2.5 microns).

The facts that in whole mounts and thick sections the neuromotor
fibers can be traced to the immediate vicinity of the basal granules
and that in thin sections actual connections can be seen are submitted
as proof that the ciliary rootlets represent the connections of the
neuromotor fibers with the basal granules.

The figures cited above also show fine fibers connected to the inner
ends of the trichocysts. These also are believed to be neuromotor
fibers innervating the defensive organelles of the body.

FIBERS OF THE CyTOSTOME AND CyTOPHARYNX

The oral groove (fig. A, or. gr.) extends from the anterior end of
the animal to the region of the posterior two-thirds of the body. As
previously described, the peripheral fibers, only, underlie the surface
of this groove. They exhibit no special modification in this region.

The eytostome is an elongated oval aperture extending obliquely
caudad from the posterior margin of the oral groove (fig. A. cyt.).
Around its margin is a row of peristomal cilia. Two fibers connect
the basal granules of these cilia with the neuromotor center. They
run one in each margin and are continued posteriorly into the
eytopharynx.

The cytopharynx begins at the anterior margin of the cytostome.
From this point to the posterior eytostomal margin it is a V-shaped
depression (pl. 33, fig. 8). Then it becomes a subeylindrical structure
except at the extreme posterior end, where there is an enlargement.
The enlargement is not perfectly spheroidal (pl. 33, fig. 11), but is
broadly indented on the oral side. The posterior extremity of the

1922] Rees: The Neuromotor Apparatus of Paramaecium 343

eytopharynx is near the posterior contractile vacuole and ends
abruptly at the endoplasm where the food vacuoles form.

There are two membranelle zones in the cytopharynx, one of
which, the anterior, was described (Maier, 1903) as an undulating
membrane. But careful studies of cross as well as longitudinal see-
tions and also of the living animal lead us to believe that this zone is
made up of a series of flat, paint-brush-like membranelles rather than
connected longitudinal rows of cilia such as would constitute an
undulating membrane (pl. 33, figs. 9, 10; pl. 35, figs. 21, 23).

The anterior zone extends from the beginning of the eytopharynx
to the enlargement. Except in the posterior region, the cilia of each
membranelle are of about equal length, but at the enlargement each
brush becomes progressively longer.

A fan of heavy fibers, each of which is of about twice the diameter
of a peripheral fiber, runs from the neuromotor center directly to the
anterior membranelle zone of the cytopharynx. Some of these fibers
may be seen in the living unstained animals as well as in most whole
mounts and cross and longitudinal sections. One gets an exceptionally
good view in thick well destained longitudinal sections (pl. 35, fig. 22).

Two of the fibers, on the right margin of the fan, are heavier than
the others. In thin cross-sections of the eytopharynx a structure
made up of basal granules is seen resembling a basal plate (pl. 33,
fig. 10). Maier (1902) describes this plate but does not mention the
fibers. The two heavy fibers above mentioned run along the margins
of each plate. All the fibers of the fan are about equidistant, almost
parallel, and do not branch. They have not been traced positively
beyond the anterior membranelle zone.

The posterior membranelle zone is less regular in outline than the
anterior, and less extensive. It was clearly seen first in a section
fifteen microns thick stained with Mallory’s. This zone begins as a
series of narrow, rounded, paint-brush membranelles just anterior to
the enlargement on the left side of the cytopharynx. Farther pos-
teriorly in the enlargement it spreads out on the indented surface,
the basal granules of its cilia being in the oral side (pl. 33, fig. 11;
pl. 35, figs. 21, 23). So far as is known, this posterior membranelle
zone has not been previously described.

Two fibers were noted above (page 342) running from the neuro-
motor center in each margin of the cytostome to the basal granules of
the peristomal cilia and on into the cytopharynx. They run into the
posterior membranelle zone (pl. 35, fig. 26) and branch profusely
even into the endoplasm beyond it.

344 University of California Publications in Zoology | Vou. 20

The neuromotor system of Paramaecium, therefore, as has been
demonstrated by the foregoing description of the neuromotor center
and the peripheral and cytopharyngeal fiber systems, is, from a
morphological point of view, a structurally adapted, coordinating
mechanism. It is easily compared with a metazoan nervous system,
being composed of fibers that connect the organelles of locomotion,
nutrition, and defense with a definite center.

THE NEUROMOTOR SYSTEM DURING DIVISION

One is surprised in reading the literature of Paramaecium to find
only two accounts of the fate of the old eytostome and eytopharynx
in dividing animals and of the origin of new ones. One of these is
quoted by Doflein (1916) from R. Hertwig (1890). The old eytostome
is represented as budding off a new one which becomes the eytostome
of the posterior daughter.

The second account is by Child (1916) who finds that the old
cytostome and cytopharynx are dedifferentiated and disappear. Jen-
nings (1908) had previously observed the same process, also the
occurrence of a slight shortening of the body in animals about to
divide. The new cytostome and cytopharynx of the posterior daughter
are redifferentiated in the position occupied by the old eytostome.
The cytostome and cytopharynx of the anterior daughter are entirely
new structures (figs. C, D, E).

The accuracy of this account I verified with only a 16-millimeter
objective and 20x oculars when animals from a rapidly dividing
culture were transferred to a Syracuse watch glass. Dividing
animals usually remain attached to some débris in the bottom of the
dish during the entire process. In one case observed, the entire
process from the time division became manifest to the separation of
the two daughter animals required forty-five minutes.

A complete account of the changes in the neuromotor apparatus
during the division process cannot as yet be given. It is certain that
the eytostomal and cytopharyngeal fibers of the anterior daughter
are new structures, as they appear very soon after the constriction
(pl. 34, fig. 20). Since the origin of a new eytostome gives rise to a
new center, it is likewise certain that the peripheral fibers of the
anterior daughter are to a very large extent newly formed. But since
the fibers, other than those of the eytopharynx, were not seen in the
stained animals in division, the extent to which dedifferentiation
and redifferentiation occurred in the peripheral fibers of the posterior
daughter has not been ascertained.

3922] Rees: The Neuromotor Apparatus of Paramaecium 345

The evidence does not indicate that the neuromotor fibers take part
in mitosis or division. It indicates rather that the behavior is very
similar to that in Euplotes (Yocom, 1918). In the latter, however,
the cytostome of the mother anima! marks the place of origin of the
eytostome of the anterior rather than the posterior daughter. The
difference here is no doubt accounted for in the posterior location of
the eytostome of Paramaecium.

D

Figs. C, D, E. Three views to show fate of old cytostome, cytopharynx,
and contractile vacuoles, and the origin of new structures in the two animals.
Slightly modified after Child. Abbreviations: v., contractile vacuoles of un-
divided animals; v.l., new contractile vacuoles; mic., micronucleus; 0. gr., oral
groove; mac., macronucleus; cyt., cytostome; cyp., cytopharynx.

THe NEUROMOTOR APPARATUS OF PARAMAECIUM COMPARED WITH THAT
OF THE FLAGELLATES AND WITH OTHER CILIATES

The neuromotor fibers of flagellates are connected with the centro-
blepharoplast complex which functions in mitosis.

Trichonympha is a flagellate covered with cilialike flagella (Kofoid
and Swezy, 1919) which are connected to the blepharoplast by a net-
work of oblique fibers. In Paramaecium the cilia are also connected
to a definite center by fibers. It is significant that in these two

346 University of California Publications in Zoology {Vou. 20

unrelated organisms whose life habits are very different similar con-
ductile elements should be evolved. The fundamental difference is
that so far as is known in Paramaecium the neuromotor system does
not function in mitosis and division and is not a part of the mitotic
apparatus, as in flagellates.

The system in Paramaecium resembles in many ways that in
Diplodiniuum (Sharp, 1914) and Euplotes (Yocom, 1920). In all
three animals a set of fibers runs from a center to the eytostomal and
eytopharyngeal organelles and also to the organelles of locomotion.

Paramaecium is unique among the animals thus far deseribed in
possessing an innervated system of defensive structures, the tricho-
eysts. It is to be regarded as a generalized member of the ciliate
eroup. The fibers are more numerous and finer and not organized
into tracts. The codrdinating center is less highly developed and
harder to differentiate.

EXPERIMENTAL

Three different experimental methods were utilized in attempting
to demonstrate that the fibrillar system of Paramaecium is conductile.

Micro-INJEcTION oF VITAL DYES

Micro-injection of vital dyes was used to determine whether the
fibers take the same stains as do nerve fibers in the Metazoa. Child’s
method of determining the axial gradient was employed (Child, .
1916) to find whether or not this gradient in Paramaecium is related
to the physiological organization that should exist in an animal with
a complex fibrillar system whose function is to receive and transmit
stimuli. The third method is that of micro-dissection similar to the
experiments of Taylor (1920) on Euplotes patella.

INTRA VITAM STAINING

Wilson (1910) summarized a method of staining nerves intra
vitam in the Metazoa with Griibler’s methylene blue. The stain was
injected into the living tissue very soon after the death of the animal.
In about twenty minutes to two hours the nerve fibers were stained.
The tissues were then fixed in saturated ammonium picrate or 7 per
cent ammonium molybdate and dehydrated quickly in 100 per cent
aleohol or glycerine.

1922] Rees: The Neuromotor Apparatus of Paramaecium 347

To demonstrate the presence of nerve fibers in Paramaecium by
modifications of this method, a one-tenth of one per cent solution of
the stain was injected into the living animals. They were then
examined under oil immersion almost continuously for periods vary-
ing up to four hours. As long as they lived, the ectoplasmie struc-
tures did not stain. The endoplasmic inclusions stained light green.
At death, the animals became opaque, so that fibers could not be seen.

Paramaecium was also placed over-night in a one-four-hundredth
per cent solution of the stain. As nearly as could be determined the
results were not different from those where micro-injection was
practiced.

AXIAL GRADIENTS

Alcohol and other narcotics, e.g., morphine hydrochlorate 1 per
cent, antipyrin 1 per cent, nicotine 49 per cent, and strychnine 1 per
cent were used rather than potassium cyanide in determining the
axial gradient (Child, 1916) for two reasons: (1) The animals are
slowed down in the narcotics so that they may be more carefully
observed. This is not the case with potassium eyanide. (2) Neres-
heimer (1902) used the above nareoties, and I desired to check on his
experiments, from which he concluded that Paramaecium does not
behave in these solutions as do animals with nervous systems.

According to the conception of gradients, the physiologically
anterior end of an animal does not necessarily coincide with the
morphologically anterior end, but the former is the region having the
highest metabolic rate. Tashiro demonstrated that a nerve fiber has
a higher metabolic rate than other structures of protoplasm. He also
proved that the gradient in an efferent nerve is from the ‘
toward the end organ. In an afferent nerve, this gradient is reversed
(Tashiro, 1917).

From the foregoing, it is clear that if the fibrillar system in
Paramaecium is conductile, the physiologically anterior end should

“eenter’’

be the region of the neuromotor center just anterior to the cytostome.
But we should also expect the end morphologically anterior to possess
a high metabolic rate because of the presence in this sensitive region
of so many fibers which conduct afferent impulses. The avoiding
reaction in particular (Jennings, 1906) results from the transmission
of contact stimuli from this anterior region and the resulting codrdi-
nated movements of the neuromotor organs, causing the animal to
reverse its direction and then to swim forward in a different course.

348 University of California Publications in Zoology [| Vou. 20

In one respect, the experiments were disappointing. Paramaecium
did not disintegrate in the manner of planarians and annelid worms.
Instead blisters appeared invariably over the posterior contractile
vacuole. Sometimes the animals died without further distortion. At
other times, the blister grew larger and larger until the ectoplasm
was disrupted and the endoplasm flowed out. For this reason, the
metabolic rate of the region of the neuromotor center was not
determined.

But it was found that the anterior cilia invariably ceased beating
first, from five to fifteen seconds earlier than the cilia of the cyto-
pharynx and posterior end. The following is a typical experiment.

EXPERIMENT NUMBER 6

11:50 a.m. Animal isolated in 6 per cent alcohol to 20 ¢.c. of which a small
drop of 1 per cent methylene blue had been added.

11:52 A.M. Circus movements to the left.

11:56 A.M. Movements slower, slight blister oyer posterior contractile vacuole.

12 mM. End over end movements very slow. Blister much larger.

12:05 P.M. Anterior cilia cease.

8 seconds later. Cytopharyngeal cilia cease.

2 seconds later. Posterior cilia cease.

When 4 per cent alcohol was used, the process often required
thirty minutes, but the events were in every ease similar to those
above described.

The animals lived in 1 per cent antipyrin about twelve minutes, in
Vo per cent nicotine about twenty minutes, in 1 per cent morphine
hydrochlorate about seventeen minutes, in 1 per cent strychnine about
five seconds. Except in the strychnine, which was used too con-
centrated, the behavior was similar to that observed in alcohol.

The experiments show that there is a gradient in Paramaecium
of a nature that one would expect if the fibers are conductile in
function.

The experiments certainly do not support Neresheimer’s conten-
tion that the organisms cannot be narecotized. The movements of
Paramaecium are appreciably slowed down in all the narcotics used.
In one case an animal remained quiet in 4 per cent alcohol for
fifteen minutes. On being disturbed, it moved about very slowly, in
sharp contrast to the rapid movements of an animal not so treated.

The behavior of Paramaecivm in these solutions of narcotics was
found to be not unlike that of annelid worms, except that Para-

maecium did not similarly disintegrate.

1922 | Rees: The Neuromotor Apparatus of Paramaecium 349

Micro-DIssecrion

It is difficult to demonstrate by the method of micro-dissection
that the fibers in Paramaecium are conductile because :

In the first place, the animal is not highly specialized, as is the
ease with Huplotes and as the locomotor organelles are smaller, their
coordinated movements are harder to see.

Secondly, the pellicle is exceedingly hard to eut. It gives way
under the pressure of the needle point without being severed and
returns to its normal position when the pressure is released. Euplotes
was found to be much easier to dissect because of its greater resistance
to the needle. Furthermore, the only fibers clearly visible in the
unstained living animal are those of the eytopharynx.

Partial transsections through the eytopharynx and hence through
these fibers were made, partial transsections also being made elsewhere
as control experiments. In addition to the above euts, the region of
the neuromotor center was cut and otherwise injured with the needle
point.

Short, rapidly tapering needles made of pyrex rods were most
effective. With a needle of this type, it was found possible to eut
animals in two.

In four cases where it could be positively determined that the
cytopharyngeal fibers had been eut, there was seen a marked differ-
ence in rate and amplitude of vibration between the membranelles
anterior to the cut and posterior to it. The movement of the posterior
membranelles was slower and through a smaller amplitude than that
of the anterior membranelles. Severe injury, on both sides of the
membranelle zone, produced no detectable disturbance so long as the
eytopharyngeal fibers remained intact. Transsections elsewhere like-
wise had no effect on the action of the membranelles. In one instance,
a wide cut, not quite reaching the cytopharyngeal fibers, was made
in the left side of the animal. No effect was detected in the beating
of the membranelles except in cases where these fibers were injured.
Verworn (1889) records similar experiments with Spirostomum.

When animals were isolated in gelatine, it was possible to observe
the ciliary vibrations very clearly. In three eases, severe injury to
the animal in the region of the neuromotor center caused a pronounced
disturbance of ciliary movement. In cone ease, no less than four
distinct groups of cilia were observed, two on each side, each moving
independently of the others. Those on one side were vibrating in

350 University of California Publications in Zoology (Vou. 20

opposite directions to those on the other. On the other hand, severe
injury to other parts of the body than the motorium did not affect
the codrdinated movement of the cilia.

Jennings and Jamieson (1902) found that pieces of Paramaecium
and other ciliates behave not unlike intact animals, except that in the
smaller pieces the movements are more feeble. These observations I
easily confirmed in the case of Paramaecium. In a small piece from
an animal isolated in gelatine not over one-fifth as large as the intact
organism, the cilia continued to vibrate normally for at least five
minutes. Five such cases were observed, three in anterior pieces and
two in posterior pieces. In no case, however, were the cilia observed
to reverse their movements in these small pieces. Intact animals in
gelatine reverse the movements as a rule every few seconds. In all
eases, however, all the cilia of the piece beat in unison.

The evidence indicates that, while the cilia may beat in the absence
of neuromotor impulses from the neuromotor center, this center does
exert an influence through the neuromotor fibers in codrdinating and
integrating the direction of vibration of the cilia as a whole.

DISCUSSION

As intimated by earlier writers (Taylor, 1920), the main objec-
tions to the idea of conduectility in fibrillar systems of the Protozoa
come from advocates of the cell theory who fail to recognize that
complexity of structure may be attained within a single cell and
without cell division and division of labor among its products. The
nucleus has been the main object of study of those who have investi-
gated Protozoa.

But no one will deny that the protoplasm of Paramaeciwm con-
duets stimuli. Else the complex behavior exhibited in the avoiding
reaction (Jennings, 1901) would be impossible. This being the ease,
the structures best adapted to conduct stimuli are those of the neuro-
motor system.

Sharp (1914) developed a clever argument in discussing the
neuromotor apparatus of Diplodinium. He ascribed to the fibers one
of three functions, that of support, of contractility, or of conductivity.
Then, by the method of elimination, he showed that the fibers are
conductile.

1922] Rees: The Newromotor Apparatus of Paramaecium 351

If we investigate the fibrillar system of Paramaecium with the
same ideas in mind, we get a more convincing proof than in the case
of Diplodinium and Euplotes. The frailness of the fine fibers pre-
cludes the idea that they are skeletal or supporting. It is a familiar
fact that Paramaeciuwm under a variety of conditions adverse to its
normal functioning contracts into a spheroidal body. But, clearly,
to get a sphere of an elongated animal the longitudinal axis must be
shortened and the transverse axes lengthened. If this change of shape
were produced by the neuromotor fibers, those whose direction is
transverse would elongate while those whose direction is longitudinal
would have to contract. In other words, they would have to function
similarly to antagonistic sets of muscles which function in response
to nervous impulses. Such codrdinated functioning of these extremely
fine fibers without innervation is unlikely.

The morphology also suggests that the fibers are conduetile. Their
fine branches, which converge from every part of the animal to the
motorium, resemble very closely similar branches in metazoan nervous
systems. Their connections with the basal granules and trichocysts
and with a center suggest these conductile and codrdinative functions
even more strongly. The set that runs in the cytostome and cyto-
pharynx connecting with the same center also suggests codrdination.
When stained with Mallory’s triple connective tissue stain, they take
the acid fuchsin. This also suggests that the fibers resemble chemi-
cally the nerve fibrils of metazoans.

The experimental evidence, while less conclusive, is still important
in several particulars. The gradient that could be demonstrated was
of the type to be expected on the basis of neuromotor function. The
transsections of the eytopharyngeal fibers and resultant lack of
coordinated movement of the membranelles are evidence which shows
that at least the eytopharyngeal fibers function in the transmission of
impulses.

SUMMARY

1. A fibrillar system may be differentiated in Paramaecium with
Mallory’s triple connective tissue stain. The fibers are very fine and
branch profusely. They take the acid fuchsin and are therefore red
by transmitted lght.

2. By means of Heidenhain’s iron haematoxylin this very complex
system of fibers has been differentiated both in whole mounts and
sections.

352 University of California Publications in Zoology — [Vou. 20

3. The cilia arise in longitudinal grooves in the pellicle. These
erooves are slightly oblique on the oral side and meet in the ciliary
suture, a line that runs obliquely through the oral surface from the
anterior to the posterior end.

4. The neuromotor system consists of two peripheral whorls of
fibers, the larger on the oral side, the smaller on the aboral side, and
of two other groups, the cytopharyngeal fibers, one running to the
anterior zone of membranelles in the eytopharynx, the other around
the margin of the cytostome to the posterior membranelle zone.
This zone has not been previously described. All the fibers of the
peripheral whorls and of the eytopharynx ramify out from the
neuromotor center at the anterior end of the cytostome.

5. This is a body at the anterior end of the ecytostome correspond-
ing to the motorium of other ciliates. This body is differentiated with
difficulty.

6. The old eytostome and eytopharynx and, so far as is known,
the neuromotor system also, are dedifferentiated prior to division.
The cytostome, cytopharynx, and neuromotor fibers of the anterior
daughter are new structures. The extent to which dedifferentiation
and redifferentiation occur has not been determined.

7. The neuromotor system of Paramaecium is of a generalized
type in conformity with the lack of specialization of parts in the
organism.

8. Griibler’s methylene blue, when injected into the animals, did
not stain the neuromotor fibers.

9. The axial gradient in Paramaecium could not be fully deter-
mined because the animals did not disintegrate. There is, however,
an anterio-posterior gradient in cessation of ciliary action along the
longitudinal axis which would be expected if the fibrillar system is
interpreted as conductile.

10. A distinet interruption of codrdinated movements is effected
when the region of the neuromotor center is injured. Cutting the
eytopharyngeal fibers interrupted the codrdimation of movement
between the membranelles anterior to the cut and those posterior to it.

11. The morphological as well as the experimental studies indicate
that the fibrillar system of Paramaecium is conduetile in function.

1922) Rees: The Neuromotor Apparatus of Paramaecium 35%

i)
w

LITERATURE CITED

BRAUNE, R.

1914. Untersuchungen tiber die im Wiederkiuermagen yvorkommenden
Protozoen. Arch. Prot., 32, 112-170, pls. 3-6.

BUTscuHut, O.
1887-89. Bronn’s Klassen und Ordnungen des Tierreichs (Protozoa), 1,
viii + 2035 pp., pls. 1-79.
CHILD, C. M.

1915. Senescence and rejuvenescence (Chicago, Univ. Chicago Press). xl
-++ 481 pp., 201 figs. in text.

DorLeEIn, F.

1916. Lehrbuch der Protozoenkunde. Hine Darstellung der Naturgesechichte
der Protozoen mit besonderer Beriicksichtigung der parasitischen
und pathogenen Formen. (Jena, Fischer), Ed. 4, xv + 1190 pp.,
1198 figs. in text.

EHRENBERG, C. G.
1830. Infusionsthierchen als vollkommene Organismen. Ein Blick in das
tiefere organische Leben der Natur (Leipzig, Leopold Voss). xviii
-+ 547 pp., pls. 1-64.

ELLs, J.

1769. Observations on a particular manner of increase in the animalculae of
vegetable infusions with the discovery of an indiffusible salt
arising from hemp-seed put into water until it becomes putrid.
Roy. Soc. London. Phil. Trans., 59, 188-152, pl. 1.

Hertwic, R.

1890. Ueber die Konjugation der Infusorien. Abh. K. Bay. Akad. Wiss.,
17, 1-83, pls. 1-4.

JENNINGS, H. S.
1906. Behavior of the lower organisms (New York, Columbia Univ.
Press). xiv + 355 pp., 144 figs. in text.
1908. Heredity variation and evolution in Protozoa. Proc. Am. Phil. Soc.,
47, 393-546, 5 figs. in text.
JENNINGS, H. §., AND JAMIESON, C.

1902. Studies on reactions to stimuli in unicellular organisms. The move-
ments and reactions of pieces of ciliate Infusoria. Biol. Bull., 3,
225-234, 4 figs. in text.

KHAINSKY, A.

1910. Ziir Morphologie und Physiologie einiger Infusorien (Paramaecium
caudatum) auf Grund einer neuen histologischen Methode. Arch.
Prot., 21, 1-6, pls. 1-3, 2 figs. in text.

354 University of California Publications in Zoology | Vou. 20

Koror, C. A., AND SwEzy, O.

1915. Mitosis and multiple fission in trichomonad flagellates. Proc. Am.
Acad. Arts and Sci., Boston, 51, 289-378, pls. 1-8, 7 figs. in text.

1919. Studies on the parasites of the termites. I. On Streblomastiz striz, a
polymastigote flagellate with a linear plasmodial phase. II. On
Trichomitus termitidis, a polymastigote flagellate with a highly
developed neuromotor system. III. On Trichonympha campanula
sp. nov. IV. On Leidyopsis sphaerica, gen. noy., sp. nov. Uniy.
Calif. Publ. Zool., 20, 1-116, pls. 1-14, 8 figs. in text.

Kouscu, K.
1902. Untersuchungen iiber die Zerflussungserscheinungen der ciliaten In-
fusorien. Zool. Jahrb., Abt. Anat. u. Ontol. der Tiere, 16, 273-
422, pls. 26-28, 5 figs. in text.
Mater, H. N.
1903. tber den feineren Bau der Wimperapparate der Infusorien. Arch.
Prot., 2, 305-824, pl. 7, 1 fig. in text.
Maupas, HE.
1883. Contribution a 1’étude morphologique et anatomique des infusoires
ciliés. Arch. Zool. exper. gén., (2), 1, 427-664, pls. 19-24.
Mercar, M. M.

1908. Opalina, its anatomy and reproduction with a description of infec-
tion experiments and a chronological review of the literature.
Arch. Prot., 13, 195-378, pls. 14-28, 17 figs. in text.

NERESHFIMER, HE. R.

1903. Die Hohe histologischen Differenzierung bei heterotrichen Ciliaten.
Arch. Prot., 2, 305-324, pl. 7, 1 fig. in text.

REES, C. W.

1921. The neuromotor apparatus of Paramecium. Am. Nat., 55, 464-468,
2 figs. in text.

1922. The micro-injection of Paramaecium. Univ. Calif. Publ. Zool., 20,
235-242,

1922. The isolation of Paramaecium with notes on the technique of micro-
injection and micro-dissection. (Manuscript.)

SHarp, R. G.
1914, Diplodinium ecaudatum with an account of its neuromotor apparatus.
Univ. Calif. Publ. Zool., 13, 42-122, pls. 3-7, 4 figs. in text.
SCHUBERG, A,
1905. Uber Cilien und Trichoeysten einiger Infusorien. Arch. Prot., 6,
61-110, pls. 4-5.
Swezy, O.

1915. Binary and multiple fission in Heramitus. Univ. Calif. Publ. Zool.,
16, 71-88, pls. 9-11.

1922 | Rees: The Neuromotor Apparatus of Paramaecium 355

TasHiro, S. ;
1917. A chemical sign of life (Chicago, Univ. of Chicago Press). vii+142
pp., 5 figs in text.

TayLor, C. V.
1920. Demonstration of the function of the neuromotor apparatus in
Euplotes by the method of micro-dissection. Univ. Calif. Publ.
Zool., 19, 403-470, pls. 29-33, 2 figs. in text.
1920. An accurately controllable micropipette. Science, n.s., 51, 617-618,
1 fig. in text.

VERWoORN, M.

1889. Psychophysiologische Protistenstudien (Jena, Fischer). viii +
219 pp., pls. 1-6, 27 figs. in text.

Witson, J. G.
1910. Intra-vitam staining with methylene blue. Anat. Ree., 4, 267-277.

Yocom, H. B.

1918. The neuromotor apparatus in Huplotes patella. Univ. Calif. Publ.
Zool., 18, 337-396, pls. 14-16, 1 fig. in text.

EXPLANATION OF PLATES

All figures are of Paramaecium, drawn with camera lucida and, unless other-
wise stated, stained with Heidenhain’s iron haematoxylin.

PLATE 32
Fig. 1. Oral view showing the oral groove, cytostome, and oral whorl of
peripheral fibers ramifying out from the neuromotor center. X 356.

Fig. 2. View of left side, showing meeting points of the anastamosing
ends of the fibers of the oral whorl with those of the aboral whorl. X 356.

Fig. 3. Aboral view showing the aboral whorl. X 356.

Fig. 4. View of left side. The anastamosing ends of the fibers of the oral
whorl meet the converging apex of the aboral whorl. X 356.

Fig. 5. Slightly oblique section, 5, thick, through the cytostome, showing
converging fibers of the oral whorl in the outer endoplasm. % 670.

Fig. 6. Oblique section through the cytopharynx, showing some of the
fibers of the oral whorl and also of the aboral whorl. X 670.

[356]

[PREES ] (PEATE 32

UNIV. CALIF. PUBL, ZOOL. VOL. 20

PLATE 33
All figures of cross-sections through the same organism. 2.5y thick. X 670.

Fig. 7. Oral groove at the beginning of the cytostome and cytopharynx,
showing ends of peripheral fibers connected with the basal granules and
trichoeysts.

Fig. 8. Through the mid-eytostome region, showing membranelle brush of
anterior membranelle zone.

Fig. 9. Through the posterior cytostome region and the cytopharynx, show-
ing membranelle brush of anterior membranelle zone.

Fig. 10. Through cytopharynx, showing membranelle brush of anterior
zone and beginning of posterior membranelle zone.

Fig. 11. Through indented portion of cytopharynx and posterior mem-
branelle zone.

Fig. 12. Through extreme posterior end of cytopharynx, showing cilia of
posterior membranelle zone partly obscured by ingested food bodies.

[358]

[REES] PLATE 33

UNIV. CALIF. PUBL. ZOOL. VOL. 20

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PLATE 34
Figs. 13-19 are successive longitudinal sections, 10u thick, through the same
organism. > 365.
Fig. 13. Through left side, showing peripheral fibers of oral whorl.
Fig. 14. Showing fibers of oral whorl and also of aboral whorl.

Fig. 15. Showing fibers of both oral and aboral whorls and section of
macronucleus.

Fig. 16. Fibers of both oral and aboral and margin of eytostome.

Fig. 17. Through the cytostome and cytopharynx, showing fibers of aboral
whorl and a few fibers of oral whorl.

Fig. 18. Section through right side of macronucleus.
Fig. 19. Through right side of animal. Fibers of both whorls in periphery.

Fig. 20. Whole mount of dividing organism, showing macronucleus, the
constriction, and in each daughter organism a new cytostome, cytopharynx,
and cytopharyngeal fibers. Mallory’s triple connective tissue stain. X 356.

[360]

UNIV. CALIF, PUBL. ZOOL. VOL. 20 [TREES] PLATE 34

PLATE 35

Fig. 21. Longitudinal section, 154 thick, through the eytopharynx, show-
ing anterior and posterior membranelle zones and indentation of eytopharynx.
Mallory’s triple connective tissue stain. X 670.

Fig. 22. Longitudinal section, 15yu thick, through the cytopharynx, show-
ing fibers of cytopharynx running from a deeply staining body and peripheral
fibers ramifying out from another body.

Fig. 23. Longitudinal section, 15, thick, through cytostome and cyto-
pharynx, showing anterior membranelle zone, part of posterior membranelle
zone and peripheral fibers. Mallory’s triple connective tissue stain. X 356.

Fig. 24. Diagonal section, 2.54 thick, through the cytostome and cyto-
pharynx, showing cilia and ‘‘ ciliary rootlets.’’ X 1160.

Fig. 25. Cross-section, 2.54 thick, showing cilia, ‘‘ciliary rootlets,’’ also
trichocysts and fibers running from them into the endoplasm. X 1160.

Fig. 26. Showing anterior face of cytopharyngeal fibers and peristomal
fibers extending obliquely caudad into the region of the posterior membranelle
zone and into the endoplasm, also the ciliary lines of part of the oral and right
sides with the ciliary suture. X 356.

Fig. 27. View of right side, showing aboral trichoeyst whorl and part of
oral trichocyst whorl. X 357.

Fig. 28. View of aboral side, showing aboral whorl cf trichoeysts. X 356.

[362]

UNIV. CALIF. PUBL.

ZOOL.

VOL, 20

[REES] PLATE 35

PLATE 36
All figures of whole mounts. X 356.

Fig. 29. View of left side, showing ciliary lines.

Fig. 30. Oral view showing ciliary lines and ciliary suture.
Fig. 31. Oral view showing oral whorl of trichocysts.

Fig. 32. Aboral view showing ciliary lines.

Fig. 33. View of left side, showing trichocysts of the oral and aboral
whorls.

[364]

Vv Fo LeU) OO oO
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31

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. On the Free, Encysted, and Budding Stages of Councilmania lafleurt, a Para- —-

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UNIVERSITY OF CALIFORNIA PUBLICATIONS
IN

ZOOLOGY
Vol. 20, No. 15, pp. 365-371 February 1, 1923

A COMPARISON
OF THE CYSTS OF ENDAMOEBA COLI AND
COUNCILMANIA LAFLEURI IN CONGO RED

BY
NATHANIEL BERCOVITZ, M.D.

Early in January, 1922, I conducted a series of experiments in
the protozoological laboratory at the University of California to
determine the permeability of the eysts of intestinal protozoan para-
sites by a number of anilin stains. During the course of the experi-
ments it was noticed that there was no staining of the cysts by aqueous
solutions of the stains until one day a number of eysts of Cowncil-
mania laflewrt Kofoid and Swezy were seen to appear red after being
exposed to Congo red. The next specimen contained cysts of Hnda-
moeba coli, and exposure to Congo red showed no reaction in the
eysts which appeared clear in the red background. A third specimen
contained cysts of Councilmania and these also took the stain when
exposed to Congo red. This seemed to be unusual, so it was decided
to make an intensive study of this phenomenon. At first it was
thought that this might be a quick way to differentiate between these
two types of amoeba, but as more eases were studied it was found
that the phenomenon was not constant. There were instances where
the Congo red had no effect on the eysts of Cowncilmania, and there
were instances where the Congo red stained cysts of Endamoeba coli
deeply.

In order to arrive at some more accurate conclusion a series of
fifty cases was studied. Fresh solutions of Congo red, 1 per cent in
normal salt, were used throughout. The effect of this stain on every
type of cyst of human intestinal parasites available was studied, but
in the eysts of Couwncilmania and Endamoeba coli only was there any

366 University of California Publications in Zoology [Vou 20

staining. Some twenty-five of the more common anilin stains in
1 per cent normal salt solution were also used on these cysts, which
included those of Endamoeba dysenteriae, large and small races;
Endamoeba nana, small race; Iodamoeba buetschlu; Chilomastix
davainei, and Giardia enterica, in addition to Councilmania and Enda-
moeba coli. In no eyst, and with no stain, was there any staining
of live eysts exeept with the Congo red on Councilmania and Enda-
moeba coli and, as already stated, this stain acted differentially on
the whole between these two.

There were a number of variables to be considered. First of all
there was the matter of determining whether the cysts were alive or
dead, as the dead eysts stained intensively with any of the dyes used.
It was soon found that in Congo red dead eysts stained a deep orange
red with nuclei showing very dark; it also became evident that in
live cysts the dye did not penetrate the cyst wall, therefore did not
stain either the cytoplasm or the nuclei.

This fact was therefore established early, that in live cysts the
dye did not stain the cytoplasm or nuclei of the cyst, but only the
eyst wall. It was also found that there were degrees of intensity of
the staining of the cyst wall, and for this reason it was decided to
designate three degrees of staining as follows: the intense shade
as ‘‘stained,’’ the light shade ‘‘pinkish,’’ and where no staining was
““unstained.’’ It was found in many of the cysts which
were ordinarily regarded as ‘‘unstained,’’ that in a powerful light a

apparent as

faint pinkish shade might be seen. This might have been due, how-
ever, to the film of staining fluid around the eyst.

The other variables were the amounts of material used and the
dye used. Obviously both of these could not be kept absolutely con-
stant, but continuous practice reduced to a minimum error due to
these factors.

After a few trials it was found that if a dye was going to act at
all on a eyst it acted immediately, and that the eyst did not stain
more intensively if exposed for a long time, even as long as two days
in the dye. So there was no error due to the time element. Variations
in room temperature did not make any difference in the results.

It is obvious that in a study like this the personal element enters
in a large way, since what would be considered stained by one might
be considered. pinkish by another, and even with the same person,
there would be variations of judgment as to the intensity of the color,
especially with different densities of the surrounding field of material

1923] Bercovitz: Endamoeba coli and Councilmania lafleuri 367

and stain. For this reason this study is not absolute, but the findings
are recorded as a general result and are liable to the variations of
personal and other elements.

Early in the study it was noticed that in stools in which Cownceil-
mania were found the cysts seemed to be more numerous than in those
containing Endamoeba coli. This feature was also studied in connec-
tion with the action of stains.

The results of the findings in the first fifty cases are recorded in
tables 1, 2, and 3. In general it was found that the eysts of Council-
mania when present are abundant in the stool, while on the other
hand those of Endamoeba coli are usually very scarce, at times not
more than one or two cysts being found on a slide. It was also found
that the tendency of the cyst wall of Cowncilmania to take the Congo
red was much more marked than in the ease of Endamoeba coli.
Furthermore, the cysts of Cowncilmania seem to be ovoidal or
ellipsoidal oftener than the eysts of Endamoeba coli.

In the first series of 50 cases there were 17 cases, or 34 per cent,
with Z. coli infection; 29 cases, or 58 per cent, with Councilmania
infection, and 4 eases, or 8 per cent, with infection of both #. coli and
Councilmania.

TABLE 1

Series J. RELATIVE ABUNDANCE OF Cysts or E. COLI AND COUNCILMANIA IN SLIDES

| Few Moderate | Many
|
TD yrs Enea Ts oF) Gol bee pene eee 64.7% 23.6% 11.8%
(ouncllmsn tases eee eee ce essa 17.2% 10.4% 72.4%

This table indicates that Cowncilmania eysts are more abundant
in stools than are the cysts of Endamoeba coli.

TABLE 2

Series I. Reaction or Cysts oF E. cont AND COUNCILMANIA TO Conco RED

Pink and Total No.

Unstained in Showing

Type of Cyst |No. of| Unstained Pinkish Stained same Color

Cases Specimen
No. % No. % No. % No. % No %
|

coliy..#.4.. 52. 17 9 | 53.1 6 | 35.4 PA lites) 0 | 0} 8 | 46.9
3 10.3) 9 31.0) 11 | 37.9) 23 | 79.3

368 University of California Publications in Zoology (Vou. 20

This table shows that the cyst wall of Cowncilmania takes the
Congo red stain more readily than that of E. coli.

In order to place these conclusions on a more accurate basis, a
second series of fifty stools was examined in which the following
factors were noted: cysts dead or alive in stool; stool formed or
liquid; actual count of cysts on slides to determine more accurately
the proportion staining, as well as abundance of cysts in these cases.

Congo red itself being a very delicate indicator, it was noted
whether the stool became dark in the Congo red, and this was used
as the test for acidity.

Where cysts were abundant one hundred were counted, and the
results with the stain noted. But in no case were more than two slides
of each specimen examined, as in many eases it would have required
examination of ten to fifteen slides to find one hundred eysts. Where
one hundred cysts were found on one slide it was considered that
the cysts were abundant. Where less than one hundred cysts were
found on one slide, but more than fifty cysts on two slides, the eysts
were considered to be moderately abundant. Where there were less
than fifty cysts on two slides the number was considered few,. and
where less than ten cysts on two slides, very few.

The results of the second series are recorded in tables 3 and 4.
In this serics 22 cases, or 44 per cent, were infected with Endamoeba
coli, and 28 eases, or 56 per cent, were infected with Cowncilmama.
No mixed infections were included in this series.

TABLE 3

Series IT. RELATIVE ABUNDANCE OF Cysts oF E. COLI AND COUNCILMANIA Cysts

Few Many

(including very (including moderately
few) abundant)
TACO rece be cree scent meee eee 86.4% 13.6%
Conneilmaniaieeree ee ere 21.5% 78.5%

The contrast between abundance of cysts in infections by HEnda-
moeba coli as compared with infections by Cowncilmania is very strik-
ing, and is of value because the cysts were actually counted. The
three groups of table 1 have been changed to two large groups as being
more accurate. Wherever there were less than fifty eysts on two slides
it was recorded as few, and where more than fifty cysts were found
on two slides the case was recorded as many.

1923] Bercovitz: Endamoeba coli and Councilmania lafleurt 369

TABLE 4
Series II. Reaction or Cysts oF ENDAMOEBA COLI AND COUNCILMANIA TO
Conco Rep
Stained Unstained Stained
(showing
Type of Cyst No. | Unstained | any color
of at all) Many Few Many Few
Cases
No.| % | No.| % | No.| % | No.| % | No % | No %
22 8 |36.3] 14 |63.7) 1 12.5] 7 |\87.5| 2 |14.5) 12 | 85
Councilmania......| 28 | 3 |10.7/ 25 |89.3] 2 66.6) B33). 240) 80.0) ty |} FAQ)

In this series eleven cases, or 22 per cent, showed no coloring of
any of the cysts. In the first series fifteen cases, or 30 per cent,
showed no coloring. In the second series the contrast between the
staining in the two types is marked. The relative difference between
percentage of cysts of each type stained is practically the same for
the two series, the second series showing an actual higher percentage
of all cysts which are stained.

In both series the diagnosis between EH: coli and Councilmania was
made after examination of slides stained with iron haematoxylin.

In the first series there were four cases where Cowncilmania and
E. coli were found in the same specimen. An attempt was made to
record the location of cysts which took the Congo red and of those
which did not; the slides in these cases were stained in haematoxylin,
in order later to find the marked eysts and thus to determine whether
they were E. coli or Cowncilmania. Inasmuch as in these four cases
both stained and unstained eysts were found, it would have been of
the greatest value if it could have been determined whether all of the
stained eysts were Councilmania and the unstained eysts E. coli, or
vice versa. However, another very curious phenomenon was observed
in these eases, in that after the Congo red had come in contact with
the eyst wall the haematoxylin did not seem to have free access to
the cyst; thus the cytoplasm and nuclei were not stained, and the
eysts could not be differentiated.

Repeated trials of this were made, and in every case the result was
the same. A large number of the cysts were distorted, and some
seemed to be more or less collapsed. The haematoxylin seemed to
cling to the cyst wall with great tenacity, as the cysts were exceed-
ingly difficult to destain. An investigation of this phenomenon is
to be reported later.

The results of the second series of fifty cases are in the main
similar to the results recorded in the first series. The incidence of

370 University of California Publications in Zoology (Vou. 20

Endamoeba coli infection is somewhat higher than in the first series
of fifty, but still considerably lower than the number of Cowncilmania
infections. As regards abundance of cysts, there are strikingly few
cysts in the great percentage of cases infected with Endamoeba coli,
while nearly 80 per cent of the cases infected with Councilmania show
abundance of cysts. In cases of E. coli infection, a smaller number
show absolutely no cysts staining at all. In many cases, however,
perhaps only two or three cysts showed any color, yet these cases were
classed as specimens showing color. In this second series Council-
mania again showed a preponderance of specimens in which eysts
take the Congo red stain.

TABLE 5

Series I anp II. ReEnatrive ABUNDANCE OF CystTS OF E. COLI AND COUNCILMANIA
IN ONE HuNDRED CASES

Cysts
Many and Moderate Few
No. % No. %
By. (COL en eee eee. eee 5 14.7 34 85.3
Councilmania...................0..... | 46 80.7 11 | 19.3

Four mixed eases of first series not recorded.

In the total series of one hundred cases there were thirty-nine
cases showing #. coli infection; fifty-seven cases showing Cowncil-
mania infection, and four eases with both Z. coli and Councilmania.

TABLE 6
ACTION OF Conco RED oN E. COLI AND CoUNCILMANIA IN NINETY-SIX PURE
INFECTIONS
E. coli Councilmania
No. of Cases % No. of Cases %
iUnstained:-ssate se 17 43.6 9 537
Stained 22 | 56.4 48 84.3

Acknowledgments are made to the California State Board of
Health for the supply of material used, to the University of California
for assistance from the grant made in aid of research on human
intestinal protozoa, and for the use of the facilities of the proto-
zoological laboratory, to the China Medical Board for a fellowship

permitting this study, and to Professor Kofoid and Dr. Swezy for
counsel.

1923] Bercovitz: Endamoeba coli and Councilmania lafleuri 371

SUMMARY

1. In infections by Endamoeba coli the eysts are present as a gen-
eral rule in small numbers; while in infections by Cowncilmania the
eysts are abundant.

2. In general the cysts of Cowncilmania take the Congo red stain
whereas the cysts of H. colt either do not take the stain, or else in most
cases take it very faintly.

3. Of all eysts of intestinal protozoa tested with some twenty-five
anilin stains, only cysts of EZ. coli and Councilmania took any of the
stains at all, and these two took only the Congo red. Furthermore,
only the cyst wall was stained, the dye apparently not passing through
the wall and thus not staining either cytoplasm or nuclei.

4. The cysts of the common intestinal protozoa when dead are
quickly penetrated by anilin dyes and the protoplasm is deeply
stained. Living cysts resist penetration by these dyes.

5. The shape of cysts of Couwncilmania in many eases is ovoidal,
or ellipsoidal, whereas cysts of Hndamoeba coli are as a rule spherical,
and also slightly smaller than Cowncilmania eysts (see Kofoid and
Swezy, 1921, a, b).

6. The condition of the stool, i.e., whether formed or unformed,
whether acid or alkaline, had no effect either upon the abundance of
the cysts or the way in which they reacted to the Congo red stain.
There was no evidence that in the more fluid stools the eysts were
less protected than in the formed stools.

PRESBYTERIAN HOSPITAL,
KATCHEK, HAINAN, CHINA.

Transmitied, December 8, 1922.

LITERATURE CITED

Koror, C. A., and Swezy, O.
1921a. On the free, encysted, and budding stages of Councilmania lafleuri, a
parasitic amoeba of the human intestine. Univ. Calif. Publ. Zool.,
20, 170-189, pls. 18-22, 3 figs. in text.
1921b. Cowncilmania lafleuri, a new amoeba of the human intestine. Proce.
Soe. Exper. Biol. Med., 18, 310-312.

Cur

bal

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1. The Life Cycle of Hchinostoma revolutum (Froelich), by John C. Johnson,
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12. On Some New Myriopods Collected in India in 1916 by C. A. Kofoid, by

Ralph V. Chamberlin. Pp. 389-402, plates 26-28. August, 1920 10.0... és

18, Demonstration of the Function of the Neuromotor Apparatus in Euplotes

by the Method of Microdissection, by Charles V. Taylor. Pp. 403-470,

plates 29-33;.2 figures in text. October, 1920 22... coe cc ecceeceeeeeeeceseneceecen a
Index, pp. 471-480.

: Studies on the Parasites of the Termites. I. On Streblomastiz striz, a Poly-
+330 5 mastigote Flagellate with a Linear Plasmodial Phase, by Charles Atwood
ee Kofoid and Olive Swezy. Pp. 1-20, plates 1-2, 1 figure in text. July, 1919

eect 2. Studies on the Parasites of the Termites. IZ. On Trichomitus termitidis, a
a Oa Polymastigote Flagellate with a Highly Developed Neuromotor System,
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ZOOLOGY

lo 16, PP. 373-300, plate 37, 1 figure in text

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rae MORPHOLOGY AND BEHAVIOR OF
Pee en nionss ARDIN DELTEILI

_ (DERRIEU AND RAYNAUD) _
PN ES wae
| CHARLES ATWOOD KOFOID anb OLIVE SWEZY

< ‘ OE

SS ssn i listigy Pi | z BY
Hh eS 3 eX ; Paty es iib> :
Gr SEP 4192

"BERKELEY, CALIFORNIA ifs
oft 6
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ZOOLOGY
Vol. 20, No. 16, pp. 373-390, plate 37, 1 figure in text August 16, 1923

ON THE MORPHOLOGY AND BEHAVIOR OF
PENTATRICHOMONAS ARDIN DELTEILLI
(DERRIEU AND RAYNAUD)

BY
CHARLES ATWOOD KOFOID anp OLIVE SWEZY

CONTENTS she
SIRI OULULC tT OY) Mae acer ne once caren octet e nee, ecuce cians oseescu-casnacastesveccs
Acknowledgments
MWratteriall. 2.3 22-2:
INTO BL AON YS a ace acess ae 378
TRGDO GIDC OTD, ecco SEE LS)
Behavior seus OOD
RraTEVaiy shl MIN SUL GIs eae ceca eee ccc ds sonatas Aeanoss Soest ceues esosn sqseeteisceztscasuesta OOO
(CURTISS aac cee SS Sr ee Ea Coe ee Re 386
Summary -....... .. 387
AP ET A EILO a CLEC C Meee sere nen coe oe soe ces oe Ee ose cna e Seco tenes ceoctoseceeetbcksue Ped eed Beers 388
TE Sraya Fea rn, OE ONS Sp ee ae 390

INTRODUCTION

The evolution of the polymastigote flagellates has progressed by
two methods: (1) increase in the number of the anterior or other
flagella, and (2) differentiation in structure and specialization in
function among the flagella. The first method has given rise ultimately
to the Hypermastigina which, in their most highly specialized genera,
are covered over their entire surface by flagella, hundreds or thou-
sands in number. The second method has resulted in the development
within the Polymastigina of organs such as the trailing, ribbon-like
flagellum of Devescovina, the undulating membrane and the axostyle
of Trichomonas, and the flagellar lines of Pyrsonympha. The
- morphological constancy in number of these fundamental organelles

374 University of California Publications in Zoology [ Vou. 20

within each of the genera and species of the Polymastigina may be
considered as established by the numerous investigations of the varied
members of this widely distributed group of (mainly) parasitic
protozoans.

The only period in the life of the individual when this morpho-
logical constancy in number is modified is the temporary phase of
promitosis. During this phase the neuromotor organelles are doubled
in number preparatory to mitosis of the nucleus and asexual repro-
duction by binary fission of the individual, a process which insures
the morphological and numerical constancy of these organelles for
the two individuals thus produced. Observed variations within the
species in the number of flagella, or of other organelles formed by
modification of flagella, are attributable to the onset of mitosis, and
may be demonstrated to be such by an analysis of the complete mitotic
process.

The importance of recognition of this deduction as a guiding
principle in the study of parasitic flagellates and of its practical
applications in the diagnosis of human flagellate infections as well
as those of other animals is emphasized by Dobell and O’Connor’s
(1921) treatment of the number of flagella in Trichomonas hominis
in their Intestinal Protozoa of Man. In this work Dobell (p. 68,
footnote) states that, ‘‘In my experience the four-flagellate form
(typical Trichomonas) is the commonest in human stools, but the
3-flagellate variety is also common. C. D.’’ They also inelude the
5-flagellate Pentatrichomonas in the synonymy of Trichomonas. The
reader is thus led to infer that Trichomonas hominis has three, four,
or five flagella.

Their erroneous treatment of this matter is not only unsound
morphologically, but in our judgment will lead to incorrect diagnoses
of infections and will deter clinical study of, and therapeutic efforts
to cure, human trichomonad infections. It tends to confuse distinct
specific entities and to obscure a possible etiological factor in human
disease by the as yet pure assumption that the intestinal ‘‘Tricho-
monas’’ in man belongs to one species only and that clinical accounts
of pathogenic conditions associated with such infections are always
to be explained by the further assumption of the coneurrence of
another unobserved etiological cause.

No review of the conflicting clinical literature on intestinal
trichomoniasis will be attempted here since it has recently been made
elsewhere by Haughwout (1918), Kofoid and Swezy (1921), and

1923 ] Kofoid-Swezy: Pentatrichomonas ardin delteili 37!

o

Dobell and O’Connor (1921), the latter decrying but by no means
establishing the imnocuousness of these infections. Intensive and
repeated clinical studies by competent clinicians, with the infecting
organism determined by competent protozoologists, will be necessary
to settle the mooted point of pathogenicity. The complex interrela-
tions in disease and the widespread occurrence of the carrier phase
in many infections capable under some conditions of inducing disease,
lead one to hesitate to accept as soundly scientific Dobell and O’Con-
nor’s sweeping assumptions of non-pathogenicity or their summary
dismissal, as being without significance, of the observations of others
as to tissue penetration and pathogenicity. More investigation, rather
than a dogmatic dismissal, is sorely needed.

In our experience the common trichomonads of the human
intestine, urogenital tract, and mouth all have four anterior, free
flagella. In case three only are seen at first, a fourth can be found
if the material is so prepared as to be adequately examined. In our
experience failure to find the fourth has been due to the belief that
three is a normal number, or to inexpertness in analysis of speci-
mens, or to the disadvantageous position of one of the four flagella,
as, for example, when it is hidden under the body. Fundamentally,
the reason lies in a failure to observe the differential behavior of one
of the anterior flagella. It is the purpose of this paper to give an
account of several infections by Pentatrichomonas ardin delteili
(Derrieu and Raynaud) in man and to direct the attention of proto-
zoologists to the distinctness of this genus and of clinicians to the
desirability of distinguishing this organism from the seemingly
innocuous and more widely prevalent Trichomonas hominis.

ACKNOWLEDGMENTS

We are indebted to Dr. L. M. Boyers, the attending physician on
our principal case, for codperation, and to the patients for prolonged
cobperation in providing material for research. Acknowledgments
are also made to the Carnegie Institution of Washington, to the Board
of Research of the University of California, to Margaret B. Fowler,
and to an alumnus of the University of California, for grants which
have made possible this investigation, as well as others, on human
intestinal Protozoa.

376 University of California Publications in Zoology [| Vou. 20

MATERIAL

In the course of 25,000 examinations of 7010 persons made by
us up to November 30, 1922, the latest date to which our statistics
have been assembled, we recognized 75 cases or 1.1 per cent infection
by Trichomonas hominis. The actual percentage of infection was
doubtless higher, since in the absence of cysts detection rests upon
finding living flagellates, and these in some types of stool do not
survive long and are usually infrequent in formed stools. In our
experience this species, except in dividing individuals, has only four
flagella.

We found three cases in which there was an infection by Penta-
trichomonas ardin delteili (Derrieu and Raynaud). The first of these
was an army officer who had resided in the Philippine Islands and
Panama, and on and near the Mexican border. Nineteen examina-
tions of this case were made over a period of nine months. In each
case Pentatrichomonas, and this flagellate only, was detected. ‘There
was a concurrent infection by Strongyloides stercoralis and by Blasto-
cystis. The patient had a previous history of dysentry, had received
treatment for amoebiasis (emetin-bismuth-iodide and neoarsphena-
mine) and had gained in weight after this treatment. Chronic
diarrhoea marked the attack here treated. No free amoebae and no
amoebic cysts of any sort were found in any of the stools examined
by us, though diligently sought.

The second ease was also an army officer who had been stationed
for thirteen months on the Western Front in France, for a time in
an area where French African colonial troops and Chinese laborers
were billeted. To date seventy-eight examinations have been made
on this case. No worm infections have been found. There are con-
current infections by Chilomastix davainei and Blastocystis. Penta-
trichomonas was not found in only thirteen of these stools. In some
instances this was due to the condition of the stool resulting from
delay in shipment of the specimen from a distance. Chilomastix was
found in twenty-seven of the seventy-eight stools and was rarely
abundant. No other animal parasite was found in these examinations.
(Shortly before our examination began the patient had been found
to have amoebiasis and had received the emetin-bismuth-iodide and
arsphenamine treatment.) No amoebae, free or eneysted, were at
any time found by us in his stools.

1923] Kofoid—Swezy: Pentatrichomonas ardin delteili 377

The third case was a female school teacher resident for eighteen
years in the Hawaiian Islands. Fifteen examinations have been made
to date of this case, all positive for Pentatrichomonas, but no other
infection, either protozoan or helminth, has been detected. There is
no clinical history of amoebic dysentery in this ease.

In all three cases there is a record of a chronie diarrhoea with
purulent, fetid, yellowish-brown stools, rarely with mucus, with but
little or no blood, and of a smooth consistency. Defecation was
frequent, sometimes as many as twenty stools a day. Semi-formed
or formed stools rarely occurred. The customary palliative measures
failed to give relief. Microscopic examination revealed enormous
numbers of Pentatrichomonas swarming in the fluid stool. In semi-
formed stools the numbers were lessened.

These three infections, all with similar symptoms and _ stools,
presented only Pentatrichomonas with five flagella, never Tricho-
monas hominis with four; at least we found only the former in every
eritical determination of the number of anterior flagella. We there-
fore conelude that Pentatrichomonas is distinet from Trichomonas.

Generic status is desirable for Pentatrichomonas in view of the
general use of the number of flagella as the basis of generic distinction
in the Polymastigina. We therefore utilize the generic name proposed
by Chatterjee (1915). The species was originally described by Der-
rieu and Raynaud (1914) as Heramastix ardin delteili from a fatal
case of diarrhoea originating in Algiers but observed in Paris. It was
also described at about the same time by Chatterjee (1915) as Penta-
trichomonas bengalensis from a ease of chronic diarrhoea at Calcutta.
Mesnil (1915a, b) noted the similarity of the two organisms, called
attention to the preoccupation of Hexamastix by a different flagellate
described by Alexeieff (1912) and recognized the availability of
Pentatrichomonas as the generic name. He held open the possibility
of specific distinctness of the Algerian and Indian species. The
nature of the similarities of the two, however, and the great varia-
bility of the species in our material make it certain, in our opinion,
that the two supposed species are identical. Chatterjee (1917)
later reported this species in 32 of 70 cases of flagellate diarrhoeas
observed at Calcutta. Wenyon and O’Conner (1917) reported one
observation of a five-flagellated trichomonad from man observed in
Egypt. Haughwout (1918, 1919, 1920) reported Pentatrichomonas
in cases of diarrhoeas at Manila and described the method by which
they engulf and seemingly digest red blood cells with which they

378 University of California Publications in Zoology —[ Vou. 20

may be associated in the stools. It appears from these records of
occurrence that Pentatrichomonas may have a wide distribution in
the tropics.

It is important that the occurrence of epidemies of trichomonad
diarrhoeas reported from Colombia by Alvarez (1916), from Argen-
tina by Vacearezza (1917), and from Arequipa by Escomel (1919),
be critically examined with reference to the presence of Penta-
trichomonas.

MORPHOLOGY

Pentatrichomonas ardin delteili has the typical organelles of the
trichomonad flagellates, namely, (1) the neuromotor system consist-
ing of the anterior flagella, undulating membrane, axostyle, blepharo-
plasts, centrosome, and rhizoplasts, and (2) the other non-fibrillar
organs, the nucleus, and the eytostome. These differ structurally, if
at all, only in minor details of proportions from those of other species
of trichomonads.

The distinguishing characteristic of the genus is the presence of
five anterior flagella. The genus Trichomonas Donné (1837) has
four and Tritrichomonas Kofoid (1921) has three.

These flagella are slender threads of uniform and equal caliber
whose length exceeds slightly that of the body in normal extension.

These anterior flagella fall into two groups as to origin and
behavior, a fact not hitherto noted and ignorance of which tends to
obscure the number of flagella. These may be designated respectively
as the single independent (ind. ant. flag.) and the three clustered
anterior flagella (clust. ant. flag., fig. A).

The two groups arise from different blepharoplasts. The indepen-
dent flagellum arises, together with the undulating membrane, the
parabasal body, and possibly the axostyle also, from the proximal
or primary blepharoplast (prim. bleph., fig. A), while the three
clustered anterior flagella arise from the more distal or secondary
blepharoplast (sec. bleph., fig. A).

Dobell and O’Connor (1921) state that there are at least three
and possibly more blepharoplasts and that the flagella appear to take
their origin from at least two of these; but they do not distinguish
the two groups of flagella nor state their relations to the blepharo-
plasts, nor that of the other organelles to these structures. Their
figures (their pl. 5, fig. 71) might be interpreted as showing two

1923 ] Kofoid-Swezy: Pentatrichomonas ardin delteili 379

blepharoplasts and a centrosome. It is possible that the granule at
the base of the clustered flagella may be subdivided, or that there
may be a granule at the tip of the axostyle, but we have been unable
to find clear evidence of more than two blepharoplasts with the
relations as above defined by us.

clust ant. fl.

sec, bleph.

prim. bleph,

. nue. rhiz,

cyt.

ax.

. food vac.

ind. ent.fl.

Fig. A. Pentatrichomonas ardin delteil. 3200. From stained preparation
of human stool. Abbreviations: az., axostyle; cent., centrosome; clust. ant. fl.,
clustered anterior flagella; cyt., eytostome; food vac., food vacuole; ind. ant. fl.,
independent anterior flagellum; marg. fil., marginal filament: kary., karyosome:
nuc., nucleus; nuc. rhiz., nuclear rhizoplast; par. b., parabasal body; post. fr. jl.,
posterior free flagellum; prim. bleph., primary blepharoplast; rhiz., rhizoplast;
sec. bleph., secondary blepharoplast; und. memb., undulating membrane.

The two blepharoplasts are joined by a very short rhizoplast and
the proximal one is connected with the centrosome (cent., fig. A) on
the nuclear membrane by a definite rhizoplast (riz., fig. A).

The centrosome (cent., fig. A; pl. 37, figs. 1, 2, 7) is asmall, deeply
staining granule on the anterior surface of the nuclear membrane
in close juxtaposition to the blepharoplasts. At certain stages of
mitosis an intranuclear rhizoplast (pl. 37, fig. 10) may be seen to

380 University of California Publications in Zoology [Vou. 20

connect the centrosome with the central karyosome as it sometimes
does in other flagellates, such as Giardia (Kofoid and Swezy, 1922).

The undulating membrane (wnd. memb., fig. A) extends from the
primary blepharoplast posteriorly in a slightly leiotropic spiral (pl.
37, fig. 4) of not over 0.5 turn at the most and extending posteriorly
about 0.8 of the length of the body to the end of the parabasal body,
where it terminates with the emergence of the marginal filament as
the posterior free flagellum. The number of undulations present in
this membrane at any one time appears to be a function of their
rate of progress. They are not always evident in fixed material.
In active free flagellates they move so rapidly as to defy estimation.
In fixed material one finds at the most (pl. 37, figs. 1, 7) eight to ten
undulations.

The membrane consists of the marginal filament (marg. fil., fig. A)
which is morphologically a flagellum originating from the primary
blepharoplast, enclosed within the outer pellicle, and terminating
distally in a free flagellum (post. fr. flag., fig. A) which is about 0.5
of the length of the body or more. It also contains a ribbon-like
extension of the periphery of the body at whose base runs the para-
basal body (par. b., fig. A). This structure is a curved rod of uniform
caliber and regular course, originating from the primary blepharo-
plast and following the course of the undulating membrane. It
terminates at the point of emergence of the posterior free flagellum.
Tt stains lightly and destains very readily in our material and is
often located with difficulty, if at all.

The axostyle (az., fig. A) is a stout rod of hyaline appearance
and uniform ealiber, which appears to originate from the primary
blepharoplast and to extend posteriorly in an axial position. Its
posterior end projects beyond the cytoplasm as a rather bluntly
pointed tip for varying distances, depending upon the amount of
cytoplasm present and upon the degree of contraction of the body.
Its free end rarely attains a length of 0.5 that of the cytoplasmic
body. Its varying degree of curvature in different individuals is
suggestive of the contractile function which we have seen and de-
seribed in Trichomonas (1915).

The eytostome (cyt., fig. A) is a well-defined and characteristic
structure. It is an elongated, broadly comma-shaped, often slightly
protuberant area (pl. 37, figs. 3, 6) on the opposite side of the body
from the undulating membrane. It extends from the anterior tip of
the body posteriorly for about 0.35 of the length of the body and

1923 | Kofoid-—Swezy: Pentatrichomonas ardin delteili 38]

tapers posteriorly asymmetrically. A dark fibrillar line forms its
margin. We are unable to find a fibrillar connection between this
margin and a blepharoplast such as we found (1920) in Chilomastiz.
It abuts directly upon the secondary blepharoplast and may have a
connection with it.

The nucleus (nuc., fig. A) varies greatly in size, ranging from
two to four microns in diameter. It is smaller directly after mitosis
in some instances (pl. 37, fig. 5). It is usually spheroidal, but
oceasionally ellipsoidal with the longer axis anteroposterior. It is
located anteriorly, never more than 0.5 its diameter from the blepharo-
plasts and usually almost in contact with them. Its membrane is
lightly enerusted with peripheral chromatin and at its center there
is often a single, spherical karyosome of varjable size (pl. 37, figs.
3, 4). In some instances there are two such chromatin masses (pl. 37,
figs. 2,7) which suggest the early prophase of mitosis. In one instance
(pl. 37, fig. 10) of a late telophase, an intranuclear rhizoplast runs
from the centrosome to the chromatin mass.

The cytoplasm of this species is generally rather coarsely vacuo-
lated, except during binary fission. The vacuoles may contain red
blood corpuscles in various stages of digestion, or clusters of bacteria
or food remnants (pl. 37, fig. 3). We have not observed in this species
the sloughing off of cytoplasmic blobs which we have previously noted
in Trichomonas (1915) and Chilomastix (1920).

The shape of the body on stained slides is stout pyriform, tapering
posteriorly with a slight asymmetrical taper anteriorly also to the
region of the blepharoplasts. It is more rotund on the side of the
eytostome. Posteriorly it tapers in an asymmetrical cone of forty-five
or more degrees. The taper at the two ends is such as almost to give
an asymmetrical biconical shape to the body, the convexity is some-
what greater on the side of the undulating membrane. In life the
length is often twice the greatest diameter, or even more, and the
asymmetry of the apex more marked than in stained individuals.

The dimensions of Pentatrichomonas in fresh stools and in cultures
vary considerably. The length of twenty-five individuals measured
from the anterior tip of the body to the posterior end of the cytoplasm,
but not to the tip of the axostyle, ranged from 9 to 20 microns, aver-
aged 14.6 and had its mode at 14 microns. The width ranged from
7 to 14 microns, average 9.3 and had its mode at 10 microns. A
rounded-up individual measured 9 by 9 microns. These measure-
ments were made on material stained in iron haematoxylin. In life

382 University of California Publications in Zoology —[Vou. 20

the length is as a rule somewhat greater, contraction undoubtedly
occurring as individuals slow down, and in fixation. The length of
the exposed tip of the axostyle was 2 to 4 microns. The diameter of
the nucleus ranged from 2 to 4 microns. The transdiameter in the
plane of the undulating membrane was greater by about one-tenth
than that at right angles. The optical cross-section was broadly pyri-
form with the narrower end at the undulating membrane.

REPRODUCTION

Binary fission (pl. 37, figs. 8-10) preceded by mitosis occurs
abundantly in stools and in cultures. We have repeatedly seen
multiple fission or somatella formation in cultures. A two-, four-,
and eight-nucleate somatella of spheroidal shape only slightly larger
than the unicellular phase with a neuromotor system attached to each
nucleus is formed. This presumably undergoes plasmotomy and forms
eight unicellular flagellates. We have never seen the least trace of
encystment in either fresh stools or cultures. Efforts to induce
encystment by slow desiccation of stools and cultures were also
fruitless.

BEHAVIOR

Observations on living Pentatrichomonas in liquid stools and
especially in the marginal zone between normal saline and iodine-
eosin stain, where movement is progressively slowed down, has enabled
us to analyze the movements of certain elements of the neuromotor
system and to trace certain stereotyped features of the behavior of
the animal, and especially to determine the differential movements
of the single independent, and the clustered anterior, flagella.

Locomotion in Pentatrichomonas is continuous in normal indi-
viduals. It is accompanied by an anticlockwise rotation, which at
times appears to be interrupted by a very brief interval of a clock-
wise direction. Progression may be in a straight line for short dis-
tances, but is generally curved and frequently interrupted by brief
intervals of circling movement in short ares of shorter radius in an
anticlockwise direction.

A quick spasmodie contraction precedes these interruptions in
speed and direction. This appears to be analogous to the motor or

1923 ] Kofoid-Swezy: Pentatrichomonas ardin delteili 383

avoiding reaction of the ciliates. It can be observed most readily in
ease of an individual temporarily anchored by the tip of the axostyle
or by the free flagellum.

A peculiar, rather regular wobbling characterizes the locomotion,
which appears to be due to a pendulation of the body about the axis
of progression which is correlated with the differential strokes of the
independent and clustered anterior flagella. As a result, the two
ends describe approximately equal cones of rotation about the spiral
axis of progression.

A persistent study of the location of the flagella in stained
preparations and during the slowing-down period in the margin of
the iodine-eosin stain in fresh smears has enabled us to distinguish the
differential strokes and to correlate them with the movement of the
undulating membrane and with the origin of the two groups from
the two blepharoplasts.

The motion of the undulating membrane, as the slowing-down
process ensues, is resolved into a series of regular, successive beats
or contractions, which traverse the membrane as a series of character-
istic undulations. These do not, however, appear to run through the
free flagellum at the end. Each beat is synchronous with the stroke
of the independent anterior flagellum. This flagellum moves inde-
pendently of the other four, in a sweeping continuous stroke, starting
from the anterior position, across the right face of the body, assuming
that the eytostome defines the ventral side. This flagellum and the
undulating membrane arise from the same primary blepharoplast and
beat synchronously.

The four remaining clustered anterior flagella all beat in a stroke
independently of the one just described. They appear to beat together
or in rapid and close succession so that in fixed material they are
often found in juxtaposition or closely spaced. They all arise from
the secondary blepharoplast.

Not only are the strokes of the derivatives of the blepharoplasts
independent but they beat at different intervals. In one individual
in the early phases of slowing down, the strokes were carefully timed.
The undulations ran at the rate of 30 beats in 10 seconds. During
this time the body was rotating at the rate of 3.5 rotations in 10
seconds. In another instance the rate was 10 beats in 10 seconds.
The independent anterior flagellum beats in the same rhythm. The
four clustered flagella of this last individual were beating at the rate
of only 4 strokes in 10 seconds. No protoplasmic lobe was in evidence.

384 University of California Publications in Zoology — [ Vou. 20

The four flagella stroke from the base with the tip lagging continu-
ously through the down and up phase, stopping for an instant in an
anterior cluster at the end of the stroke. They pass over the region
of the undulating membrane during the stroke. The first effect is a
short jerk of the body posteriorly as the stroke begins, followed by
a spiral anticlockwise turn of the body in a short are and a forward
thrust.

It is obvious that only once in its ecireuit will the independent
flagellum pass the clustered flagella. It follows that during the most
of the time it is at some distance from the other and if fixed suddenly
would, as a rule, lie apart from the four flagella. This we find is its
usual position on our stained slides. A knowledge of this fact is of
assistance in determining the number of flagella. Failure to observe
it may lead to overlooking the fifth flagellum.

Another characteristic of this and of other trichomonad flagellates,
often seen in moribund or slowed-down individuals, is the formation
of a stumpy or finger-like protoplasmic protrusion originating near
the anterior end of the body and progressing posteriorly beyond
the middle in diminishing prominence. The organism described by
Castellani (1905) as Entamoeba wndulans is doubtless based on this
phenomenon in a moribund trichomonad flagellate.

The exact nature of this process has not hitherto been determined.
Dobell and O’Connor (1921) deseribed it as occurring after ‘‘the
organism loses its flagella and other organs.’’ As we have observed
it, it certainly may occur when all the organelles are present and
functioning, though slowly. It is highly probable that it is the mani-
festation in slowing-down individuals of the spasmodic contraction
or motor reaction of normal locomotion, but it takes place so quickly
that it escapes observation as such in the rapidly moving organism.
Since it varies greatly in extent when it can be observed in operation,
it is quite possible that it is exaggerated in extent in moribund
individuals. It is not a pseudopodium either morphologically or
functionally.

Its form is that of a blunt process with rounded tip and spreading
base. Its height varies but is greatest near the middle of the body
or just anterior to this level. It may attain a height equal to one-half
the transdiameter. In other cases the lobe is so broad at the base that
it gives a subtriangular outline to the body.

The axostyle within the cytoplasm is so difficult to detect in life
that one cannot determine whether or not it shares in this movement.

1923] Kofoid-Swezy: Pentatrichomonas ardin delteili 385

The probability that it is concerned both with the motor reaction and
with the protoplasmic lobe should be borne in mind in the study of
these phenomena.

This lobe is not formed in, nor is it a phase of, the undulating
membrane. We have repeatedly watched it in individuals in which
the undulating membrane was still functioning. It is formed on the
side opposite to that membrane and its rhythm is much slower than
that of the membrane. In one observed instance the undulating
membrane had a rhythm of 30 beats in 10 seconds while the lobe had
one of but 6 beats in the same time.

SURVIVAL IN WATER

This parasitic flagellate may survive for some time in liquid stools
at room temperatures. The period of survival in different stools of
the same persons is not the same, possibly because of varying bacterial

«

growths. The flagellates often disappear in 3 to 5 days, but in one
instance a liquid stool kept at room temperature, 65°—70°, retained
living Pentatrichomonas from October 4 till October 27, a period of
24 days. Sterilized tap water, rain water, and creek water are less
favorable for the survival of these fagellates, though not immediately
destructive. The flagellates disappear in these fluids within three
days following inoculation with fresh stools. This period is sufficient
as to length of time for dispersal to and infection of other hosts and
may afford a basis for epidemies in cases of infected water supplies.

In normal saline, three cultures survived for 8, 10, and 15 days
respectively. On two vaselined slides at laboratory temperatures,
flagellates in a stool diluted with normal saline multiplied and
remained active for 5 to 10 days and died out in 13 and 25 days
respectively.

386 University of Califorma Publications in Zoology [| Vou. 20

CULTURE

Pentatrichomonas may be cultivated with facility in 10 per cent
serum of the rabbit or guinea pig in Locke’s solution at room tempera-
ture. Seven cultures started on September 29 in guinea pig serum
ran until October 28 with a great abundance of flagellates. One
culture was used for preparations on that date. One had died out
by November 7, two more by the thirteenth, one by the twentieth, two
by the twenty-fifth, and the last one lingered till January 24, a period
of 118 days. The flagellates survived in the original stool only six
days. A culture in 10 per cent rabbit serum in Locke’s solution
started on October 4, subeultured on October 27 and on November 9,
survived without later subcultures till February 14, 98 days after
the last subculture.

Using stools from this case Mrs. E. H. Wagener has cultured this
organism successfully in 10 per cent human serum from Wassermann
samples at 37°25 C, at present writing for 134 days, with 40 trans-
plants at intervals of approximately 3 days. They thrive best in
neutral serum (PH 7.4) and die out at 6.2 and 8.2 respectively. Of
late these cultures are growing in a ‘pure mixed’ culture with B. coli.

Dobell and O’Connor (1921) discredit Escomel’s (1913) state-
ment that he had cultivated Trichomonas hominis by inferring, with-
out the least evidence, that this investigator mistook free-living
flagellates in his cultures for Trichomonas. They also discredit
Lynch’s (1915) suecessful culture of Trichomonas in acid broth, with
the statement, ‘‘Lynch claims’’ and proceed to dogmatize as follows:
‘At the present time, however, it is not possible to cultivate this
organism with certainty in any medium. All attempts which we
ourselves have made have been failures.’’ Fortunately ex-cathedra
statements and negative results are easily evaluated.

We have cultivated Trichomonas hominis in fecal suspensions by
Boyd’s method and in 10 per cent guinea pig serum in normal saline,
with transplants at intervals of 3 to 5 days, for several months.

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Kofoid—Swezy: Pentatrichomonas ardin delteili 387

SUMMARY

Pentatrichomonas ardin delteili is a trichomonad flagellate of
man distinet from Trichomonas hominis. It has five anterior flagella
while 7. hominis has but four. The number of these flagella is eon-
stant, except as doubled in the premitotie phase, and is characteristic
in the two genera. The anterior flagella fall into two groups arising
from separate blepharoplasts. Four clustered flagella arise from the
secondary or distal blepharoplast. The remaining single flagellum
arises from the primary or proximal blepharoplast, together with the
axial filament of the undulating membrane, the parabasal body, and
the axostyle. The two blepharoplasts are united by a rhizoplast
which continues to the centrosome on the anterior face of the nuclear
membrane.

These two groups of motor organelles move in independent
rhythms. The independent flagellum and undulating membrane of
the proximal group run a more rapid rhythm, beating in one ease
of a slowed-up individual 10 strokes every 10 seconds, while the four
clustered flagella of the distal group beat only 4 strokes to 10 seconds.
Only once in the stroke of the independent flagellum does it lie con-
tiguous to the four clustered ones. This fact tends to cause obscurity
as to the number of flagella in this and in other trichomonads. <A
knowledge of it will assist in accurate diagnosis of trichomonad
infections.

Pentatrichomonas eats red blood eorpuscles in stools and in eul-
tures. It persists for 5 days in the stool in the laboratory, for 3 days
in water, for 15 days in normal saline, for 118 days in 10 per cent
rabbit serum in Locke’s solution. It can be continuously cultivated in
10 per cent rabbit, guinea pig, and human serum in Locke’s solution
at room and body temperatures with transplants at intervals of 3
days. Its optimum PH is 7.4.

It has been observed by us in three human cases, each with tropical
residence or contacts, and each with similar symptoms of continuous,
chronic, persistent diarrhoea resistant to treatment. To determine the
pathogenicity of human intestinal infections, clinical study and
accompanying accurate determination of the infecting species are
required.

ZooLOGIcAL LABORATORY,

UNIVERSITY OF CALIFORNIA.
Transmitted July 9, 1923.

388 University of California Publications in Zoology (Vou. 20

LITERATURE CITED

ALEXEIEFF, A.
1912. Sur quelques protistes parasites intestinaux d’une Tortue de Ceylon
(Nicoria trijuga). Zool. Anz., 40, 98-105, 3 figs. in text.
ALVAREZ, A. V.
1916. Trichomonosis intestinal. Tesis, Bogota, pp. x+99, 3 pls.
CASTELLANI, A.
1905. Observations on some Protozoa found in human faeces. Centralbl. f.
Bakt. Parasit. u. Inf., I. Abth., 38, 66-69, 5 figs. in text.

* CHATTERJEE, G. C.

1915. Ona five flagellate Trichomonas (n. sp.) parasitic in man. Indian Med.
Gaz., 50, 1-9, 1 pl.

1917. Note on flagellate dysentery. Indian Jour. Med. Res., 4, 393-401,
pls. 25-29.

DERRIEU, G., and RayNaup, M.

1914. Dysenterie chronique 4 flagellé nouveau. Bull. Soc. Path. Exot., 7,
571-574.

DoBELL, C., and O’Connor, F. W.

1921. The intestinal Protozoa of man (London, John Bale Sons and Daniels-
son), pp. ix+211, 8 pls., 2 figs. in text.

Donne, A.

1837. Recherches microscopiques sur la nature des mucus et la matiére des
divers écoulemens des organes genito-urinaires chez 1’homme et chez
la femme (Paris, 1’Auteur), pp. iv+71, 1 pl.

EScoMEL, E.

1913. Sur la dysenterie a Trichomonas 4 Arequipa (Pérou). Bull. Soe. Path.
Exot., 6, 120-122.

1919. La Tricomonosis intestinal (Lima, Sammarti), 78 pp., 3 pls.

Havucuwout, F. G.

1918. The tissue invasive powers of the flagellated and ciliated Protozoa with
especial reference to Trichomonas intestinalis. A critical review.
Philippine Jour. Sci., (B), 13, 217-259, 1 fig. in text.

1920. Some current problems in protozoal dysentery. China Med. Jour., 1920,
1-15.

Havucuwovt, F. G., and De Leon, W.

1919. On the ingestion of erythrocytes by Pentatrichomonas sp. found in a

case of dysentery. Philippine Jour. Sci., (B), 14, 207-218, 1 pl.
Kororp, C. A.

1920. A eritical review of the nomenclature of human intestinal flagellates,
Cercomonas, Chilomastix, Trichomonas, Tetratrichomonas, and Giar-
dia. Univ. Calif. Publ. Zool., 20, 145-168, 9 figs. in text.

Korom, C. A., and Swezy, O.

1915. Mitosis and multiple fission in trichomonad flagellates. Proce. Am. Acad.
Arts and Sci., 51, 289-378, pls. 1-8, 7 figs. in text.

1920. On the morphology and mitosis of Chilomastiz mesnili (Wenyon). A
common flagellate of the human intestine. Univ. Calif. Publ. Zool.,
20, 117-144, pls. 15-17, 2 figs. in text.

1923] Kofoid—Swezy: Pentatrichomonas ardin delteili 389

1921. The flagellate infections of the human digestive tract. Nelson’s Loose
Leaf Encyclopedia of Living Medicine, 4, 365-398, 6 pls.

1922. Mitosis and fission in the active and eneysted phases of Giardia enterica
(Grassi) of man, with a discussion of the method of origin of
bilateral symmetry in the polymastigote flagellates. Univ. Calif.
Publ. Zool., 20, 199-234, pls. 23-26, 11 figs. in text.

LyncH, K. M.

1915. Trichomoniasis of the vagina and the mouth. Cultivation of the causa-
tive organism and experimental infection. (A preliminary com-
munication.) Am. Jour. Trop. Dis. and Prev. Med., 2, 627-634.

MESNIL, F.

1914. Review of Derrieu and Raynaud’s ‘‘Dysenterie chronique a flagellé
nouveau’’ in Bull. Soc. Path. Exot., 7, 571. Bull. Inst. Pasteur,
12, 968.

MESNIL, F.

1915. <A propos du flagellé nouveau décrit par MM. Derrieu at Raynaud. Bull.

Soe. Path. Exot., 8, 574-575.
VACCAREZZA, R. F.

1917. Tricomonosis intestinal. Anales Inst. Modelo Clin. Med. (Buenos

Aires), 2, 1-37, 15 figs. in text.
WENYON, C. M., and O’Connor, F. W.

1917. Human intestinal Protozoa in the Near East (London, John Bale, Sons

and Danielsson), 218 pp., 4 pls.

EXPLANATION OF PLATE

PLATE 37

All figures of Pentatrichomonas ardin delteili (Derrieu and Reynaud) X 2500.
Material fixed in hot Schaudinn’s fluid and stained in iron haematoxylin. Draw-
ings made with Abbe camera lucida.

Fig. 1. Individual with undulating membrane in lateral position, cytostome
below with chromatin diffused throughout the nucleus. Note detached position
of the anterior independent flagellum.

Fig. 2. ‘Ventral’ view with eytostome uppermost and undulating membrane
below. Note curvature of axostyle, centrosome on the nuclear membrane, and
two karyosomes, probably preparatory to mitosis.

Fig. 3. Lateral view with very large nucleus, large karyosome, and food
vacuole with contents.

Fig. 4. Lateral view with remnants of food particles in the cytostome and
chromatin encrusted nuclear membrane.

Fig. 5. Individual which had recently ingested a red blood corpuscle. From
life.

Fig. 6. Lateral view of contracted specimen with much curved axostyle, and
diffusely stained nucleus.

Fig. 7. Lateral view with protuberant cytostome. Note centrosome on the
anterior face of the nuclear membrane, two karyosomes in the nucleus, and the
straight axostyle.

Figs. 8, 9, and 10. Binary fission with two nuclei and a common cytostome,
each with its independent neuromotor system.

[390]

[ KOFOID—SWEZY ] PLATE 37

, CALIF, PUBL. ZOOL. VOL. 20

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UNIVERSITY OF CALIFORNIA PUBLICATION S—(Continued)

The Life Cycle of Echinostoma revolutum (Froelich), by John C. Johnson.
Pp. 338-388, plates 19-25, 1 figure in text. May, 1920 000 a

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Ralph V. Chamberlin. Pp. 389-402, plates 26-28. August, 1920 0... =,

Demonstration of the Function of the Neuromotor Apparatus in Euplotes
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Index, pp. 471-480.

Studies onthe Parasites of the Termites. I. On Streblomastizx striz, a Poly-
mastigote 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

Studies on the Parasites of the Termites. II. On Trichomitus termitidis, a
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by Charles Atwood Kofoid and Olive Swezy. Pp. 21-40, plates 3-4, 2
piteg ry wefigs eflsrrizy.q rose Hs ait LS hs Cpe sets cana vase SIC ep ABEERIN ESA 9) anak LPM ME A AM

Studies on the Parasites of the Termites. ITI. On Trichonympha campanula
sp. nov., by Charles Atwood Kofoid and Olive Swezy. Pp. 41-98, plates
m2; 4 fisnres'in ‘text:. Tuly,: 1919) i ee eee) a

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nov., sp: nov., by Charles Atwood Kofoid and Olive Swezy. Pp. 99-116,
plates 13-14,:1 figure in text. - July, 1919 10k ee tha

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Pp. 117-144, plates 15-17, 2 figures in text. April, 1920 22. AS
A Critical Review of the Nomenclature of Human Intestinal Flagellates,
Cercomonas, Chilomastiz, Trichomonas, and Giardia, by Charles A. Kofoid.
Pp: 145-168, ‘9: fisures in text. ‘/dume,-1920 225 eee ee
On the Free, Encysted, and Budding Stages of Councilmania lafleuri, a Para-
sitic Amoeba of the Human Intestine, by Charles Atwood Kofoid and
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Mitosis and Fission in the Active and Encysted Phases of Giardia enterica
(Grassi) of Man, with a Discussion of the Method of Origin of Bilateral
Symmetry in the Polymastigote Flagellates, by Charles A. Kofoid and
Olive Swezy. Pp. 199-234, plates 23-26, 11 figures in text. March,

SAUNT DO tes st AE NE ES ge ON Eg Ne
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Pp. 243-800, plates 27, 28, 15 figures in text. May, 1922 Www...

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IN

ZOOLOGY
Vol. 20, No. 17, pp. 391-400, 14 figures in text August 16, 1923

THE PSEUDOPODIAL METHOD OF FEEDING
BY TRICHONYMPHID FLAGELLATES
PARASITIC IN WOOD-EATING
TERMITES

BY
OLIVE SWEZY

In the food-taking habits of the Protozoa we find as great a
diversity as is found in other features of their life history or
morphology. In those groups having a holozoie type of feeding, two
general methods of taking food particles into the body may be dis-
tinguished. In the forms showing a definite polarity food is taken
in at or near the anterior or head end, in common with the method
general in the Metazoa. When a distinct pellicle or a structurally
differentiated ectoplasm is present, as in the flagellates and ciliates,
an oral aperture or cytostome is generally found, at least in those
forms which ingest solid food particles.

In the amoeboid type of protozoan, with little or no evidences
of polarity, food may be taken in at any point in the body. This
method of feeding is the common one among the Rhizopoda, the
former one being typical of the Ciliata and Flagellata.

In the remarkable and highly complex group of flagellates inhabit-
ing the digestive tract of the termites, at least two genera of flagellates,
Trichonympha and Leidyopsis, are found which combine the type of
feeding of the Rhizopoda with the highly differentiated ectoplasm
characteristic of the higher Ciliata. This fact, combined with the
very striking polarity exhibited by these flagellates, distinguishes
this group from most, if not all other Protozoa in its food habits.

In the first published description of the trichonymphids by Leidy,
in 1881, mention is made of the great number of solid food particles

392 University of California Publications in Zoology | Vou. 20

found within the body, and the total lack of evidence of the manner
of ingesting them. Later investigators have been equally at a loss to
account for their presence with the lack of a visible cytostome. Kent
(1884), however, decided that an oral aperture was present at the
base of the cone-like anterior portion of the body, opening into a
narrow oesophageal tract which extended to the digestive cavity at
the posterior portion of the body.

Porter (1897) was unable to confirm Kent’s observations and
offered another solution of the problem, but with little evidence to
support it. He found cross-sections of the body of Trichonympha
which showed deep folds of the body wall, invariably containing cilia
in which wood particles were often entangled. He says that
most of the cilia are probably afterwards withdrawn from the fold but the lips
of the fold become so closely applied to each other that the ligneous particles
are left behind in the depth of the fold. I believe that the lips afterwards fuse

together, that the walls of the infolded portion then disappear, and that the
food thus finally becomes entirely enclosed in the body protoplasm of the parasite.

Other investigators have not been more successful in solving the
mystery of food-taking in the trichonymphids, nor has our own
earlier work (Kofoid and Swezy, 1919) on these flagellates solved the
problem. This has been due probably to the extreme delicacy of
these organisms and the brief duration of their activities under the
microscope in ordinary fluids, such as physiological salt solution. We
have, however, been more fortunate in the study of the tricho-
nymphids from a colony of Termopsis angusticollis (?) from Santa
Cruz, California.

These termites had been living in the laboratory in a block of wood
in a stone crock closely covered since January, 1920. In spite of
the fact that no moisture had been added to the crock during that
time and no provision made for ventilation, the termites were found
in vigorous condition when removed for examination.

For study of the lying organisms the largest termites were
selected, those in which the abdomen seemed to be somewhat swollen.
The entire intestinal tract was removed and placed upon a slide which
had been ringed with vaseline. The wall of the intestine was carefully
opened and spread apart and a coverslip placed over it. Usually in
the largest individuals there was sufficient liquid in the intestine to
make the addition of physiological salt solution unnecessary, and best
results were obtained in such cases. Trichonympha has been kept
alive and in active motion in slides prepared in this manner for two

1923 | Swezy: Pseudopodial Method of Feeding 393

to six hours. In physiological salt solution and various other media
that have been tried, the body of the flagellate tends to round up
immediately, becoming greatly distended and dissolving a few minutes
after being placed on the slide.

These termites contained a species of Trichonympha and one of
Leidyopsis in great abundance. Both were found to be actively
feeding and full details of the process could be watched under the
microscope.

A slide prepared in the manner described above presents a remark-
able picture of ceaseless activity and turmoil when viewed under a
lf, objective with a low ocular. So narrow is the space between
the bodies of the flagellates that often flagellar activity is entirely
obscured and the resulting picture shows only a tangled mass of
writhing protoplasmic bodies, the movements and appearance of
which defy description. Near the outer borders of such masses
the number of individuals becomes somewhat lessened and here their
movements may be followed with greater ease and certainty.

The outstanding feature in such a picture is the extreme mobility
of the flagellate’s body. When worming its way through a crowded
mass of its fellows, the change of outline is incessant, with such
changes reflected in the shape of the nucleus and protoplasmic con-
tents of the posterior part of the body. The endoplasm in the anterior
end seems to partake less of this movement, though this may be due
to the fact that it is very finely granular and contains no particles
that would catch the eye by their movement. The anterior portion
of the body, or that covered by the differentiated ectoplasm (Kofoid
and Swezy, 1919a), is in constant motion, waves passing spirally in
an antero-posterior direction. These movements are irregular, some-
times being of great frequency and amplitude, particularly when
an obstacle is met, at other times becoming slower, with only a slight
rise and fall of the surface. The posterior portion is usually more
passive and changes its shape only to meet the requirements of its
environment. When adverse conditions on the slide have slowed
down the motion of the flagellate, the protoplasmic movements cease
entirely, but the flagella continue their rippling motion for some time.

Leidyopsis shows the same types of movements described for
Trichonympha, but the area of differentiated ectoplasm being much
smaller, both relatively and actually, its mode of progression is much
less striking than that of the larger flagellate. It exhibits two distinet
shapes of body. The first (fig. 1) is elongate with the posterior

394 University of California Publications in Zoology | Vou. 20

portion somewhat tapering or narrowly rounded. The second (fig. 3)
is that of a shallow bell which, viewed from the anterior end (fig. 2),
presents a circular or nearly circular outline. Its movements in this
stage remind one strongly of those of a jellyfish, depending not so
much on its flagella as on its complicated system of myonemes (Kofoid
and Swezy, 1919b) by means of which the surface is kept in constant
vibration. When in this condition its movements do not result in

Figs. 1-3. Leidyopsis sp. 1. Figure showing typical form. The part of the
body posterior to the line showing width of ectoplasm is covered only by thin film
of ectoplasm. X 400. 2. Bell-shaped individual looked at from the anterior end.
3. Same individual viewed from the side. Figures 2 and 3 were drawn from
living specimens. X 300.
much change of position. The edges of the bell do not remain passive
but push out in httle exploratory movements.

If a food particle comes in contact with the lower surface of the
bell it may be thrown off by the activities of the body, or it may be
caught by the mobile ectoplasm of that region, which seems to become
somewhat sticky. In the latter case the flagellate proceeds to engulf
the object, even if it is of considerable size. More frequently the
mobile edges of the bell may become attached to a food particle and
initiate the engulfing process.

The first point of contact between the flagellate and the food
body, usually a particle of wood, may be very small (fig. 4), but it is

1923] Swezy: Pseudopodial Method of Feeding 395

held quite firmly, a necessary requirement in the turmoil going on
around it if the animal is to feed at all. This appears to be done by
means of secretions of the ectoplasm at the point of contact. A email
pseudopod of clear ectoplasm may now be seen moving along the

Fig. 4-9. Leidyopsis sp., drawn free-hand from living specimens. Figure 4
shows beginning of process of ingesting food body. Figure 5 shows a somewhat
later stage. Note long clear pseudopod. Figures 6 to 9 give successive stages in
the ingesting of a food body, process occupying about one hour. Note clear
pseudopods creeping along wood particles. X 300.

surface of the wood (fig. 4). The pseudopod may continue along
one side at first (fig. 6), or the ectoplasm may move out in a ring
around the particle, drawing it into the body by one end, dependent

apparently on the location of the point of its first contact. When
this point is at the side the pseudopods may extend in either direction

396 University of California Publications in Zoology — [ Vou. 20

along the length of the wood. When the protoplasmic body comes in
contact with the end, that is completely engulfed first.

The length of time necessary for the complete intake of any
particle could not be learned with exactness, owing to the tendency
of the strong light used as illumination for the microscope to slow
up protoplasmic movements other than those used in locomotion. In
the individual shown in figures 4 to 6, the time of completing the
process was found to be one hour. This individual was not held in
the field continuously but was returned at short intervals, with only
partial light in the interims. When first brought into the light the
pseudopods could be seen moving forward with slow creeping motion.
In figure 5, a flagellate is shown with one long pseudopod extending
out to a branch of the woody particle that is being engulfed. This
pseudopod was formed when that branch touched the surface of the
body, contact being made at only one point but that strong enough
to draw out the strand of clear ectoplasm when the surrounding
obstacles foreed the piece of wood farther away from the body (see
also fig. 13).

The protoplasm shows remarkable elasticity as well as tenacity.
One flagellate was seen with a piece of woody material about its own
size attached by a pseudopod 10» in length. In swimming it met
obstacles which its own mobile body easily passed, but the unyielding
wood was caught and the pseudopod was drawn out to about five times
its original length. After great struggles the wood passed the
obstacles and in a few seconds the pseudopod shortened to less than
its original length, drawing the wood with it.

The body may become almost completely filled with woody particles
(fig. 11), since the act of engulfing one piece does not prohibit the
attempt to capture a second one at the same time by another part
of the body. Only rarely is a flagellate seen without many food
bodies within the cytoplasm. These seem to he in close contact with
the endoplasm in most cases but occasionally may be enclosed by a
well-marked food vacuole. Many of the particles within the body
of the flagellate show sharply defined outlines, while others, by their
vague, structureless appearance, give evidence of the progression of
digestive operations.

No evidences of selection have been found in the feeding of these
flagellates. The host termite is a wood-eating species and the
intestinal contents consist almost entirely of woody particles. These
may be of any size from minute particles to others several times the

1923] Swezy: Pseudopodial Method of Feeding 397

length of the flagellates. The larger pieces are engulfed as freely
as smaller ones. Occasionally Leidyopsis may be found to have
mastered a piece of wood so long that the body of the flagellate
becomes stretched out several times its own usual width, forming a
grotesque caricature of its customary appearance (fig. 10). It is
also not uncommon to see pieces so long that the ends project beyond
the two sides of the body.

Figs. 10-11. Leidyopsis sp., drawn from living specimens. Note huge size
of food bodies ingested. X 300.

The smaller flagellates, such as Trichomitus, may become the prey
of both Trichonympha and Leidyopsis. An individual of the latter
genus was noted with an engulfed Trichomitus that was rapidly
rotating in a vacuole in the posterior part of the body. This indi-
vidual was watched for about one hour when its movements had
become slowed down. The vacuole did not have the appearance of
the ordinary food vacuole but seemed to have been formed by the
activities of the Trichomitus.

Particles may also be ejected from the body in the posterior
region. The ectoplasm here lacks the structural differentiation char-
acteristic of the anterior portion of the body as well as the ciliary
coating of flagella (fig. 1). No evidences of an attempt to engulf
or eject food particles through the differentiated ectoplasm of the
anterior region have been seen.

398 University of California Publications in Zoology [Vou 20

The feeding habits of Trichonympha are similar to those described
above for Leidyopsis. A change in the shape of the body occurs
but is not so striking as in Leidyopsis. The posterior portion only
partakes in this change, often losing its elongate rounded form and
becoming wider, giving to the flagellate a bell-shape with the lower
margin considerably swollen. The long flagella on the posterior
portion of the differentiated ectoplasm extend backward with their
tips interlacing in the ordinary form. When the lower part of the

Figs. 12-14. Trichonympha sp., drawn from living specimens, showing various
stages in the ingesting of food bodies. Note short clear pseudopods in figure 12
and the long one in figure 13. X 200.

bell becomes wider they do not form such a close covering of the
posterior portion and food particles may be more easily swept into
contact with the hungry ectoplasm of that region. The same result
is obtained when the posterior portion becomes drawn out beyond
the tips of the flagella. It seems probable that this latter is the
usual form that the flagellate assumes when actively feeding. In
many individuals the posterior portion of the body containing food
bodies occupies about one-third to one-fourth the entire length of
the body (figs. 12, 13). Individuals may show these proportions
reversed. One flagellate was measured which was 560 in length
and only 140n from the anterior tip to the posterior margin of the
nucleus. The remaining portion was literally filled with food
particles.

1923 | Swezy: Pseudopodial Method of Feeding 399

The flagella do not seem to take any part in the capture of food
bodies, though it may be that particles becoming entangled in the
flagella are more easily captured by the ectoplasm. When contact
has been made the same procedure follows as in Leidyopsis, the body
often becoming distorted in its attempts to take in disproportionately
large pieces (fig. 14).

The relatively large amount of food material used by these
flagellates may be directly proportioned to their activities, since these
exceed, in energy expended in meeting the impact of their environ-
ment, the activity of any other forms known to the writer. More
remarkable is the method of feeding, since they are flagellates which
combine the highest degree of ectoplasmic development to be found
in the Protozoa, covering with the cilia-like flagella from one-third
to two-thirds or more of the body surface, with the thin mobile ecto-
plasm characteristic of the Rhizopoda over the remaining portion of
the body. The degree of development of the anterior part of the
body is undoubtedly the result of environmental conditions (Kofoid,
1923) and, in the absence of a cytostome anteriorly, the amoeba-like
ingesting of food at the posterior end seems also to be the result of
the same factors.

SUMMARY

Trichonymphid flagellates parasitic in the digestive tract of wood-
eating termites do not feed by means of their abundant anterior
flagella, nor by means of an anterior gullet as proposed by Kent
(1884), nor at any point on the flagellated area as suggested by Porter
(1897), but by means of pseudopodia formed on the posterior region
of the body. These pseudopodia are peculiary adhesive, clinging to
the particle of wood which they touch, and slowly creeping out over
these particles until they completely engulf them. Particles double
the diameter of the body may be engulfed.

ZOOLOGICAL LABORATORY,
UNIVERSITY OF CALIFORNIA.

Transmitted May 1, 1923.

400 University of California Publications in Zoology [Vou. 20

LITERATURE CITED

Kent, S.
1884. Notes on the infusorial parasites of the Tasmanian white ants. Papers
and Proe. Roy. Soe. Tasmania, 1884, 270-273.

Koro, C. A.
1923, Parasitism and evolution. Address at National Academy of Sciences,
April 23, 1923. MS.

Kororp, C. A., and Swezy, O.
1919a. Studies on the parasites of the termites. III. On Trichonympha cam-
panula sp. noy. Univ. Calif. Publ. Zool., 20, 41-98, pls. 5-12, 4 figs.
in text.
1919b. Studies on the parasites of the termites. IV. On Leidyopsis sphaerica
gen. nov., sp. nov. Jbid., 20, 99-116, pls. 13-14, 1 fig. in text.
Leipy, J.
1881. The parasites of the termites. Jour. Acad. Nat. Sci., Philadelphia, (2),
8, 425-450, pls. 51-52.
Porter, J. F.
1897. Trichonympha and other parasites of Termes flavipes. Bull. Mus.
Comp. Zool., Cambridge, 31, 48-68, pls. 1-6.

13.

12.

(13,
aft 14.
45.

16.

piladaitir ff

UNIVERSITY OF CALIFORNIA PUBLICATIONS— (Continued)

The Life Cycle of Lchinostoma revolutum (Froelich), by John C. Johnson.
Pp. 338-388, plates 19-25, 1 figure in text. May, 1920

. On Somé New Myriopods Collected in India in 1916 by C. A. Kofoid, by

Ralph V. Chamberlin. Pp. 389-402, plates 26-28. August, 1920 0.0... of
Demonstration of the Function of the Neuromotor Apparatus in Euplotes
by the Method ef Microdissection, by Charles V. Taylor. Pp. 403-470,
plates .29-33,)2 figures in text. ‘October, 1920 oo.ccco lec ccilcenneceeseteees a
Index, pp. 471-480.

Studies on the Parasites of the Termites. I. On Streblomastiz strix, a Poly-
mastigote Flagellate with a Linear Plasmodial Phase, by Charles Atwood
Kofoid and Clive Swezy, Pp. 1-20, plates 1-2, 1 figure in text. July, 1919

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
1o¥ep0) cycle b cla A cb .q capes av bk aM Ke Ht i cnn BANU Hla SO Gy akan cer RP as ae eRe

Studies on the Parasites of the Termites. III, On Trichonympha campanula
sp. nov., by Charles Atwood Kofoid and Olive Swezy. Pp. 41-98, plates
Bel 2b 4 fisures intext Duly; LIL ee ead 8 oes us

Studies on the Parasites of the Termites. IV. On Leidyopsis sphaerica gen.
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$ 4 rs ol. 20, Nos. 18 and 19, pp. 401-430, plates 38-39 October 31, 1923

METHODS OF OBTAINING AMOEBA-FREE
_ RATS FOR EXPERIMENTAL INFECTION
WITH INTESTINAL AMOEBAE

3 BY
a JOHN F. KESSEL

_ EXPERIMENTAL INFECTION OF RATS AND
“MICE WITH THE COMMON INTESTINAL
| - AMOEBAE OF MAN |

* ‘ BY
NE yes JOHN F. KESSEL

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UNIVERSITY OF CALIFORNIA PUBLICATIONS
IN

ZOOLOGY
Vol. 20, No. 18, pp. 401-408 October 31, 1923

METHODS OF OBTAINING AMOEBA-FREE RATS
FOR EXPERIMENTAL INFECTION WITH
INTESTINAL AMOEBAE

BY
JOHN F. KESSEL

CONTENTS

Routine examination of normal faeces
Examination of faeces of rodents after administration of a purgative ... 403
Incidence of infection at different ages
Waric TST OTIS yee se meen ens ee eS A Ae
Literature cited

BO 2 bP

In an investigation carried on recently by the writer an attempt
was made, first, to determine the different species of intestinal amoebae
found in culture rats and mice, and second, to establish, in culture
rats and mice, experimental infections of the common intestinal
amoebae of man and of the amoebae common to rats and mice.
During these experiments methods for hastening the evacuation of
the intestinal Protozoa in the faeces have been developed which it is
thought will prove of value to other investigators in similar fields.

The writer is greatly indebted to Professor C. A. Kofoid of the
Department of Zoology in the University of California for suggestions
and guidance throughout the whole investigation of which this paper
records a part.

A. ROUTINE EXAMINATION OF NORMAL FAECES

Examination of human faecal material for the presence of intes-
tinal Protozoa has shown that it is necessary to examine samples taken
on several successive days in order to make accurate and complete
diagnosis of the organisms present. No experimental work has as
yet been done to determine the degree of certainty of the results of
such examinations of human faeces, but statistics show that the

402 University of California Publications in Zoology [Vou. 20

greater the number of samples examined, the greater the percentage
of infections found (see Kofoid, 1920). In the Department of Para-
sitology, California State Board of Health, in Berkeley, six routine
examinations of human faeces without finding infection are made
before a case is pronounced as probably negative. Dobell (1917 and
1920) has also pointed out the advisability of making six routine
examinations before a human case is pronounced uninfected and in
1917 states that ‘“‘the expectation for the average infected case is
that it will be found positive twice in every five examinations.”’

Routine examination of the normal faeces of rats and mice has
shown this method to be less accurate with these animals than is
probably the case with man. Two reasons may be given for this fact.
First, the number of amoebae found in the rodents is considerably
less than is found in man, and, second, the active stages of the intes-
tinal Protozoa found normally in rats and mice inhabit the small
intestine and caecum in the main and are seldom found in the large
intestine, while in man, amoebie infections, including their cysts, are
common in the colon.

Brug (1919) kept records of routine examinations for amoebae
in the normal faeces of six rats and coneluded that this method is
not thoroughly reliable. His results are included in table 1 for the
sake of comparison with similar conclusions which were drawn early
in the investigation by the present writer.

Tt will be seen from the accompanying table that only 16.3 per cent
of all the examinations made gave positive results, though all the
animals examined were known to be infected with amoebae. Brug
(1919) states that ‘‘the cysts may be found for several days in sue-
cession, then for several days, weeks or months in succession nothing
may be found.’’ Similar results, but not for intervals so long as
months, were obtained in the present investigation. Further, the
number of cysts, when present, may vary greatly. At times only
one eyst may be found on the entire cover glass preparation, while
at other times as many as three or four cysts may be found with the
4 mm. objective in one field of the microscope. As a rule, cysts only
were found in the faeces, though on three occasions motile amoebae
were present. These facts point to the conclusion that there is no
regular periodicity in the discharge of intestinal amoebae in these
rodents.

It is evident from these data that the small number of examin-
ations sufficing for the detection of intestinal Protozoa in man would

1923] Kessel: Infection with Intestinal Amoebae 403

be inadequate in making a diagnosis for rats and mice. It is further
seen that the number of examinations necessary to determine the
exact infections present, or to make it relatively certain that an
animal is free from a given infection, would extend over an almost
prohibitive period of time in experimental work. It was therefore
decided to evacuate the amoebae in the faeces through administration
of a purgative.

TABLE 1

RESULTS OF ROUTINE EXAMINATION OF NORMAL RODENT FAECES FOR
INTESTINAL AMOEBAE

Rats examined Time in Number of Times Times %
by Brug months examinations) positive negative aS

Rat No. 5 105 8 97 7.6
Rat No. 5 88 13 75 14.7
Rat No. 3 91 8 83 8.8
Rat No. 2 37 7 30 19.0
Rat No. 2 26 1 25 3.8
Rat No. 2 28 2 26 oA

Motala! ates 375 39 336 10.6

Rats and mice

examined by Kessel
Rat No. 3 22 9 13 40.9
Rat No. 4 2 10 1 9 10.0
Rat No. 3 22 10 12 45.5
Rat No. 2 28 2 26 fall
Rat No. 2 28 9 19 32.1
Mouse Nos incest: 2 15 6 9 40.0
Mouse No. 2.. Phe as 2 14 6 8 43.0
Mouse No. 3.......... 2 14 4 10 28.6

MOLAIS ences. : 153 47 106 30.6
Grand totals................ 528 86 442 16.3

B. EXAMINATION OF FAECES OF RODENTS AFTER
ADMINISTRATION OF A PURGATIVE

Castor oil mixed with food was first tried as a purgative. The
faeces were evacuated successfully, but the oil droplets in the smear
were confusing because of their resemblance, in optical properties, to
amoebic cysts, especially when seen through a low power microscope.
So commercial epsom salt was used instead. At first this was mixed
with the drinking water and given to the rats in their water bottles.
As the animals refrained from drinking the mixture, except when
especially thirsty, the salt was mixed with food.

[ Vou. 20

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1923] Kessel: Infection with Intestinal Amoebae 405

Stale bread was soaked in a solution of water saturated with the
magnesium sulphate. Hungry rats usually ate this mixture readily,
though the mice often refused to eat it until they had been offered
nothing else for several days. If the rats ate readily of the bread and
salts at 6 p.m., a sufficient quantity of semi-fluid material was assured
for examination during the next day. Rats which gave no reaction
from the salt the morning after being fed the prepared bread were
placed together in a separate cage and redosed, and were regarded
aS unexamined rats. Rats which gave a positive reaction to the
purgative were given a lighter dose of salt the second night. A second
examination of faecal material was made the following day, after
which the animals were autopsied and examination was made of the
intestinal contents at different levels of the digestive tract.

In making the smears a drop of normal salt solution was placed
near one end of the slide and about half an inch toward the middle
of the slide was placed a drop of Donaldson’s iodine-eosin stain. The
faecal material was then smeared, first in the saline portion and then
in the iodine-eosin portion. In placing the cover glass on the smear,
contact was made first with the saline half of the smear. The motile
forms are easily detected in the normal saline solution and the cysts
more easily seen in the iodine-eosin. Cover glasses 12 mm. square
were used and two smears were made on the same slide. A 5%
ocular was used first with a 16 mm. objective for determining the
presence of the organisms. Then the 4 mm. objective was used for
more detailed study and for determining the species of organism
present.

As the writer’s investigation concerned amoebae only, no account
was taken of other Protozoa in the first series of animals examined.
In the second series examined other Protozoa were noted when present.
Results from the examination of 128 rats are shown in table 2.

From the above table it will be noted that in one hundred and
twenty-eight rats examined, amoebae were found in the caecum in
only four rats where they had not been detected previously in the
faeces. Three of these cases had been examined on one day only
and the amoebae probably would have been detected had a second
examination been made. The fourth infection in the caecum only
was a very light infection. The four cases of Trichomonas found in
the caecum only were also exceptionally light infections. Amoebae
were found twice in the ileum while Trichomonas was found four

406 University of California Publications in Zoology [Vou. 20

times. The numbers found were small and it is probable that these
may have been forced from the caecum by the peristalsis induced by
the purge.

It should be noted here that during this investigation rats which
were known to harbor infections of amoebae and which showed no
amoebae in faeces collected without the administration of a purge,
invariably showed amoebae in the caecum at autopsy, which followed
immediately after faecal examination, though amoebae were not found
in the colon at autopsy.

The general conclusions to be drawn from the above facts are as
follows:

1. Faecal examination of rats on two consecutive days after they
have been purged with epsom salt is a comparatively safe method for
detecting the presence of amoebae and Trichomonas muris in these
animals. :

2. Amoebae common to rats and mice and Trichomonas muris
commonly inhabit the caecum of these animals.

3. Giardia muris, Hexamitus muris, and probably Chilomastiz
inhabit the small intestine normally, and their presence in rodents
cannot always be detected upon examination of the faeces after
administration of epsom salt.

Hegner (1923) has found a much higher percentage of rats
infected with Giardia muris and Hexamitus muris than has been
found in this laboratory. It thus seems that the incidence of
infection for different Protozoa may vary greatly in different rodent
colonies.

During the present investigation it has been found that it is much
more difficult to work with mice than with rats. In the first place,
mice will not eat the mixture of bread and salts so readily as the
rats will, and in the second place, it is more difficult to collect semi-
liquid faeces from mice than from rats. No table to determine the
accuracy of the epsom salt method for examining mice for Protozoa
has been compiled, but from a few examinations made it is concluded
that on aecount of the difficulty encountered in working with mice
the method cannot be relied upon as it can in the case of rats.

1923] Kessel: Infection with Intestinal Amoebae 407

C. INCIDENCE OF INFECTION AT DIFFERENT AGES

In choosing animals for experimental infections it is advisable to
select those most susceptible to infection. The present investigation
has shown that the incidence of amoebic infection among culture
rats in this laboratory is 34 per cent. The rate of infection is much
higher in middle-aged animals than it is in very young animals or
in very old animals as is shown in table 3.

TABLE 3

INCIDENCE OF INFECTION OF AMOEBAE IN CULTURE RATS AND MICE AT
DIFFERENT AGES

Mice
Age
Positive Negative Total % Positive
Under two months.......... f 2 vi 9 22.0
Two to four months........ ner F 16 15 31 51.6
About ten months........ sree 12 28 40 30.0
Rats
Age
Positive Negative | Total | % Positive
Under two months.................. ; 9 44 53 17.0
Two to ten months..................... 54 42 96 56.3
Overiten months ::.....0.....5.0.0-2.-+ 3 40 43 AO)

From this table it is seen that animals between the ages of two
and ten months show by far the highest rate of infection. The infer-
ences are that young animals, before weaning, either because of their
milk diet (Kessel, 1923b) or because of the minimum degree of
exposure to infection, have not established an infection ; that the older
animals have, in some way and to some extent, cleared themselves of
infection; that the animals midway between these two ages not living
on a diet of milk exclusively, are subject to constant exposure, and,
since they have not had time to establish an immunity, show the
highest degree of infection.

In this investigation, in most cases, old amoeba-free animals failed
to become infected when fed cysts by mouth, while young amoeba-
free animals fed on the same material established an infection (see
Kessel, 1923a and b).

It may therefore be concluded that the most advantageous time
to choose rats for feeding experiments is shortly after weaning or
at about the age of two months. Among rats of this age will be found
the greatest number free from amoebic infection and also those rats
with the greatest susceptibility to infection.

408 University of California Publications in Zoology [Vou. 20

D. CONCLUSIONS

1. Routine examination of normal rodent faeces for discovering
intestinal Protozoa is a tedious and unreliable method.

2. Stale bread soaked in a saturated solution of magnesium sul-
phate in water is a convenient purgative to administer to rats.

3. Examination on two consecutive days of the faeces from 128
purged rats, procured by this method, after which the rats were
autopsied and the intestinal contents examined, showed that this is a
relatively safe method for the detection of amoebic and trichomonad
infections in rats.

4. Young rats show the greatest susceptibility to infection and
are the most satisfactory for infection experiments.

ZooLoGicaAL LABORATORY,
UNIVERSITY OF CALIFORNIA.
Transmitted July 11, 1923.

EK. LITERATURE CITED
Brue, 8. L.

1919. De entamoeben van de rat. Geneesk. Tijd. Nederl. Indié, 59, 1-15,

pls. 1-2.
DoBELL, C.

1917. Reports upon investigation in the United Kingdom of dysentery cases
received from the Eastern Mediterranean. I. Amoebic dysentery
and the protozoological investigation of cases and carriers. [ Brit-
ish] Medical Research Committee, Special Report Series, No. 4 (Lon-
don, Joseph Causton and Sons, Ltd.), pp. 1-85.

DoBE.u, C., and O’Connor, F. W.

1921. The intestinal Protozoa of man (London, John Bale, Sons and Daniels-

son, Ltd.), v+211 pp., pls. 1-8, 2 figs. in text.
HEGNER, R. W.

1923. The effects of changes in diet on the incidence, distribution and num-
bers of certain intestinal Protozoa of rats. Am. Jour. Hyg., 3,
180-200, pl. 4.

KESSEL, J. F.

1923a. Experimental infection of rats and mice with the common intestinal
amoebae of man. Univ. Calif. Publ. Zool., 20, 409-430, pls. 38-39.

1923b. The distinguishing characteristics of the parasitic amoebae of culture
rats and mice. Ibid. (In press.)

Koror, C. A., and Swezy, O.

1920. On the prevalence of carriers of Endamoeba dysenteriae among sol-
diers returning from overseas service. New Orleans Med. and Surg.
Jour., 73, 4-11.

Kororp, C. A., Kornuauser, S. I., and Swezy, O.

1919. Intestinal parasites in overseas and home service troops of the U. 8.

Army. Jour. Am. Med. Assoe., 72, 1721-1724.
WENyYON, C. M.

1907. Observations on the Protozoa in the intestine of mice. Arch. f. Prot.,

Suppl. 1, 169-201, pls. 10-12.

=

UNIVERSITY OF CALIFORNIA PUBLICATIONS
IN

ZOOLOGY
Vol. 20, No. 19, pp. 409-430, plates 38-39 October 31, 1923

EXPERIMENTAL INFECTION OF RATS AND
MICE WITH THE COMMON INTESTINAL
AMOEBAE OF MAN

BY

JOHN F. KESSEL

CONTENTS
PAGE
A. Introductory and historical..............000.0.0.. ; : 410
Ere Hleedin PeXpOCTIM CIS 5..-.-.....<ccseeceestecsceseseuessetenecsarers aes ee se AL
I. Materials and meth adae: bree Rceicics eokecn: ; 411
eee ab lest am du conclusionssrse ev iarvtetesecererdsisnreseveesssnee teers sctsees . 412
Table 1. Rats fed with E. dysenteriae........ 3 413
Table 2. Mice fed with H. dysenteriae and C. Taplow: 414
Table 3. Rats fed with ZL. coll............. 415
Table 4. Rats fed with C. lafleuri, E. nana, anal L. patachiin 416
Table 5. Rats fed with Z. coli, and C. lafleuri.......... 417
Table 6. Rats fed with Z. dysenteriae, E. coli, aad C: lafleurs abt ained
from other rats.......... hoes levaeuisi 419
Ill. Effect on cysts of passage Shag iieestives Ree the . 419
Table 7. Results from autopsying rats and mice fifteen hours after
feeding cysts of E. dysenteriae and C. lafleuri.......-.ccccce 420
OP ISCUSSI ON eee oS falas ccccceneseneeee ane eave ; 421
I. Specificity of parasites to a given hosts 2 : 421

II. Factors involved in establishment of a ps parent infection by amoebae 422

III. Application of cross-infection ee from the point of view of
medical zoology... :

IV. Morphological eeciance of pars sarees in cross- eanfection eenmenta 424

D: PSYC TUA AYE a. ce cece emaecsacao ce HEEaENe
SPM GETAUUTE: CLLCC! ayeecee cose ece-cve--cns--e . 426
PME atesraridlexplAanatlONS:c..c0..-cerccecccevecrsssesecescconcesnsessncactse-nronussusse 428

410 University of California Publications in Zoology [Vou. 20

A. INTRODUCTORY AND HISTORICAL

The present investigation was first attempted to determine whether
or not rats and mice could be experimentally infected with Hnda-
moeba dysenteriae (Councilman and Lafleur). Most of the attempts
to infect rodents by feeding them with eysts of H. dysenteriae recorded
in literature prior to this time have been unsuccessful (see Kruse
and Pasquale, 1894; Werner, 1908; Dale and Dobell, 1917; Wenyon
and O’Connor, 1917; and Chatton, 1917, 1918).

Lynch (1915), however, has recorded spontaneous and experimental
infection of rats in Charleston, South Carolina. He made his diag-
nosis of amoebae entirely from the motile forms and took no note
of the fact that there are intestinal amoebae normal to rats and mice,
the motile forms of which might easily be confused with EH. dysen-
teriae, as both have hyaline pseudopodia. For an aecount of the
amoebae found in rats and mice see Grassi (1881), Wenyon (1907),
Brug (1919), Rudovsky (1921), and Kessel (19230). See Wenyon
and O’Connor (1917) and Dobell (1919) for an account of the
pseudopodia of EH. dysenteriae. Lynch published no figures and
while the clinical symptoms he describes indicate the presence of
acute infections of H#. dysenteriae, it seemed possible that his rats
might have died of some other infection common to rats, as implied
in the criticisms of Chatton (1917) and of Dobell (1919).

In 1919, Brug reported what he thought was an infection of
E. dysenteriae in two wild rats in Java and further coneluded that
he had been successful in establishing an experimental infection of
E. dysenteriae from man in a rat of the species Mus rattus. His work
seems to be conclusive, yet he entertains some doubt as to whether
the species he found in the rats and EH. dysenteriae of man are
identical.

Baetjer and Sellards (1914) reported successful inoculation of
guinea-pigs with H. dysenteriae, lesions of the bowel being found
at autopsy. Chatton (1918) concluded that guinea-pigs may be
infected with H. dysenteriae either by feeding cysts or by anal injec-
tions and that the infections, once established, are restricted to the
caecum.

Huber (1909) sueceeded in infecting rabbits by feeding them
eysts of #. dysenteriae and the examination of the rabbits at autopsy
showed a localization of amoebie ulcers in the caecum.

1923 | Kessel: Experimental Infection of Rats and Mice 411

The species of intestinal amoebae common to man, other than
E. dysenteriae, have never been experimentally established in rats
and mice. The present investigation also included an attempt to
infect these rodents with Cowncilmania lafleuri Kofoid and Swezy
and to infect rats with E. coli (Loésch), Endolimax nana (Wenyon
and O’Connor), and Jodamoeba biitschlii (Prowazek).

It was apparent from the first that a detailed study of the amoebae
normal to the rat and mouse was essential before critical experimental
infections could be carried out satisfactorily. Accordingly, this work
was undertaken first, and to date three species of intestinal amoebae,
Councilmania muris (Grassi) Kessel, Cowncilmania decumani (Rudov-
sky) Kessel, and Endamoeba ratti sp. nov., have been found in rats
and mice and conclusions relating to their distinguishing character-
isties have been recorded in a separate paper (Kessel, 1923b).

For assistance and guidance in the present work the writer is
glad to make acknowledgment to Professor C. A. Kofoid, under whose
supervision the investigation has been carried on, to Professor E. L.
Walker, who has been kind in giving counsel and advice, and to
Dr. Olive Swezy, whose suggestions have been of great value. Miss
Inez Smith, technician of the Division of Parasitology, California
State Board of Health, has generously cooperated in supplying
material from that laboratory for use in the feeding experiments.

B. FEEDING EXPERIMENTS
I. Mareriats AND MrerHops

The rats used for the feeding experiments have been obtained
from the rat colony of the Department of Zoology, University of
California, and belong to the species Rattus norvegicus (Erxleben).
For a history of the colony, see Kessel, 1925).

The rats chosen for experimental infection were young rats, about
two months of age. It was found during the experiment that male
rats are more suited to this type of work since it is easier to collect
faeces from them than from the females.

Rats known to be free from amoebic infection of any type, and
other rats known to harbor an infection of Cowncilmania common
to the rat, were used. Amoeba-free rats were isolated by the epsom
salt method described by Kessel (19230). These rats were kept
separate from those known to be infected with amoebae.

412 University of California Publications in Zoology (Vou. 20

The material used for feeding was obtained from the laboratory
of the Division of Parasitology of the California State Board of
Health, which is under the direction of Professor C. A. Kofoid. The
diagnoses were made first by that laboratory and later authenticated
by the writer before feeding the rats. The morphological character-
istics of the amoeba common to man were compared with the descrip-

tions and figures of Kofoid, Kornhauser, and Swezy (1919), Dobell
' (1919), Kofoid and Swezy (1919), and Kofoid (1923). As it was
not always possible to obtain a sufficient quantity of faecal material
to feed a large number of rats, the interval between feeding in some
instances is considerable. In feeding; bread was soaked in water
until it became quite soft. The water was then squeezed out and a
small handful of bread was placed in each feeding dish. The faecal
material was well mixed with water in another dish until it was very
liquid in consistency. A portion of this was then poured over the
bread in each dish and the contents well stirred. There was no con-
stant proportion of faecal material to bread, for the amount of faecal
material obtainable varied. The rats ate well of all mixtures fed even
though the bread was always colored by the faeces.

After the feeding experiments were completed and an interval
of two weeks or longer had passed, the rats were examined, always
after being given a dose of epsom salt. Fresh smears were examined
in iodine-eosin and permanent smears were stained with iron
haematoxylin.

II. Tasries AND CONCLUSIONS

The results of the feeding experiments are displayed in tables,
and a short discussion follows of the conclusions drawn from each
table.

DISCUSSION AND CONCLUSIONS FROM TABLE 1

1. Hight out of twenty-nine, or 27.6 per cent of all the rats fed,
became infected with E. dysenteriae.

2. Hight out of ten, or 80 per cent of all amocba-free rats fed,
became infected with EH. dysenteriae. This may have been due in
part to their being non-infected rats, and in part to their having
received an extra feeding of EH. dysenteriae cysts on January 5 which
the other rats did not receive. It will be noticed in tables 4 and 5
that a greater percentage of amoeba-free rats became infected than
was the case with rats having a previous amoebie infection with rat
amoebae.

1923 ] Kessel: Experimental Infection of Rats and Mice 413

3. Of the eleven rats that became infected with EH. dysenteriae
(see also table 4), four died between the time of the last feeding,
January 17, and June 12.

TABLE 1

RESULTS OBTAINED FROM FEEDING Rats witH Cysts oF ENDAMOEBA DYSENTERIAE

Rat Feedings Examinations Au-
Infection with cysts of May 16, 18, 21, 22, |topsy Remarks
not E. dysenteriae 23, 25, 26 and June
known from man June 3 4
No. 1 0 0
2, 0 0
3 April 16, 18, 0 0
and 21, 1921 Motile Trichomonas, smaller
4 0 0 than Tritrichomonas of rat,
apparentlyT richomonas
hominis.
Amoeba- Jan. | Feb. | April) June 3
free 31 28 1 8
No. 5 0 OF} 50 0
6 ar || ae |) Se |) se
o ap || sei) ar fl) SF
(es mucus in stool
Jan. 31. Died Mar. 2 of
8 Oct. 20, 1922 +)+/)—-;);—-/)+ lung infection. Had large
Nov. 1, 1922 ulcer of colon.
9 Jan. 2, 1923 ae |) se || ar | se
10 Jan. 5, 1923 +)4+/)4+)—-]+ Bloody mucus in_ stool
Jan. 17, 1923 Jan. 31.
11 +)/4+})+]—-]+ Died May 20 of lung infee-
\ tion.
12 ONO OF 0
13 qe | Se || Se |) ar
14 ae || Se lar || ae
Infected
with
amoebae
of rat
None of the rats previously
infected with amoebae
Nos. Oct. 20, 1922 common to the rat be-
15 Nov. 1, 1922 0 0o;-|- 0 came infected with JH.
to Jan. 2, 1923 dysenteriae. The infection
29 Jan. 17, 1923 of rat amoebae remained
positive in ten of the fif-
teen cases.

4. For the most part no tendency to diarrhoea was noticed among
the rats; on the contrary the faeces were harder than normal, thus
showing a condition of constipation.

5. The four rats that died gradually became thin and the hair
was ruffled several days before death. An intestinal ulcer was found
in the colon of No. 8 while in Nos. 10, 11, and 62 no lesions were
found, though a mucoid condition was noticeable. In each ease there

414 University of California Publications in Zoology [Vou. 20

was a severe lung infection which was the apparent cause of the
death of the rats. Smears and sections made from the infected parts
of the lungs revealed no amoebae.

6. The type of lung infection found in these rats is the most
common cause of death among rats in the rat colony in the Depart-
ment of Zoology. During the period in which the four of the eleven
rats infected with H. dysenteriae died, one rat infected with E. coli
and three from the colony of one hundred and fifty uninfected rats
died of the same lung disease. Thus the mortality among the eleven
rats infected with H. dysenteriae was four and the mortality among
all the other experimental and uninfected rats of the rat colony, or
a total of two hundred and twenty-five rats, was also four. It seems
probable that the resistance of the rats infected with E. dysenteriae
was so lowered that they were more susceptible to the lung infection.
Tt is significant in this connection to note that a rabbit fed cysts of
E. dysenteriae by Chatton (1918) died of a lung infection.

7. The course of the infection of H. dysenteriae among the rats
in this experiment was the chronic rather than the acute type which
seems to be the common type of amoebic infection found in kittens.
Seven of the rats infected with B. dysentertae were in apparent health
at the time the last examination was made, though they all showed
the presence of cysts.

8. Motile amoebae, mononucleate cysts, binucleate cysts, and four-
nucleate cysts of EH. dysenteriae were recovered from the rats, and
these were without any apparent morphological or racial change from
their previous status in man (pl. 38, figs. 7-12).

TABLE 2

RESULTS OBTAINED FROM FEEDING WHITE Mick witH Cysts oF E. DYSENTERIAE
AND OF COUNCILMANIA LAFLEURI

Mouse Examinations |Autopsy

Adult, Feedings Remarks

amoeba- Feb. | Feb. | Feb.| Mar.

free 3 9 18 2

No. 1 0 0 0 0
2 0 0 0 0
3 se | se |) ar aE Cysts and motile
4 0; O 0 0 E. dysenteriae. Mouse normal.
5 January 11, }| 0 0 0 0
6 13and14 )| 0} 0 0 0
it +/+) + + Cysts and motile
8 0 0; 0 0 Councilmania lafleuri. Mouse
9 0) 0 0 0 normal.
10 0 @ || O 0

1923 ] Kessel: Experimental Infection of Rats and Mice 415

DISCUSSION AND CONCLUSIONS FROM TABLE 2

1. As no young animals were available for this experiment, only
adult amoeba-free mice were used.

2. One mouse only became infected with E. dysenteriae and only
one with Councilmania laflewri.

3. As shown by Kessel (1923b) adult animals are more difficult
to infect with amoebae than young ones, and it is probable that the
low percentage.of infection in this series of amoeba-free mice is due
to the advanced age of the animals. This indicates that in some eases
a resistance or immunity to infection is established in old animals.

4. The eysts recovered were apparently normal and the species
had undergone no morphological change though passed through the
rodent host (pls. 38-39).

TABLE 3

RESULTS OBTAINED FROM FEEDING RATS WITH Cysts OF ENDAMOEBA COLI

Rat Feedings of Examinations |Autopsy
cysts of E. coli x Remarks
Infection from man Jan. Jan Jan.
unknown 18 25 29
No. 30 oo + a E. coli
31 + + + Councilmania sp. of rat
32 ++ + ob Mixed
33 + -E + E. coli
34 ++ + + Councilmania sp. of rat.
35 Dee. 19 of + “fb FE. coli
36 Jan. § 0 + + Councilmania sp. of rat
37 0 0 0
38 0 0 0
39 + =t= + E. coli

DISCUSSION AND CONCLUSIONS FROM TABLE 3

1. No amoeba-free rats were on hand when this feeding experiment
was begun, so young rats were chosen, the infection of which was
unknown. As they had only recently been weaned it is probable that
the percentage of infection was low.

2. The Councilmania found in the final examinations was in each
case one of the species, at that time undetermined, common to the
rat, and it is concluded the rats had this infection when fed with
E. coli.

3. An infection of E. coli only was recovered from four of the ten
rats, while a mixed infection of E. coli and Councilmania common to
the rat was found in one rat. Thus five out of ten or 50 per cent
of the rats fed with E. coli established an infection.

416 University of California Publications in Zoology [Vou 20

4. The motile forms showed characteristic granular pseudopodia
and the cysts were identical in size and morphological characteristics
with the eysts fed to the animals (pl. 38, fig. 3).

TABLE 4

RESULTS OBTAINED FROM FEEDING RATS WITH CysTS OF COUNCILMANIA LAFLEURI,
ENDOLIMAX NANA, IODAMOEBA BUTSCHLII, AND ENDAMOEBA DYSENTERIAE

Rats Examinations
Feedings Autopsy Remarks
Amoeba- Jan. Feb. June
free 23 24 8
. Councilmania lafleuri
No. 40 ar ap aie ae Endolimax nana
. Todamoeba biitschlii
41 0 0 OPO
42 0 0 0 0
43 + ar + C. lafleuri
44 =F =F 3 aF C. lafleuri
45 => + + + C. lafleuri
46 ae + ae C. lafleuri
47 sin aE + C. lafleuri
48 0 0 0) 0
E. dysenteriae
49 + oF = Endolimax nana
Todamoeba biitschlit
50 ar = ae + C. lafleuri
51 AF = aR C. lafleuri
52 November 14 + + ao C. lafleurt and Giardia
53 December 2 + + + C. lafleuri
54 January 5 a a + E. dysenteriae
Infected
with
amoeba
of rat
No. 55 0 0 0
56 0 0 0
57 0 0 0
58 0 0 0
59 0 0 0
60 + ob _ C. lafleuri
61 0 0 0
62 + + + + E.. dysenteriae
63 0 0 0 { Died of lung infection,
June 12
64 0 0 0

DISCUSSION AND CONCLUSIONS FROM TABLE 4

1. Eleven of the twenty-five rats, or 44 per cent, fed with cysts of
Councilmania laflewri became infected.

2. Ten of the fifteen amoeba-free rats, or 66 per cent, fed with
eysts of Cowncilmania laflewrt became infected.

3. Two of the twenty-five rats, or 8 per cent, fed with cysts of
Endolimax nana and Todamoeba biitschlii became infected.

1923 | Kessel: Experimental Infection of Rats and Mice 417

4. Cysts of Edamoeba dysenteriae were recovered from three rats
of this series, although the presence of such eysts had been overlooked,
but later found, in the stained slides of the human faecal material fed
to these animals. <A light infection in man was thus detected by the
feeding experiment. :

TABLE 5

RESULTS OBTAINED FROM FEEDING RATS WITH CysTS FROM A MIXED INFECTION OF
ENDAMOEBA COLI AND COUNCILMANIA LAFLEURI

Examinations
state Feedin,
e. ‘eedings oan ae = Autopsy Remarks
tres 23 24 7
No. 65 + + + + C, lafleuri
66 at + + | {Mixed £. Coli and C.
lafleuri
67 se + + +f Mixed £. coli and C.
lafleuri
68 0 0 0 0
69 0 0 0 0
70 ae ab: + + C. lafleuri
71 at oe + + C. lafleuri
72 alt ae + + C, lafleurt
73 ae ae aL + re E. coli and C.
lafleuri
74 ae at + + C. lafleuri
——_ December
Infection 12 and 14
unknown
No. 75 0 0 OR ieee
76 aL a ate + C. lafleuri
77 aL + + + E. coli
Nos. i
78 to 0 0 0 0
85
No. 86 ave at aL + E. coli
87 dt de ak + E. coli

5. Of the three rats infected with FE. dysenteriae, one died of a
lung infection on June 12. The other two on that date showed cysts
in the faeces but were in apparent normal health.

6. The faeces containing the cysts of H. dysenteriae were fed to
the rats January 5. As the rat faeces contained cysts of EH. dysenteriae
in fair numbers on January 23, or eighteen days later, it may be
concluded that an infection of E. dysenteriae may be established in
rats within eighteen days after feeding them with the amoeba cysts.
It is highly probable that the infection was well established several
days before this time though an examination was not made.

418 University of California Publications in Zoology [| Vou. 20

7. A Giardia, resembling Giardia enterica, was recovered from
one of the rats in this series. As no cysts of this flagellate had been
detected in the feeding material, the infection in the stools used must
have been very light.

8. The eysts of Councilmamia lafleurt, Endolimax nana, Iodamoeba
bitschli, and Endamoeba dysenteriae recovered from this series of
rats were without any apparent racial or morphological change.

9. The rats which beeame infected with C. laflewri, Endolimar
nana, and Iodamoeba biitschlu remained in apparent normal health.

DISCUSSION AND CONCLUSIONS FROM TABLE 5

1. Twelve out of twenty-five, or 48 per cent of the rats fed a
mixed infection of E. coli and Counciimania laflewri, became infected
with both or one of these amoebae.

2. Eighty per cent of the amoeba-free rats became infected while
only 27 per cent of the rats of unknown previous infection acquired
the amoebae. It is probable that the ones that became infected were
amoeba-free at the time of feeding.

3. The motile amoebae of C. laflewri were easily distinguishable
by their hyaline pseudopodia while the motile amoebae of E. coli
showed the characteristic granular pseudopodia. The cysts of both
species were also morphologically characteristic after being passed
through the rat, and presented no apparent structural change.

DISCUSSION AND CONCLUSIONS FROM TABLE 6

1. EL. dysenteriae, E. coli, and C. laflewri, respectively, may be
transferred from rats experimentally infected with each of these
amoebae to uninfected rats.

2. The cysts and motile forms of the amoebae may be recovered
without any apparent morphological change after transfer.

3. None of the rats infected in this experiment suffered any
apparent pathogenic consequences during the time of the experiment.
This indicates that amoebae that have passed through a second series
of rats are no more virulent than those passed directly from man

to rats.

1923 ] Kessel: Experimental Infection of Rats and Mice 419

III. Errect on Cysts or Passace THroucH DicEstive TRACT

During this investigation the faecal material of the rats and mice
has been examined on a number of occasions twenty-four and forty-
eight hours after feeding the animals with human faecal material.
There is no constancy as to the appearance of normal or disintegrated

TABLE 6

RESULTS OBTAINED FROM FEEDING Cysts oF E, DYSENTERIAE, E. COLI, AND
COUNCILMANIA LAFLEURI, RESPECTIVELY, OBTAINED FROM INFECTED
Rats To OTHER AMOEBA-FREE RATS

rae Examinations
Material Feedings a 2 cia a Remarks
Amoeba- April} May| May
free 21 3 4
No. 90 0 0 0
91 Ee usenerice } O05) 20
92 rom Rat b +/+] + Cysts only
93 No. 10 } +)+)4+ Motile forms
| and cysts
94 Oi, 0) 10
ae +)+)+ Motile amoebae
9 eral: } | Racca material ae se he Pee ore
97 ftom Bat j 4 Make ree {jt+y+] 4+ Mot Hie amoebae
ae Bits April 15 5 ; ‘i and cysts
99 +/+ )+ Motile amoebae
100 Se iam | oars Motile amoebae
and eysts
101 OF O80
102 c lafieuri | +i/+]+ Motile amoebae
rom Rat and eysts
103 No. 40 +) 4 )+ Motile amoebae
and eysts
104 +) 4+ )+ Motile amoebae
and cysts

human amoebie cysts in the rat faeces. Cysts which are apparently
unmolested by the passage through the rodent have been recovered
while other cysts in different stages of disintegration have been
detected. In the examinations made 44 per cent contained no cysts
of any type, 40 per cent contained dead or degenerate cysts, and
16 per cent contained live cysts. This indicates a difference in
viability of the cysts.

Two young amoeba-free rats and three young amoeba-free mice
were fed a mixture of barium sulphate, milk, and human faecal

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1923 ] Kessel: Experimental Infection of Rats and Mice 421

material containing cysts of E. dysenteriae and (. lafleuri. It was
possible, on account of the presence of the barium sulphate, to deter-
mine that the substance fed passed to all parts of the digestive tract.
The animals were autopsied fifteen hours after feeding and exam-
inations were made of the intestinal contents at different levels. The
table on page 420 records the results.

DISCUSSION AND CONCLUSIONS FROM TABLE 7

1. Both dead and normal cysts of human intestinal amoebae may
be found shortly after feeding in any part of the intestinal tract of
the experimental rats and mice.

2. Motile amoebae were found most commonly in the caecum,
none being found in the small intestine. Motile forms were found
only once in the colon. This indicates that the caecum is the region
where the amoebae escape from the cyst, especially as a budding cyst
of Cowncilmania laflewrt was found here, there being but one nucleus
left in the eyst.

3. The motile amoebae encountered varied greatly in size and
were rounded up in most instances. They often presented an uneven
margin and abnormal vacuolation, indicating that disintegration was
in progress. It is probable that this apparent excessive rate of dis-
integration of motile amoebae is the result of the process of adjust-
ment to the environment of the new host. Some normal amoebae
were found.

C. DISCUSSION
I. Sprciriciry oF PARASITES TO A GIvEN Host

In the history of parasitology two opposing opinions have been
evident ; one, to regard parasites as being restricted to specific hosts,
and the other, to regard as possible in nature the transfer of a para-
site from one species of host to another. The first tendency has led
to the formation of a great many new species on the basis of their
habitat and has minimized, in the minds of the scientific world, the
danger of transference of parasite from host to host. Until recently,
the second tendency has been greatly underestimated, much of the
experimental work recorded indicating that the transference of a
given parasite from one species of host to another is often difficult
to accomplish. ;

422 University of California Publications in Zoology (Vou. 20

Certain experimental studies cited in this paper, together with the
results of this investigation, lead to the conclusion that transfer is
possible at least in certain instances or wnder given conditions. With
reference to E. dysenteriae, all attempts prior to 1915 to infect rats
with this species of amoeba failed. In 1915 Lynch appears to have
succeeded in infecting rats with EH. dysenteriae. Sinee 1915 four
other attempts have been made to infect rats and mice with this same
species of amoeba, but, prior to the present investigation, only one
has been successful, and that with one rat only. It seems likely that
the failures have been due to using an insufficient number of animals,
especially of young, amoebae-free individuals.

In the present investigation successful infection of rodents with
E. dysenteriae has been accomplished in thirteen rats and one mouse,
with #. coli in seventeen rats, with Couwncilmania lafleuri in twenty-
three rats and one mouse, with Endolimaxr nana in two rats, and with
Todamoeba biitschli m two rats (pls. 38-39).

This successful transference to rodent hosts of the amoebae com-
monly parasitic in the digestive tract of man is evidence in favor of
a certain degree of promiscuity of infection of a given species of
parasite in several species of host, provided the conditions for sue-
cessful infection are met.

II. Factors INvoLveD IN ESTABLISHMENT OF PARASITIC INFECTION
By AMOEBAE

The varying factors in establishing an infection of amoebiasis
in the rodent host appear to be (1) the viability of the cysts used
in feeding, and (2) the age of the rodent host, together with (3)
its active resistance to an amoebic infection.

In some cases large numbers of lying amoebic cysts were fed
to young rats without suecessful infection, e.g., Rats 1, 2, 3, and 4
in table 1, while in other cases small numbers of amoebic cysts
were fed to a series of rats one time only and successful infection
resulted, e.g., Rats 49, 54, and 62. It seems probable that the cysts
in the second case were more viable than those in the first case.

Young amoeba-free rodents have acquired an amoebic infection
in 80 per cent of the cases fed, while young rodents known to harbor
an amoebic infection of amoebae normal to rats and mice have
acquired a further amoebic infection in only 15 per cent of the cases.
Thus, young amoeba-free rodents are more susceptible to amoebic
infection than young rodents with a known amoebic infection.

1923 | Kessel: Experimental Infection of Rats and Mice 423

Young amoeba-free rodents have acquired an infection of amoeba
in 80 per cent of the cases fed, while old amoeba-free rodents have
acquired an infection in only 20 per cent of the cases. This, together
with the facts that 27 per cent of rats of known amoebic infection
have later been known to rid themselves of the infection (Kessel,
1923b) and that the rate of amoebic infection is much lower in old
rodents than in middle-aged rodents (Kessel, 1923a), indicates that
as the animals progress in age they are able to establish an active
resistance to amoebic infection.

III. Appnication of Cross-INFECTION EXPERIMENTS FROM THE
Pornt oF Virw or Mepicau Zooioay

The fact that the amoebae common to the intestinal tract of man
have been successfully established in rats and mice is important, first,
from the standpoint of preventive medicine. Infection of rats and
mice with the amoebae of the human digestive tract has been sue-
cessful in 55 per cent of all the attempts made, while the cross-
infection of these rodents with amoebae common to themselves has
been successful in 53 per cent of all the attempts made. In the
experimental work of this investigation we thus see that even slightly
greater success has attended attempts to infect rats with amoebae
common to man than with the amoebae common to rodents (see
Kessel, 1923b).

The fact of transfer of human infections to rats and mice throws
heavy suspicion upon these rodents as facultative carriers of the
causative organisms of amoebiasis in man and supports the findings
of Lynch (1915) and Brug (1919). Whether the amoebae thus
experimentally infected into rodents still retain their pathogenicity
for man, it is still impossible to state, but the fact that they may
be passed from one rodent to another indicates that the viability
of the amoebae is not destroyed by passage through one rodent, and
that an infection once established among these rodents may spread
to other rodents.

The second important application to experimental medicine of
facts determined in this investigation is that the young rats may
be used successfully in further investigation relating to pathological
conditions resulting from amoebiasis. Experimental infection of
kittens with £. dysenteriae has resulted in an acute form of amoebiasis
(Dobell, 1917) and the amoebae recovered are thought to be abnormal

424 University of California Publications in Zoology [Vou 20

in appearance. As our rats have carried the infection of E. dysen-
teriae for five months, it seems highly probable that amoebiasis in
rats is inclined to become chronie, as it is in man.

IV. MorpHonoaican CoNsTANCY OF PARASITES IN Cross-INFECTION
EXPERIMENTS

While many protozoologists hold to the theory that the different
species-of amoebae common to the digestive tract of man are constant
in their morphological characters, respectively, yet there is an idea
among certain pathologists that the morphological characteristics of
amoebae may alter as the amoebae are transferred from one environ-
ment to another.

During this investigation Hndamoeba dysenteriae, Endamoeba
colt, Councilmania lafleuri, Endolimax nana, and Iodamoeba biitschlit
have been successfully transferred from man to rodents. During the
period of infection, which was five months in the case of E. dysenteriae
and four months in the case of the other species of amoebae, no
perceptible morphological change was noted in any of the species
used in the experiments. These facts afford valuable evidence in
favor of the morphological constancy of given species of amoebae,
at least of the failure of immediate change through transfer.

D. SUMMARY

1. Infections of five species of human intestinal amoebae, End-
amoeba dysenteriae, Endamoeba coli, Councilmania lafleurt, Endo-
limax nana, and Iodamoeba biitschlii have been experimentally
transferred to rats by feeding them human faeces containing cysts
of these amoebae.

2. Infections of E. dysenteriae and C. lafleurt have been experi-
mentally transferred to mice by feeding them human faeces containing
eysts of these amoebae.

3. The percentage of rodents that have become infected with these
five amoebae common to man, is greater among young amoeba-free
animals than among old amoeba-free animals, or among young animals
known to possess an infection of amoeba common to the rat and
mouse.

ee eee

1923 | Kessel: Experimental Infection of Rats and Mice 425

4, Infections of EL. dysenteriae, EF. coli, and C. lafleuri have been
experimentally transferred from rats harboring experimental infee-
tions of these amoebae to other young amoeba-free rats by feeding
them the faeces of the infected rats.

5. The amoebae transferred from man to the rodent host have,
in every case, presented no apparent morphological or racial change
during the period of the experiment.

6. Four of the rats infected with EL. dysenteriae and one infected
with £. coli died of an apparent lung infection during the experiment.
During the same period, three rats uninfected with human amoebae
from the rat colony of one hundred and fifty animals died of the
same lung infection. This fact indicates that rats infected with
E. dysenteriae have a lowered resistance and are more susceptible
to the lung infection than normal rats.

7. One of the four rats that died with EH. dysenteriae showed an
intestinal ulcer.

8. Infections of EH. dysenteriae in rats and mice apparently
assume a chronic form instead of an acute form as is reported in the
case of kittens.

9. Both dead and normal eysts of human intestinal amoebae may
be found for some hours after feeding in any part of the intestinal
tract of experimental rats and mice. This indicates a difference in
the viability of the eysts.

10. The findings of this investigation afford evidence in favor
of the morphological constaney of species of parasitic amoebae, under
conditions of change of host, and of the possibility of transfer of
one species of parasitic amoebae from one species of host to another.
Consequently heavy suspicion is thrown on the rat and mouse as
possible carriers of the causative organisms of human amoebiasis.

ZOOLOGICAL LABORATORY,
UNIVERSITY OF CALIFORNIA.

Transmitted July 12, 1923.

426 University of California Publications in Zoology (Vou. 20

EK. LITERATURE CITED

Baetser, W. A., and SELLARDS, A. W.

1914. The behavior of amoebic dysentery in lower animals and its bearing
upon the interpretation of the clinical symptoms of the disease in
man. Bull. Johns Hopkins Hosp., 25, 237-241.

Brug, 8. L.

1919. De entamoeben van de rat. Geneesk. Tijd. Nederl.-Indié, 59, 1-15,

pls. 1-2.
CuHaTton, E.

1917. L’éclosion des kystes et les premiers stages de 1’évolution de 1’amoebe
dysentérique humaine chez le chat. Bull. Soc. Path. Exot., 10,
834-841.

1918. L’amibiase intestinale expérimentale du cobaye 4 Entamoeba dysen-
teriae. Ses caractéres speciaux: localization caecale, absence de
dysenterie, importantes réactions hyperplasiques. Arch. Inst. Pasteur
Tunis, 10, 137-156.

CouUNCILMAN, W. T., and Laruevur, H. A.

1891. Report in pathology. I. Amoebic dysentery. Johns Hopkins Hosp.

Rept., 2, 395-548, pls. 1-7.
Date, H. H., and DosEtt, C.

1917. Experiments on the therapeutics of amoebic dysentery. Jour. Pharm.

and Exper. Therap., 10, 399-459.
DosELL, C.

1919. The amoebae living in man (London, John Bale, Sons and Danielsson,

Ltd.), vi+ 155 pp., 5 pls.
GrassI, B.

1881. Intorno ad alcuni Protisti endoparassitici ed appartenati alle classi dei
flagellati, lobosi, sporozoi, sciliati. Atti Soc. Ital. Sci. Nat., 24,
135-224, pls. 1-4.

HUBER.

1909. Untersuchungen tiber Améebendysenteriae. Ztschr. f. klin. Med., 67,

262.
KartTu.is, 8.

1891. Einiges tiber die Pathogenese der Dysenterieamében. Centralbl. f.

Bakt., 9, 365-372.
KESSEL, J. F.

1923a. Methods of obtaining amoeba-free rats for experimental infection with
intestinal amoebae. Univ. Calif. Publ. Zool., 20, 401-408.

1923b. The distinguishing characteristics of the parasitic amoebae of culture
rats and mice. Ibid. (In press.)

Koror, C. A.

1923. Amoeba and man. Univ. Calif. Chronicle, 25, 140-174 and 291-312,

pls. 1-6.
Koron, C. A., and Swezy, O.

1921. On the free, encysted, and budding stages of Councilmania lafleuri, a
parasitic amoeba of the human intestine. Univ. Calif. Publ. Zool.,
20, 160-198, pls. 18-22, 3 figs. in text.

1923 | Kessel: Experimental Infection of Rats and Mice 427

Kororp, C. A., KoRNHAUSER, S. I., and Swezy, O
1919. Criteria for distinguishing the Lndamoeba of amoebiasis from other
organisms. Arch. Int. Med., 24, 35-50, 43 figs. in text.
Kruse, W., and PasquaLe, A.
1894. Untersuchungen iiber Dysenterie und Leberabscess. Ztschr. f. Hyg.,
16, 1-148, pls. 1-6.
LyncH, K.
1915. The rat as a earrier of dysenteric amoeba. Jour. Am. Med. Assoc.,
65, 2232-2234,
Rupovsky, F. A
1921. tiber RattenamObiase. Wien. tierarzt. Monatssch., 8, 1-9, pl. 7.

WEnNyon, C. M.
1907. Observations on the Protozoa in the intestine of mice. Arch. f. Prot..
Suppl. 1, 169-201, pls. 10-12.
WEnyon, C. M., and O’Connor, F. W.
1917. Human intestinal Protozoa in the Near East (London, Wellcome Bureau
of Scientific Research), pp. 1-218, pls. 1-4.
WERNER, H.
1908. Studien tiber pathogene Amében. Arch. f. Schiffs- u. Tropenhyg., 12,
419-436, pls. 1-6.

F. EXPLANATION OF PLATES

All figures are drawn from faecal smears fixed in hot Schaudinn’s fluid and
stained in iron haematoxylin. The magnification in all tases is 2500 diameters.

PLATE 38

Fig. 1. Mononucleate cyst of Jodamoeba biitschlii in human faecal material
fed to rats.

Fig. 2. Mononucleate eyst of Iodamoeba biitschlii recovered in faecal material
of rat.

Fig. 3. Motile’- Hndamoeba coli with granular pseudopodium recovered from
rat.

Fig. 4. Binucleate cyst of Endolimax nana in human faecal material fed
to rats.

Fig. 5. Binucleate cyst of Endolimax nana recovered in faecal material of rat.

Fig. 6. Motile Councilmania lafleuri, with hyaline pseudopodia, recovered
from rat.

Fig. 7. Mononucleate cyst of Lndamoeba dysenteriae in human faecal material
fed to rats.

Fig. 8. Mononucleate cyst of HLndamoeba dysenteriae recovered in faecal
material from rat.

Fig. 9. Four-nucleate cyst of Endamoeba dysenteriae in human faecal
material fed to rats.

Fig. 10. Binucleate cyst of Hndamoeba dysenteriae in human faecal material
fed to rats.

Fig. 11. Binucleate cyst of Hndamoeba dysenteriae recovered in faecal
material from rat.

Fig. 12. Four-nucleate cyst of ELndamoeba dysenteriae recovered in faecal
material from rat.

[428]

UNIV. CALIF. PUBL. ZOOL. VOL, 20 [KESSEL] PLATE 38

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PLATE 39

Fig. 13. Eight-nucleate eyst of Endamoeba coli in human material fed to rats.

Fig. 14. Eight-nucleate cyst of Endamoeba coli recovered in faecal material
from rat.

Fig. 15. Eight-nucleate eyst of Endamoeba coli recovered in faecal material
from rat.

Fig. 16. Eight-nucleate eyst of Endamoeba coli in human faecal material
fed to rats.

Fig. 17. Binucleate cyst of Councilmania lafleuri recovered in faecal material
from rat. Large glycogen vacuole, surrounded with chromatoidal bodies. Upper
nucleus in telophase, showing chromosomes and karyosome having separated.
Lower nucleus in prophase.

Fig. 18. Eight-nucleate eyst of Endamoeba coli recovered in faecal material
from rat. :

Fig. 19. Hight-nucleate cyst of Councilmania lafleuri in human faecal material
fed to rats.

Fig. 20. Hight-nucleate cyst of Cowneilmania lafleuri recovered in faecal
material from rat.

Fig. 21. Eight-nucleate cyst of Cowncilmania lafleurt in human faecal material
fed to rats.

Fig. 22. Hight-nucleate cyst with bud of Councilmania lafleuri recovered in
faecal material from rat. Note the somewhat massed type of karyosome which
may be confused by some with the typical nucleus of £. coli.

Fig. 23. Eight-nucleate eyst of Councilmania laflewri recovered in faecal
material from rat.

[430]

RAESSEE RUATE 2S

UNIV. CALIF, PUBL. ZOOL. VOL. 20

|

f $ UNIVERSITY OF CALIFORNIA PUBLICATIONS—(Continued)
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bets AME Sty OAS eck er LOIN ee Re I Sota adh eee hee Spe cne seme stem Wagar 25
18. Methods of Obtaining Amoeba-free Rats for Experimental Infection with

Intestinal Amoebae, by John F. Kessel. Pp. 401-408.
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Amoebae of Man, by John F. Kessel. Pp. 409-430, plates 38-39.

Nos. 18 and 19 in one cover. October, 1923 ....000.2-..222o22eceeiesete tele 50

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Pp. 431-445, 19 figures in text. October, 1923
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IN

| it ZOOLOGY /
, No. 20, es 431- 445, 19 figures in text October 31, 1923

THE GENUS COUNCILMANIA, BUDDING
_ INTESTINAL AMOEBAE PARASITIC
IN MAN AND RODENTS

. t ee :

u vs Pas
Aa Ha ; hy ae

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IN
ZOOLOGY
Vol. 20, No. 20, pp. 431-445, 19 figures in text October 31, 1923

ON THE GENUS COUNCILMANIA, BUDDING
INTESTINAL AMOEBAE PARASITIC
IN MAN AND RODENTS

BY
CHARLES A. KOFOID, OLIVE SWEZY, anp JOHN F. KESSEL

CONTENTS

PAGE
TEPER RCO IYO KOS a eee 431
Councilmania Kofoid and Swezy 1921 .. 432
WNomencla tures rccs--cc.c<.s2--2---- eae sseeceSobsnesartes tt cea cesse-uetastis isu ssiesvaeesscetuesntassers 433
Councitlmania laflewrt Kofoid and Swezy 1921 -..-2 —-......e anne eee eee 435
Councilmania muris (Grassi) .. 438
Councitmania decumant (Rudovsky) Kessel ...........--.--2.2.---22-ceesesesceeeeeeeeeeeeee 439
INC VELOUSPCCLOSH Ole COUR CELI ONG or nanan as anc cn nce ane nde onsen een teense esas neateetasees 443

Summary om
TETA HEUER oCSYe SAYS 9 ee eee eS 445

INTRODUCTION

The parasitic intestinal Protozoa of the mammals, including man,
reveal many suggestions of an evolutionary origin from stocks repre-
sented in comparable parasites of the lower vertebrates. This rela-
tionship is exhibited by the fact that every protozoan genus of this
faunal assemblage of the intestine of man is represented either by
other species in the lower vertebrates or by closely related or more
primitive genera. These genera may even extend to invertebrate
hosts; for example, Endamocba is found not only in mammals but
also in the cockroach, while Craigia (Paramoeba) occurs also in
Sagitta. The trichomonad flagellates have an as yet little known,
but certainly wide distribution in both vertebrates and invertebrates.
The most complex derivative of this group, the genus Giardia, has

432 University of California Publications in Zoology [Vou. 20

not been found below amphibians in the vertebrate scale, and
Pentatrichomonas, the most complex of the trichomonads proper of
mammals, has thus far been found only in man.

The discovery by Kofoid and Swezy (192la, b) of a type of
amoeba in man generically distinct from those previously described
from the vertebrates, naturally raises the question as to whether or
not species of this genus occur in other mammals.

As a result of the investigations of Kessel (1923a, b, c) as Univer-
sity Fellow, 1922-1923, in our laboratory, two additional species of
Councilmania have been distinguished in culture mice and rats of
our laboratory colony. Experimental rearing of these species by
transfers to amoeba-free hosts and the like transfer and successful
culture of the human parasitic Protozoa, including Endamoeba coli
and Couwncilmania lafleuri (see Kessel, 1923b), have enabled us to
verify, supplement, and extend our observations on this genus. It
is the purpose of this paper to present a brief summary of these
results and relate them to the findings of Kofoid and Swezy (1921a, b)
and’ to their subsequent work on Cowncilmania shortly to be pub-
lished in fuller detail.

Acknowledgments are made to the Research Board of the Univer-
sity of California, to the Carnegie Institution, and to friends of the
University contributing to the support of this work, for funds which
have made the investigation possible. Much of our material has been
obtained in routine examinations made in the Division of Parasitology
of the California State Board of Health, in part by Miss Inez Smith.

CouNCILMANIA KoroIp AND Swezy 1921

This genus is distinguished by a group of characteristics which
no other parasitic amoeba, or group of parasitic amoebae presents.
These are: (1) the karyosome is centrally located (in the resting
phase of the nucleus in the cyst), dispersed in numerous subequal,
diserete granules; (2) the nuclear membrane in cysts is generally
faint, and not often heavily or continuously encrusted with peri-
pheral chromatin; (3) the cysts bud in the caecum or colon of the
host by the escape of uninucleate amoebulae from a definite pore;
and (4) the pseudopodia are ectoplasmic, broad, rounded, very
hyaline, are formed very suddenly, and may persist as clear lobes
for extended periods of time. The three known species are all para-
sitic. The active stages are found in the caecum and colon and the
cysts also occur (in the rodent hosts) in the caecum. The cysts,

1923] Kofoid—Swezy—Kessel: On the Genus Councilmania 433

either with or without budding, occur in the stools of the host, though
not regularly. In rodent hosts they are much more sporadic than
in the human one.

The type species is Cowncilmania lafleuri Kofoid and Swezy
(1921). Other species may be expected to be found in the wild
rodents.

This species prior to 1921 was confused with Endamoeba coli
(Loesch) Sehaudinn, and since its publication has been included in
the synonymy of F£. coli by Wenyon (1922), Brumpt (1923), and
others, we believe incorrectly and without adequate, critical reéxam-
ination of material obtained under critical conditions.

NOMENCLATURE

Amoeba muris was originally described by Grassi (1881) from
rats and mice, but only in the motile stages. His account affords
no basis for distinguishing between the two forms Kessel (1925qa)
has found in rats and mice. However, Wenyon’s (1907) figures and
account of the cysts of the amoeba which he designates as Amoeba
muris are sufficiently detailed to afford a basis for the specific recog-
nition of one of the two species. He described but one species in
the mouse and gives no account of the amoebae of the rat. His
aecount apparently applies wholly, or in the main, to one species,
but in his plates there are at least three figures of cysts (his pl. 10,
figs. 20, 33, 34) which clearly differ in nuclear structure from the
others delineated by him. He makes no reference to these differences
in his text or in his diagram of Amoeba muris. His references to
the figures do not deal with any of the distinctive features of these
three cysts, namely, the peripheral chromatin blobs which characterize
the nucleus of Councilmania decumani (Rudovsky).

We conclude, therefore, that Wenyon’s paper (1907) deals pri-
marily with the species muris and thus emends Grassi’s account and
sufficiently establishes its characteristics, to wit, budding in the cysts
and the absence of large chromatin blobs of peripheral chromatin on
the nuclear membrane in the cysts. The budding process, the often
faint nuclear membrane, and the diffused karyosome which char-
acterize Councilmania are recognizable in his figures, though not
always portrayed with accuracy of detail, especially the numerous
prophases of mitosis. We believe that three of his figures (his pl. 10,

434 University of California Publications in Zoology [Vou 20

figs. 20, 33, 34) are of Councilmania decumani. These have coarse
blobs of peripheral chromatin on the nuclear membrane, and the
nuclei are somewhat smaller than the general run in C. muris. It
appears from this that Wenyon (1907) found in mice the two species
which we have found, but recognized only one of them, C. muwris.

Brug (1919), working on the amoebic infections of the wild Mus
rattus of Java, encountered two amoebae (possibly three). To one
of these he gave the name Entamoeba muris, the other he ealled
Entamoeba tetragena. This last he found in a natural infection in
a wild rat and succeeded in one instance in inducing an infection
in the rat by feeding cysts from human stools. His figures and dis-
cussion convinee us that he was dealing in these instances with an
infection by Endamoeba dysenteriae (Councilman and Lafleur).

His figures (his pl. 1) of the amoeba called by him Entamoeba
muris clearly portray an amoeba with a few coarse blobs of peripheral
chromatin on the nuclear membrane in the encysted stages in stained
material. Two of his figures (17 and 18) are indeterminate and one
only (fig. 15) may possibly belong to Councilmania muris. The others
are clearly referable, not to C. muris, but to C. decumani Rudovsky
(1921).

In 1921 Rudovsky failed to find Endamoeba muris (Grassi) Wen-
yon in mice but described an amoeba which he found in 139 wild rats
as Entamoeba muris decumani. This amoeba is correctly character-
ized as having thin nuclear membrane, small karyosome, and a small
amount of chromatin. He noted the difficulty in getting a typical
glycogen reaction of the contents of the vacuole in the binucleate
stage when the glycogen vacuole has its greatest volume. He figures
2-, 4-, 8-nucleate cysts, not very clearly, but with sufficient detail to
be recognizably distinct from Wenyon’s (1907) main type which he
(Rudovsky) cites as EH. muris. He does not figure, and apparently
did not find the species muris. We conclude that this species of
Rudovsky (1921) is identical with one we find in both rats and
mice and that it is different from the one which Wenyon regarded
as Grassi’s Amoeba muris.

Wenyon (1907) seemingly found both species (but recognized
only one) in the mouse. Brug (1919) seemingly found both in the
rat (but recognized only one) and applied Grassi’s name to it
although it differed from the species to which Wenyon applied the
name. Rudovysky (1921) found only one species in the rat but recog-
nized its differences from the species muris.

.

1923] Kofoid—Swezy—Kessel: On the Genus Councilmania 435

We have found both species in both rats and mice of our laboratory
colonies, and refer them to the genus Cowncilmania because both bud
in the encysted stage, and have distributed karyosomes, at least at
some stages.

The following summary of the characteristics of the three known
species belonging to the genus Councilmania will facilitate their
determination.

CoUNCILMANIA LAFLEURI Koroip AND Swezy 1921

The motile amoeba is characterized by explosive thrusting out of
broadly rounded ectoplasmie pseudopodia, one, often two, less fre-
quently three at one time. In life these are absolutely clear and
hyaline and may linger unchanged for some time before the endo-
plasm moves in the same direction. They are often shorter and
broader than shown in figure 1. The line of separation between endo-
plasm and ectoplasm is sharply marked. The nucleus has in this
phase a well developed film of peripheral chromatin, which may
retract in scattered blobs, five to eight in optical section. Inside of
this is a zone traversed by granular radii, the outer part of which is
more granular and the inner forms a clearer halo about the centrally
located karyosome. The karyosome is not solid and spherical but has
irregular margins indicating that it is an assemblage of granules.
These are more closely packed together in the motile stage than in
the cysts. The endoplasm contains food vacuoles with bacteria, the
eysts of other protists, and, at times, red blood corpuscles.

The cysts occur in the 1-, 2-, 4-, and 8-nucleate stages (figs. 2-5)
in the stools, but the earlier phases are best found in the liquid
stool after a saline purge. The glycogen vacuole is best seen in the
binucleate cyst (fig. 2) when it crowds the nuclei to the periphery.
It may be single or multiple and may resist prolonged iodine staining
at times. It stains in sections in Best’s carmine. As it disappears,
the chromatoidal bodies (fig. 2) form as flakes and splinters about
its periphery and in later phases concentrate in a centrally located
bundle (figs. 4, 5) with ragged or irregular ends.

The nuclei of the earlier encysted stages often appear in pre-
mitotic phases. In some of these (fig. 2) eight chromosomes and a
meridional intradesmose can be demonstrated. The most typical
nuclei in the resting phases can be best seen in the 8-nucleate cysts
(figs. 4,5). These are characterized by a slight amount of peripheral
chromatin (fig. 4), or none at all (fig. 5), and the intermediate zone
is often very clear so that the nuclei stand out in the cytoplasm as

+

436 University of Califorma Publications in Zoology [Vou 20

Figs. 1-5. Cowncilmania laflewri Kofoid and Swezy (1921). » 2500. From
camera lucida drawings of amoebae from smears of human faeces fixed in hot
Schaudinn’s fluid and stained in iron haematoxylin.

Fig. 1. Motile amoeba showing hyaline pseudopodium, sharp demarcation of
ectoplasm and endoplasm and nucleus with central, massed karyosome, with ragged
outline indicating massing of granules; clear zone, spoke radii, and peripheral
chromatin.

Fig. 2. Binuecleate cyst with subeentral empty glycogen vacuole, with adjacent
accessory vacuoles and flakes of chromatoidal substance developing on the peri-
phery of the vacuoles. The two nuclei are in the early anaphase with polar centro-
somes, joined by a meridional intradesmose. Eight pairs of chromosomes are in
transit toward the poles of the spindle.

1923] ~Kofoid—Swezy—Kessel: On the Genus Councilmania 437

clear spheres. The karyosome is large, central, and composed of
diserete granules, sometimes densely massed, sometimes dispersed
in a granular sphere or in a more or less complete ring. This dis-
persal takes on a variety of aspects and is modified by the approach
of mitosis when the karyosome migrates to the nuclear membrane and
divides. The two moieties migrate to the poles of the spindle and
spin out the meridional intradesmose between them. Except during
this period of migration and mitosis the karyosome tends to assume
a central location in the nucleus rather than an excentrie one. The
excentricity, if present, is rarely marked. The nuclei of the 2-, 4-,
and 8-nucleate cysts have an average and range of 6 microns, 5-6
microns, 5.3 microns, 4-5.6 microns, 3.5 microns, 3-4 microns respec-
tively in diameter. The cysts are spheroidal, or ellipsoidal, often the
latter, with the longest diameter not over 1.1 the shortest one. The
eysts are generally 16 to 20 microns in longest diameter and range
from 8 to 39 microns. The cysts reach an 8-nucleate phase in the
bowel, and rarely go to a 16-nucleate condition before the budding
process begins. This is brought about by the opening of a minute
circular pore through the cyst membrane at or near the end of
the centrally located chromatoidal mass whose substance is dissolved
in the eytoplasm which eseapes through the pore and causes it to
stain more deeply. The nuelei slip out with the cytoplasm and
amoebulae, each with a single nucleus, successively detach themselves
from the eyst. Mitosis may proceed within the eyst during the
process.

Small amoebulae occur in stained smears adjacent to budding
eysts and empty cysts may sometimes be found in the stool. Chromo-
plile strands or ridges in the peripheral cytoplasm (figs. 6, 7) in a
single or a tripartite are are formed prior to the opening of the pore
and appear to be associated with the chromatoidal body and the
subsequent location of the pore. The habitat is presumably the colon
and caecum. Cysts have been found by us in the contents of the
duodenal bucket by the Lyon method. In artificially infected culture
rats this species occurs in the caecum.

Fig. 3. Four-nucleate cyst with disappearing glycogen vacuole, traces of
chromatoidal substance, four nuclei with dispersed granular, centrally or sub-
centrally located karyosomes, and considerable peripheral chromatin.

Fig. 4. Eight-nucleate cyst with nuclei with less peripheral chromatin and
massed ring-shaped, central karyosomes, and centrally located chromatoidal body
with ragged ends.

Fig. 5. Budding eyst with eight nuclei, chromatoidal substance contributing
to the outpouring eytoplasm, pore at the end of the chromatoidal body. Karyo-
somes ring-shaped and centrally located, nuclear membrane with little peripheral
chromatin.

438 University of California Publications in Zoology * (Vou. 20

CoUNCILMANIA MURIS (GRASSI)

The motile forms of the amoebae belonging to the genus Cowncil-
mania found in rats and mice cannot easily be distinguished in fresh
smears from the motile forms of C. laflewri except by size. The aver-
age size of rounded motile C. decwmani and C. muris is 19 microns
while C. laflewri may attain a size of 63 by 35 microns. The pseudo-
podia are, without exception, hyaline in structure. This fact is borne
out by all the previous investigations of the amoebae of rats and

7

Figs. 6 and 7. Cowncilmania lafleuri Kofoid and Swezy 1921. 2500. From
camera lucida drawings of amoebae from smears of human faeces fixed in hot
Schaudinn’s fluid and stained in iron haematoxylin.

Fig. 6. Initial stage of emergence of bud. Tripartite chromophile strands
below with the pore at the tip of the upper one. The deeply stained cytoplasm
is beginning to emerge from the pore. The nuclei have centrally located, massed
or ring-shaped karyosomes. . >

Fig. 7. Eight-nucleate cyst with broad, rather diffuse chromophile strand or
ridge (below). Note the depression at the surface. It is not a wrinkle of the
cyst wall but a cytoplasmic differentiation. The chromatoidal body is vertical,
is greatly reduced in size, and is enveloped in a clear zone. Its lower end points
toward the middle of the chromophile ridge. The nuclei are in early prophases
with small karyosomes on or near the membrane. The karyosomes are in most
instances divided and the intradesmose is in process of formation. When not
divided the karyosome at this stage of mitosis is usually peripheral in location.

mice, namely, by Grassi (1881), Wenyon (1907), Brug (1919), and
Rudovsky (1921). For a detailed description of the amoeboid move-
ment of the amoebae, see Kessel (1923c).

The normal habitat of this amoeba is the caecum, where it feeds
upon bacteria, yeasts, and on other Protozoa. So far as known, it is
non-pathogenic to the rat.

1923] Kofoid-Swezy—Kessel: On the Genus Councilmana 439

In specimens stained with iron haematoxylin the nuclear structure
of C. muris is distinctive. The nuclear membrane is very thin and
is seldom, if ever, encrusted with chromatin granules. The karyosome
is characterized by the mass of 5 to 8 dispersed granules, arranged
centrally or subeentrally. This diffuse condition of the karyosome is
characteristic of every cyst in a resting phase encountered in this
species (fig. 12).

A glycogen mass is commonly found in the 1-, 2-, and 4-nucleate
cysts (figs. 15-17). In these stages, phases of mitosis are usually
present. Mitosis is similar in this species to the process in other
species of parasitic amoebae in that the nuclear membrane remains
intact during division and that an intradesmose connects the centro-
somes of the spindle. The number of chromosomes found in C. muris
is six (fig. 11).

The 8-nucleate cyst is the typical or characteristic cyst of this
species. The nuclei in the resting stage are characterized by a very
thin nuclear membrane, often very difficult to find, its position being
determined by the limits of the cytoplasm surrounding the nucleus.
The karyosome retains the typical dispersed condition found in the
nucleus of the motile forms.

The average diameter of the nuclei in the two-nucleate cysts of
this species is 5.5 microns, the range being 5 to 6 microns; of the 4-
nucleate cysts the average diameter of the nuclei is 4.8 microns, the
range being 4 to 5.2 microns; while in the 8-nucleate cysts the average
diameter of the nuclei is 3.4 microns, the range being from 2.75 to 3.75
microns.

Chromophile ridges are commonly present in the 8-nucleate cysts
in this species (fig. 12). The chromatoidal bodies are typically
bundles of splinters with jagged ends (figs. 10,13). In the 8-nucleate
cyst the point of the chromatoidal body is generally found. in the
region of the pore. Budding occurs in this species in a manner
similar to the type of budding found in C. laflewri (fig. 13).

The cysts vary from 14 microns to 19 microns in diameter, with
an average of 15.8 microns. It occurs in both mice and rats of our
culture colony.

CoUNCILMANIA DECUMANI (Rupovsky) KESSEL

The motile forms of (. decumani are very similar to the motile
forms of (. muris in that the vacuolation and food materials are
identical and the pseudopodia are broadly rounded and hyaline in
structure. There is also no marked difference in size.

440 University of California Publications in Zoology [Vou. 20

13

Figs. 8-13. Councilmania muris (Grassi, 1881) Kessel, emend. 1923. 2500.
Camera lucida drawings of amoebae from smears of rat faeces fixed in hot
Schandinn’s fluid and stained in iron haematoxylin.

Fig. 8. Motile amoeba showing hyaline pseudopodium and posterior tooth-like
projection. Note the marked differentiation between endoplasm and ectoplasm.
Nucleus showing karyosome of dispersed chromatin granules, and thin nuclear
membrane with no encrusted chromatin.

Fig. 9. Mononucleate cyst, showing glycogen mass surrounded by small
chromatoidal bodies. Nucleus with thin membrane and dispersed granules in a
darkly staining region. Two of the granules are connected by an intradesmose.

Fig. 10. Binucleate cyst showing glycogen mass in center and chromatoidal
body at edge. Nuclei in anaphase, showing spindle and six chromosomes.

1923 Kofoid—Swezy—Kessel: On the Genus Councilmania 441

The nuclear structure of the motile stages is, however, quite
different in the individuals stained with iron haematoxylin where the
karyosome, instead of appearing as a number of dispersed granules,
oceurs as a sphere or a crescent (fig. 15). The nuclear membrane is
slightly heavier than the nuclear membrane of C. muris and is com-
monly encrusted with discrete chromatin granules (fig. 15).

This massing of chromatin material in blobs on the nuclear mem-
brane in the eysts is a characteristic of this species, such a condition
being common in some stages of nuclear division, and in the resting
stage, but not at the metaphase (fig. 17). The karyosome of this
species is more massed than the karyosome of C. mwris or C. lafleurt,
but shows a tendency to dispersion throughout by minute irregu-
larities on its margins. The karyosome is never so small nor so
characteristically massed as the karyosome of Hndamoeba coli or
E. dysenteriae.

The stages of mitosis are similar to those of other parasitic
amoebae (fig. 17). Four chromosomes, including one minute one,
are characteristic for this species.

The typical resting nucleus is seen in figure 18. It is characterized
by a thin but distinct nuclear membrane upon which several heavy
chromatin blobs are encrusted. The number may vary from two to
six or eight, but usually four chromatin blobs are present. The
karyosome is excentric, larger than the karyosome of Endamoeba coli,
and shows a tendency to dispersion.

The nuclei of Cowncilmania decumani are on the whole smaller
than the nuclei of C. muris. Their average diameter in the binucleate
eysts is 4.6 microns and the range 4 to 5.2 microns; while the average
diameter in the 4-nucleate cysts is 3.8 microns, the range being from
3.1 to 4.4 microns, and the average diameter in the 8-nucleate cysts
is 2.8 microns, the range being from 2.5 to 3 microns.

This species of amoeba is poorer in chromatoidal material than
C. muris, the presence of masses of chromatoidal splinters being the

Centrosomes are connected by an intradesmose. The nuclear membrane is thin
with no encrusted chromatin.

Fig. 11. Four-nucleate cyst, one nucleus in metaphase and the other three in
prophase. Note dispersed condition of chromatin granules of nuclei in prophase,
The membranes are characteristically thin with no encrusted chromatin.

Fig. 12. Eight-nucleate cyst showing typical chromophile ridge. The nuclei
show the characteristic, central, dispersed karyosome, and thin nuclear membrane
with no encrusted chromatin.

Fig. 13. Budding cyst, showing tripartite chromophile ridge and exceptionally
large chromatoidal mass with splintered ends. The karyosomes are dispersed
granules and the nuclear membranes are faint.

442 University of California Publications in Zoology {Vou. 20

Figs. 14-19. Councilmania decumani (Rudovsky, 1921) Kessel, emend. 1923.
x 2500. Preparations same as in figures 8-13.

Fig. 14. Motile amoeba showing hyaline, rounded pseudopodia with sharp
demarcation between ectoplasm and endoplasm; nucleus with somewhat massed
erescentrie karyosome in darkened halo and chromatin granules encrusted on
nuclear membrane.

Fig. 15. Uninueleate cyst with no glycogen mass but with a number of
small, spherical chromatoidal bodies. The karyosome is located in a darkly stain-
ing halo containing four small grannles. Nuclear membrane is encrusted with
heavy chromatin blobs.

Fig. 16. Binucleate cyst with glycogen mass on the margin of which are
chip-shaped chromatoidal masses. Nucleiin early prophase, the karyosome break-
ing into two portions connected by an intradesmose. Characteristic peripheral
chromatin encrusted on nuclear membrane.

1923 ] Kofoid—Swezy—Kessel: On the Genus Councilmania 443

exception rather than the rule. Small masses with irregular ends
(fig. 19) or chips or spheres of chromatoidal substance (fig. 15, 16)
are commonly encountered.

The budding cyst of C. decumani is characterized by the absence
of chromatoidal material and by few chromophile ridges, only one
small ridge having been found in this species to date. Buds, however,
are common and are produced by the same method of reproduction
in this species as in the others belonging to this genus.

The normal cysts range from 12 to 20 microns in diameter but
one large 16-nucleate cyst has been found, measuring 22 microns.
The average diameter of the cysts of this species is 15.5 microns. This
species occurs in both mice and rats of our culture colony.

KEY TO SPECIES OF COUNCILMANIA

Cysts spheroidal to ellipsoidal, 8-34, generally 16-20 microns in longest diameter;
in 8-nucleate cysts the nuclear membrane is generally very faint with little
peripheral chromatin; diameter of nuclei 3.5 microns; karyosome central, com-
posed of numerous coarse, irregular granules; 8 chromosomes ................ lafleuri

Cysts generally spherical, 15.8 (13-19) microns in diameter; very little peripheral
chromatin at any stage in the nuclei of the cysts; nuclear membrane very
faint; diameter 3.4 (2.75-3.75) microns; karyosome central composed of 5-7
dispersed fine, uniform granules; 6 chromosomes ..................-...----------------- muris

Cysts spherical, 15.5 (12-22) microns in diameter; peripheral chromatin in several
rather large, distinct blobs; nuclear membrane distinct, diameter 2.8 (2.5-3.0)
microns; karyosome exccntric, composed of massed granules, 4 chromosomes -...

decumani

Fig. 17. Four-nucleate cyst, two nuclei in prophase, showing karyosome break-
ing up and peripheral chromatin blobs. The other two nuclei are in early anaphase
showing the spindle, the four chromosomes having just divided. The centrosomes
are connected by an intradesmose.

Fig. 18. Eight-nucleate eyst showing clustered chromatoidal bodies. The
nuclei are characteristic resting nuclei of this species with excentric karyosome
more massed than in C. muris but with a tendency to dispersion and with char-
acteristic chromatin blobs on nuclear membrane.

Fig. 19. Eight-nucleate cyst with no chromatoidal bodies and with bud in
early formation. Note absence of chromophile ridge. The nuclei show the typical
structure common to this species.

444 University of California Publications in Zoology [Vou. 20

SUMMARY

The genus Cowncilmamia is characterized by clear, hyaline pseudo-
podia, a nuclear membrane usually not heavily encrusted with peri-
pheral chromatin, a karyosome that in the resting phase in the eyst
is composed of numerous discrete granules, and by a budding process
in which uninucleate amoebulae escape from the cyst in the caecum
or colon of the host by a definite pore. The pore is formed near the
end of the chromatoidal mass and on or near the chromophile ridge.

The type species is C. laflewri found in the human intestine. In
this species little peripheral chromatin is found on the nuclear
membrane, and the karyosome is composed of numerous coarse
granules. Eight chromosomes are found at mitosis.

C. muris is found in rats and mice. The karyosome is central
and composed of 5 to 7 dispersed, fine, uniform granules. Six chromo-
somes are present during mitosis. No chromatin blobs occur on the
nuclear membrane.

C. decumani is also found in rats and mice. The nuclear mem-
brane is clearly marked with several rather large, distinct blobs, and
a karyosome composed of massed granules. Four chromosomes are
found at mitosis.

ZooLoGicaAL LABORATORY,
UNIVERSITY OF CALIFORNIA.

Transmitted July 28, 1923.

1923] Kofoid-Swezy—Kessel: On the Genus Councilmania 445

LITERATURE CITED
Brua, 8. L.
1919. De entamoeben van de rat. Genees. Tijdschr. Nederlandsch-Indié, 59,
1-16, pls. 1-2.
Brumpt, E.
1922. Précis de parasitologie (Paris, Masson), ed. 3, xv+1216 pp., 5 pls.,
736 figs. in text.
Grassi, B.
1881. Intorno ad aleuni protisti endoparassitici ed appartenenti alle classi
dei flagellati, lobosi, sporozoi e ciliati. Atti Soc. Sci. Nat., 24,
135-224, pls. 1-4.
KESSEL, J. F.
1923a. The distinguishing characteristics of the intestinal amoebae of culture
rats and mice. Univ. Calif. Publ. Zool. (In press.)
1923b. Experimental infection of rats and mice with the common intestinal
amoebae of man. Ibid., 20, 409-430, pls. 38-39.
1923c. Methods of securing amoeba-free rats for experimental infection with
intestinal amoebae. Jbid., 20, 401-408.

Koro, C. A., and Swezy, O.
1921a. Councilmania lafleuri, a new amoeba of the human intestine. Proc.
Soe. Exper. Biol. Med., 18, 310-312.
1921b. On the free, encysted, and budding stages of Councilmania lafleuri, a
parasitic amoeba of the human intestine. Univ. Calif. Publ. Zool.,
20, 170-189, pls. 18-22, 3 figs. in text.
Rupovsky, F.
1921. Ueber Rattenamoebiase. Weiner tieriirz. Monatsschr., 8, 636-646, pl. 7.
Wenyon, C. M.
1907. Observation on the Protozoa in the intestine of mice. Arch. Prot.,
Suppl. 1, pp. 169-201, pl. 10-12, 1 fig. in text.
1922. On three supposed new Entamoeba of Man. Trop. Dis. Bull., 19, 19-22.

(H 11.
12.
13.

VoL 20. 1.

2.

a

7.

8,

hs
10.

11.

12.

13.

x 14,

15.
16,

17.

18.
19,

20.

2

ry

UNIVERSITY OF CALIFORNIA PUBLICATIONS—(Continued)

The Life Cycle of Echinostoma revolutum (Froelich), by John C, Johnson.
Pp. 338-388, plates 19-25, 1 figure in text. May, 1920 0.
On Some New Myriopods Collected in India in 1916 by C. A. Kofoid, by

Ralph V. Chamberlin. Pp. 389-402, plates 26-28. August, 1920 _........ St

Demonstration of the Function of the Neuromotor Apparatus in Euplotes
by the Method ef Microdissection, by Charles V. Taylor. Pp. 403-470,
plates 29-33, 2 figures in text. October, 1920 22... eteeeeeeceeee =

Index, pp. 471-480.

Studies on the Parasites of the Termites. I. On Streblomastiz striz, a Poly-
mastigote 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

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 Invtexti Duly: LONG sos ec ie rR Ne ee

Studies on the Parasites of the Termites, III. On Trichonympha campanula
sp. nov., by Charles Atwood Kofoid and Olive Swezy. Pp. 41-98, plates
5-12, 4 figures AYN Lex bay LY yD OL sr CS Me PO em ee ei

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 fienre!m Texte Muryy DID Bac oes RN

On the Morphology and Mitosis of Chilomastiz mesnili (Wenyon), a Common
Flagellate of the Human Intestine, by Charles A. Kofoid and Olive Swezy.
Pp. 117-144, plates 15-17, 2 figures in text. April, 1920 202

A Critical Review of the Nomenclature of Human Intestinal Flagellates,
C€ercomonas, Chilomastiz, Trichomonas, and Giardia, by Charles A. Kofoid.
Pp. 145-168,"9 figures in text. June, 1920 -..--o2:e td

On the Free, Encysted, and Budding Stages of Councilmania lafleuri, a Para-
sitic Amoeba of the Human Intestine, by Charles Atwood Kofoid and
Olive Swezy. Pp. 169-198, plates 18-22, $ figures in text. June, 1921 ..._.

Mitosis and Fission in the Active and Encysted Phases of Giardia enterica

(Grassi) of Man, with a Discussion of the Method of Origin of Bilateral
Symmetry in the Polymastigote Flagellates, by Charles A. Kofoid and
Olive Swezy. Pp. 199-234, plates 23-26, 11 figures in text. March,
1922 ....

The Micro-Injection of Paramaecium, by Chas. Wm. Rees. Pp. 235- 242.

ON ast PB 2 Pg ae ata Cn Pc Bt ARES Se «ila ORES REN tae aD er PE ren MO WOW a Fh AS

On Balantidium coli (Malmsten) and Balantidium suis (sp. nov.), with
an account of their neuromotor apparatus, by J. Daley McDonald.
Pp. 243-300, plates 27, 28, 15 figures in text. May, 1922 ..0 00

Mitosis in Endamoeba dysenteriae in the Bone Marrow in Arthritis de-
formans, by Charles Atwood Kofoid and Olive Swezy. Pp. 301-307,
7 figures in text.

Endamoeba dysenteriae in the Lymph Glands of Man in Hodgkin’s Disease,
by Charles A. Kofoid, Luther M. Boyers, M.D., and Olive Swezy. Pp. 309-
$12, 4 figures in text.

Nos. 11 and 12 in one cover. April, 1922 2.2.22. cc cece eee eeaeeeeeee ne

Mitosis in the Encysted Stages of Hndamoeba coli (Loesch), by Olive
Swezy. Pp. 313-332; plates, 29-31. May, 1922 .2....-.0. nee

The Neuromotor Apparatus of Paramaccium, by Charles William Rees.
Pp. 333-364; plates 32-36, 5 figures in text. November, 1922.0...

A Comparison of the Cysts of Hndamoeba coli and Councilmania lafleuri in
Congo Red, by Nathaniel Bercovitz, M.D. Pp. 365-371. February, 1923.

On the Morphology and Behavior of Pentatrichomonas ardin delteili (Der-
rieu and Raynaud), by Charles Atwood Kofoid and Olive Swezy. Pp.
373-390, plate 37, 1 figure in text. August, 1923.20.20. cece

The Pseudopodial Method of Feeding by Trichonymphid Flagellates Para-
sitic in Wood-eating Termites, by Olive Swezy. Pp. 391-400, 14 figs. in
COX toy SAT EUS: SLO DS rc Be PS a ae a Oe

Methods of Obtaining Amoeba-free Rats for Experimental Infection with
Intestinal Amoebae, by John F. Kessel. Pp. 401-408.

Experimental Infection of Rats and Mice with the Common Intestinal
Amoebae of Man, by John F. Kessel. Pp. 409-430, plates 38-39.

Nos. 18 and 19 in one cover. October, 1923 _.....22.2...2o2.-eeteet eee

On the Genus Couwncilmania, Budding Intestinal Amoebae Parasitic in Man
and Rodents, by Charles A. Kofoid, Olive Swezy, and John F. Kessel.
Pp. 431-445, 19 figures in text. October, 1923 —.2...2..2 eee eee

Morphology and Binary Fission of Menoidium incurvum (Fres.) Klebs.
Pp. 447-476, plates 40-41, 2 figures in text. October, 1923-22.

.

1,00

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Pp.i43-47;,) Mlarchy1019) So ts ee a Be Ok Fo ae Saar a
$3. Notes on the Natural History of the Bushy-tailed Wood Rats of California,
by Joseph Dixon. Pp. 49-74, plates 1-3, 3 figures in text. December, 1919
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5. A Study of the California Jumping Mice of the Genus Zapus, by A. Brazier
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Cunningham Miller. Pp. 293-328, plates 13-17, 6 figures in text. Novem-
ery AO ese oe a ee AEE Sale oD ie eis, TE es ae
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Stylarioides papillosa, sp. nov., a New Annelid from the San Diego Region,
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Kofoid Bucket in 1922, by Winfred Emory Allen. Pp. 435-445, 5 figures
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445-458. -Ochober, O23 het! eae 2 a ee ees eee ee
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UNIVERSITY OF CALIFORNIA PUBLICATIONS

ZOOLOGY
Vol. 20, No. 21, pp. 447-476, plates 40-41, 2 figs. in text

November 10, 1923

MORPHOLOGY AND BINARY FISSION OF
MENOIDIUM INCURVUM (FRES.) KLEBS

Introductory and historical.
Material and technique.........

General morphology.................
Neuromotor system..........

Gullet and reservoir.....
She mrel Cvs Seseese-ceee.-s cst:
Habits and activities........
Nuclear division..............
Resting stage.................
Prophase............. 2
Metaphase.................
ENTE) ESTES secmencactneeces ;
Melophase see...
Binary fission.................-
Paradesmose........
Gullet and reservoir...
WIR CUIBSION meena.
The resting nucleus.......
The endosome...............
prophase metres ee as-e
Metaphase................
Anaphase and telophase..
NS RUTITGU AYER cnet apnccee nee ac Ee

Wintrensitmme (cited ey css:-ese-ceee.c.t2cee-
Explanation of plates... 0.0.0...

BY

RICHARD P. HALL

CONTENTS

448 University of California Publications in Zoology [Vou. 20

INTRODUCTORY AND HISTORICAL

Early in the spring of 1921 there appeared in great abundance,
in one of my aquaria, a small euglenoid flagellate, which has been
identified as Menoidiwm incurvum (Fres.) Klebs. This form was
first described by Fresenius (1858) as Rhabdomonas incurva. Stein .
(1878) later found this species but considered it to be merely a
““jugendliche Form”’ of his Astasia proteus (= Distigma proteus
Ehrb.). Seligo (1885) found, in the same culture, Rhabdomonas
incurva, Astasiopsis distorta Duj., and a so-called intermediate form.
Influenced by the earlier statement of Stein he concluded that, since
he found no division stages of his Rhabdomonas, this flagellate was a
stage in the life history of Astasiopsis. However, both Biitschli
(1883-1889) and Klebs (1883) later recognized Rhabdomonas incurva
as a distinet species; finally, Klebs (1892), on the basis of the resem-
blance between Menoidium pellucidum Perty and Rhabdomonas in-
curva Fresenius, included the latter in the genus Menoidium Perty:

Ivh moéchte jetzt die Gattung weiter fassen als Perty, Stein und ich selbst es
frither gethan haben, indem ich die Rhabdomonas incurva hinzuziehe, da in der
That der Unterschied zwischen dieser Art und Menoidium pellucidum viel geringer
ist als derjenige zwischen einzelnen Huglena-resp. Phacus-Arten. Die Gattung
Menoidium umschliesst dann die starren, eingeisseligen, Astasia-ahnlichen Flagel-
laten.

The species Menoidiwm incurvum is described by Klebs (1892)
as follows: :

Korper cylindrisch, an beiden Enden abgerundet, meist etwas gekriimmt.
Plasmamembran mit weit von einander stehenden Liangsstreifen versehen. Lange,
16-21; Breite, 7-8.

Klebs’s deseription coincides with my own observations, with the
addition that these flagellates always contain a number of nearly
colorless paramylum bodies, clumped together usually at the anterior
end, although sometimes present at both ends (pl. 40, figs. 1 and 6).
Since nuclear division and binary fission have not heretofore been
described in this flagellate, I shall attempt to discuss these processes
in this paper.

I am indebted to Dr. Robert C. Rhodes of Emory University, at
whose suggestion this investigation was undertaken, for his advice
and assistance; also, I wish to thank Professor Charles A. Kofoid,
in whose laboratory this work was completed, for his invaluable
criticisms and suggestions.

1923 ] Hall: Binary Fission of Menoidium incurvum 449

MATERIAL AND TECHNIQUE

Abundant material was obtained from an aquarium at Emory
University, Atlanta, Georgia. The flagellates became dominant early
in March, 1921, and were still plentiful in July. The aquarium in
which the form occurred measured about 36 by 18 by 24 inches, and
was filled to a depth of five or six inches with a mixture of mud,
sand, sawdust, old leaves, and straw. A small turtle and several
large tadpoles had been placed in the aquarium several weeks before
the appearance of the flagellates, and were kept there continuously.
Menoidium appeared to be uniformly distributed throughout the
aquarium, there being no marked accumulation at the surface.

Several new cultures have been started at the University of Cali-
fornia from samples of the original material sent me from Emory
University. Menoidiwm has been present in these cultures fairly
constantly for the past year and a half, although never in such
abundance as in the first aquarium. Various attempts have been
made to obtain pure cultures in peptone and beef extract solutions,
boiled hay infusions, and different combinations of these, but none
was markedly successful. Some of the Protozoa which have occurred
in the cultures in association with Menoidiwm are: Euglena, Petalo-
monas, Cryptoglena, Amoeba, Difflugia, Arcella, Chilomonas, Coleps,
Stylonychia, Euplotes, Loxophyllum, Centropyxis, Cyclidium, and
Colpidium. In addition several species of small Crustacea, rotifers,
diatoms, and green algae have usually been present.

In fixation, both the cover-glass flotation and the centrifuge
methods have been used to accumulate material in quantity; the best
results have been obtained by centrifuging. As fixing agents, strong
and weak solutions of Flemming’s fluid, and Schaudinn’s fluid (both
hot and cold solutions) have been used; Schaudinn’s sublimate-alcohol
has been more satisfactory. All staining has been done by the cover-
glass method. In the flotation method as well as with centrifuged
material, the cover-slips were smeared with a thin film of Mayer’s
egg albumen fixative; in the case of material killed after centrifuging,
the organisms were pipetted onto the smeared covers and then stained
in the usual way. Among the stains tried in addition to alcoholic
and aqueous iron-alum haematoxylins are: Delafield’s haematoxylin,
the mixtures of Mallory and Mann, Benda’s safranin light-green,

450 Unwersity of California Publications in Zoology [Vou 20

polychrome blue and orcein, Prenant’s triple; and as counterstains
after haematoxylin: eosin, erythrosin, light green, methyl green,
methylene blue, Congo red, Bordeaux red, orange G, and safranin.
As nuclear stains, none has been as satisfactory as the aqueous and
aleohohe iron-alum haematoxylins; of the counterstains, Bordeaux
red, eosin, methylene blue, orange G, and light green have produced
good results.

As special stains for rhizoplasts and basal granules of flagella, a
weak solution (0.1 per cent) of Bordeaux red, as reconimended by
Rhodes (1919), and the solution of Gicklhorn (1921) have been used
before staining in iron-haematoxylin. The organisms were stained
for twelve to thirty-six hours in either of these solutions and then
transferred directly to iron-alum. Both stains are good, but the use
of Bordeaux red has been especially advantageous.

GENERAL MORPHOLOGY

Menoidium incurvum is characterized by a rigid, elongate bean-
shaped body (pl. 40, fig. 1), ranging in length from 15 to 25. Even
under a 4 mm. objective the surface of the living flagellate shows
regularly spaced longitudinal striations which are some distance
apart (pl. 40, figs. 1 and 6). As determined by a study of optical
cross-sections in stained material, the number of these varies from
ten to fifteen for flageilates whose nuclei are in the prophase or
resting stage; the greater number of those examined possess fourteen
striations. The number of these striations is correlated with the
circumference of the organisms; as the flagellate enlarges with the
approach of binary fission, the number is seen to be increased (pl. 40,
fig. 2). Posteriorly the striations converge to a point (pl. 40, fig. 3) ;
anteriorly they extend to the margin of the gullet (pl. 40, fig. 4). In
optical cross-sections (pl. 40, fig. 5) their thickness is approximately
the same as that of the region between them, so that they probably
do not represent appreciably thickened parts of the pellicle.

The endoplasm contains a variable number of plastids (pl. 40,
fies. 1 and 6), ranging from practically colorless te a very light green;
these were not stained in any of the stains used and are probably
paramylum bodies, since they appear tinged with yellow when
treated with solutions of iodin. In the living flagellates these plastids.

1923 | Hall: Binary Fission of Menoidium incurvum 451

are clumped together at either one or both ends of the animal (pl. 40,
figs. 1 and 6), usually only at the anterior end and never altogether
at the posterior end; in no case have they been observed to fill the
entire body of the flagellate.

THe NrvurRoMoTOR SysTEM

Since the introduction of the conception of a neuromotor system
(Kofoid and Christiansen, 1915a, b, Kofoid, 1916), subsequent investi-
gations (Swezy, 1916; Boeck, 1917; Kofoid and Swezy, 1919a, b, 1920,
1922) have demonstrated the presence of such a system in many differ-
ent flagellates. The neuromotor system consists essentially of the
flagellum, the blepharoplast with its connected structures, and the
rhizoplast extending from the nucleus to the blepharoplast, thus form-
ing an integrated system composed of the nucleus and locomotor
organelles. In addition there is, at the base of the nuclear rhizoplast,
an extranuclear centrosome, the center of the system.

The system in Menoidium is simple: there is a single flagellum
ending in a blepharoplast, from which a rhizoplast extends to the
nucleus (fig. A; pl. 40, fig. 9). The blepharoplast is located at the
bottom or side of a vacuole-like enlargement, the reservoir, at the
posterior end of the gullet (fig. A). The nuclear rhizoplast is very
delicate and can be seen in well-stained material only ; Bordeaux red-
iron haematoxylin preparations are most favorable for such obser-
vations. So far it has been impossible to trace the rhizoplast in
flagellates in which the nucleus is at the posterior end. In many
forms, other than euglenoids, there is an extranuclear centrosome
at the base of the rhizoplast. In Huglena agilis (fig. B) a similarly
located granule is easily detected in Bordeaux red-iron haematoxylin
preparations; in Menoidium (fig. A) such a granule has been deter-
mined in similar preparations by the use of green-filtered mono-
chromatic light; however, this granule in Menoidiwm has been found
in comparatively few of the flagellates examined, since it is very small
and does not always stain heavily. Thus it appears that the neuro-
motor system of at least some of the euglenoids is essentially similar
to that of Giardia (Kofoid and Swezy, 1922), Trichomonas (Kofoid
and Swezy, 1915a) and related forms, in that they possess an extra-
nuclear centrosome at the base of the rhizoplast. Structures such as
those described by Wager (1899) at the level of the eyespot in Euglena
are not present in Menoidiwm.

452 University of California Publications in Zoology [Vou. 20

GULLET AND RESERVOIR

The gullet-reservoir structure in Menoidium incurvum (fig. A) is
similar to that described for Huglena (Wager, 1899; Haase, 1910;
Hamburger, 1911), Astasia (Béla?, 1916), Peranema (Hartmann and
Chagas, 1910), Heteronema (Rhodes and Kirby, MSS), Eutreptia
(Steuer, 1904), and Copromonas (Dobell, 1908). The gullet, open-
ing somewhat eccentrically at the anterior end, is apparently con-

Fig. A. Menoidium incurvum, diagrammatic camera lucida sketch: bleph.,
blepharoplast; cary., endosome; cent., extranuclear centrosome; chrom., chromatin
granules in resting nucleus; cyt., gullet; fl., flagellum; nuc., nuclear membrane;
par. b., paramylum bodies; post., reservoir; rhiz., rhizoplast. X 2100.

Fig. B. Euglena agilis, camera lucida drawing: bleph., blepharoplast; cent.,
extranuclear centrosome; cyt., gullet; f., flagellum; par., paradesmose; par. b.,
paramylum bodies; rhiz., rhizoplast. X 2500.

tinuous with a reservoir situated usually near the nucleus (fig. A). A
contractile vacuole in connection with the reservoir, as described in
Copromonas (Dobell, 1908), Euglena viridis (Wager, 1899), and
Eutreptia (Steuer, 1904), has not been observed in Menoidium. Since
no ingestion of food particles has been observed and no typical food
vacuoles, which are characteristic of holozoic forms, have been seen,
the function of the cytostome and gullet is evidently not that of
ingestion of solid food. Khawkine (1886) has suggested that the

1923 | Hall: Binary Fission of Menoidium incurvum 453

gullet of Huglena is for the admission of liquid food. Wager (1899),
however, considers this hypothesis as unproved, and states that the
gullet and reservoir are excretory organelles, serving as outlet for
substances discharged by the contractile vacuole. The regular oceur-
rence of a number of paramylum bodies at the anterior end in
Menoidium suggests that the gullet is in some way connected with
metabolism, possibly through the admission of liquid food; for the
liquid medium would probably enter the gullet unless there is some
undiscovered means of keeping the cytostome closed. But whatever
the function of the gullet and reservoir, the structure of these organ-
elles is essentially similar in the holozoic, the saprozoic, and the holo-
phytie euglenoids.

Tur NucLEeus

The nucleus of Menoidiwm is usually situated near the center of
the body, although frequently posterior and sometimes at the extreme
posterior end. In structure the nucleus (fig. A) is quite typical for
the euglenoids. There is a homogeneous, heavily-staining endosome
(the ‘‘Binnenkérper’’ of Doflein and Tschenzoft) located usually near
the center; the term endosome is used in the sense ascribed to it by
Minchin (1912) and is not to be confused with the ‘‘entosome’’ of
Prowazek (1901). Around the endosome, in the resting nucleus
(pl. 40, fig. 7), chromatin granules occur apparently in the nodes
of a linin network; in mitosis, the chromatin forms distinct chromo-
somes surrounding the endosome. In a few cases twelve have been
counted, but this is only a tentative determination since the exact
limits of the chromosomes are usually difficult to determine. A fairly
distinet nuclear membrane is present, and persists throughout mitosis.

HABITS AND ACTIVITIES

The method of nutrition in Menoidiwm is probably saprozoic, since
the evidence at hand favors such a conclusion; observation of the
living organism has failed to reveal any ingestion of solid food
particles, and no such particles have been seen in stained material.
Lemmermann (1913) also regards Menoidiwm incurvum as ‘‘meso-
saprob.’’ Menoidiwm is preyed upon by the Euplotes, Stylonychia,
and Amoeba which have usually been present in the cultures. The
amoebae are especially voracious, often engulfing several of the
flagellates, which persist for some time during digestion.

454 University of California Publications in Zoology [Vou. 20

The locomotion of Menoidium is characteristic, since the smooth
gliding type of movement found in many euglenoids is lacking. There
is a rotation to the right, but this is accompanied by an oscillation
of the body on its minor axis, producing an apparently awkward type
of locomotion; yet this locomotion is evidently effective enough, since
the flagellate is capable of traveling more rapidly than the much
larger Euglena that occurs with it.

Although eneysted forms are mentioned by Lemmermann (1913),
the evidence for their occurrence in the cultures under observation
is inconclusive. Several possible cases have been found in stained
material, but owing to the fact that the cultures have contained
several other flagellates positive identification of these cysts as those
of Menoidium has not been possible. In this connection there might
be mentioned a peculiar case of an individual which apparently
contained three normal nuclei, one in the center of the body, the
second anterior, and the third posterior, to the first; since this was
the only case discovered, its significance has not been determined.

NUCLEAR DIVISION
Tue ReEsTING STAGE

The resting nucleus of Menoidiwm incurvum (pl. 40, fig. 7) con-
tains a more or less centrally located endosome, around which the
chromatin occurs in the nodes of a linin network. With the use of
counterstains, such as Bordeaux red, after haematoxylin, the network
is lightly stained, while the chromatin masses are deeply stained,
This granular condition of the chromatin merges gradually into that
of the coiled chromatin threads of the early prophase, so that it would
be exceedingly difficult to determine just when the prophase begins.

Contrary to the observations of Bélar (1916), Hartmann and
Chagas (1910), and Haase (1910) on various euglenoids, the endo-
some of Menoidium shows no centriole at this or any other stage of
mitosis. My haematoxylin preparations vary from deeply stained
to almost totally destained; also a number of anilin nuclear stains
and counterstains after haematoxylin have been used; yet none of
these brings out any structure resembling a centriole.

1923 ] Hall: Binary Fission of Menoidium incurvum 45

Or

PROPHASE

With the initiation of the prophase, the scattered chromatin
granules appear to become organized into fine thread-like structures,
so that soon the semblance of a fine spireme is produced (pl. 40, fig. 8).
It has not been determined with any degree of certainty how many
threads this ‘spireme’ contains, but in figure 8 of plate 40 are shown
what appear to be the ends of several threads, indicating the presence
of more than one. The fine chromatin threads gradually shorten and
thicken until, finally, distinct chromosomes are formed. Although
it is possible that such structures exist, there is no evidence whatever
for the emergence of chromosome pairs from the resting stage into
the prophase, as described by Tschenzoff (1916) for Huglena. At
first these chromosomes are coiled in a seemingly haphazard fashion,
but they soon assume a somewhat radial arrangement with respect
to the endosome (pl. 40, figs. 9 and 10). The number of these chromo-
somes of the prophase has not been definitely determined, since the
exact endings of all of them cannot be made out; tentatively their
number may be stated as approximately twelve. Except in a very
few instances the chromosomes have not appeared granular, as
described for Euglena (Tschenzoff, 1916; Dehorne, 1920) and Hetero-
nema (Rhodes and Kirby, MSS). It is quite possible, however, that
such is the structure of the chromosomes of Menoidium; their com-
paratively small size renders eritical observation difficult.

With the advance of the prophase, the finer threads become trans-
formed into the thicker chromosomes of the later stages (pl. 40,
fig. 11). In the meantime, the endosome has begun to elongate in
preparation for the metaphase, while the chromosomes still appear
radially arranged with respect to the axis of the endosome.

METAPHASE

With further elongation of the endosome, splitting of the chromo-
somes apparently begins (pl. 40, figs. 12 and 13; pl. 41, figs. 14 and
15). In such stages the chromosomes appear thinner and more
numerous than those of the late prophase. Although the evidence is
not entirely conclusive, the split apparently begins at one end of a
thick chromosome and continues toward the other, resulting in the
formation of a V-shaped structure (pl. 40, fig. 13; pl. 41, fig. 15).
During the splitting process the free ends of the V’s begin to migrate

456 University of California Publications in Zoology [Vou 20

toward opposite poles of the endosome. In the next stage observed,
the chromosomes are arranged in the so-called equatorial plate (pl. 41,
figs. 16, 17, 20, and 21), in which they are grouped around the endo-
some parallel to its long axis. From the observed behavior of the
chromosomes, it seems probable that the equatorial plate is formed
by an unfolding of the V-shaped chromosome pairs, the free ends
of which have migrated to opposite poles of the endosome while the
other ends remain attached in the plane of the equator.

ANAPHASE

In the early anaphase (pl. 41, fig. 18) the daughter chromosomes
begin to separate in the equatorial plane, resulting at a slightly later
stage (pl. 41, fig. 19) in complete separation of the two groups of
daughter chromosomes. Further elongation of the nucleus now
begins, with the chromosomes still retaining their parallel relation
to the endosome. Constriction of the nucleus into two daughter
nuclei occurs and these begin to pull apart, still connected by the
middle portion of the endosome (pl. 41, figs. 22 and 23). This con-
necting portion becomes thinner and thinner as the nuclei become
further separated, until finally constriction is completed at the end
of the anaphase (pl. 41, fig. 24). The nuclear membrane remains
intact throughout this stage.

TELOPHASE

After the connecting thread of the endosome has broken (pl. 41,
fie, 24), the daughter nuclei begin to round up (pl. 41, fig. 25). The
two new endosomes shorten and reassume their spherical shape, and
the parallel arrangement of the chromosomes is replaced by a spiral
organization comparable with that of the prophase. This reorgan-
ization process is continued until typieal resting nuclei are formed
in the daughter flagellates with the completion of binary fission.

There is no adequate evidence here for a precocious splitting of
the chromosomes in either the anaphase or the late telophase, as in
Euglena (Tschenzoff, 1916). If such were the ease, the metaphase
phenomena would involve merely the separation of previously formed
daughter chromosomes; but in Menoidium incurvum the actual split-
ting seems to occur in the metaphase.

1923 ] Hall: Binary Fission of Menoidium incurvum 45

~]

BINARY FISSION

Binary fission is initiated by division of the blepharoplast, rhizo-
plast, and granule, or extranuclear centrosome, at the base of the
rhizoplast; later two flagella are seen. Whether the second flagellum
arises from the old by splitting, or as a new outgrowth from the
second blepharoplast with the old flagellum retained by the first,
could not be determined. Although outgrowth of the new flagellum
is to be expected, there is some indication of splitting beginning at
the blepharoplast and proceeding distally (pl. 40, figs. 10 and 12).
Jt is more probable, however, that this appearance is produced by
outgrowth of one flagellum in close conjunction with the other.
A similar ease is described by Kofoid and Christiansen (1915))
in Giardia, where the new flagella arise as outgrowths from the
blepharoplast, each new flagellum following the intracytoplasmic path
of the corresponding old one and emerging at its side. In Menoidium
the two flagella are at first closely associated, as shown in figure 14
(pl. 41), in which the two blepharoplasts are still close together and
the two flagella appear almost fused in most of their course through
the gullet.

In Heteronema acus, Rhodes and Kirby (MSS) find that the new
flagella arise as outgrowths. Dobell (1908) states that in Copromonas
the old flagellum is drawn in on the approach of mitosis and, after
the division of the blepharoplast, a new flagellum grows out from
each daughter blepharoplast. Steuer (1904) records a splitting of
the flagella in Hutreptia. Hartmann and Chagas (1910) describe
the outgrowth of a new flagellum from each daughter blepharoplast
in Peranema. Schiissler (1917) states that in Scytomonas the old
flagellum may be retained by one daughter blepharoplast and the
other produced by outgrowth, or that both flagella may arise by
outgrowth as described by Dobell (1908). Thus, from the published
accounts it would seem possible that the new flagella may be formed
differently in the various euglenoids, but this is rather improbable ;
the origin of new flagella by outgrowth seems best supported.

Several observers (Wager, 1899; Hamburger, 1911) have described
the flagella of various euglenoids as bifureating, after entering the
reservoir, into two fibers, each of which ends in a basal granule or
blepharoplast. Haase (1910) figures the flagellum as bifureating
at the level of the eyespot, and the resulting fibers as passing back-

458 University of California Publications in Zoology [Vou. 20

ward where they end posterior to the nucleus in a single basal granule.
Since there are cases in Menoidium which present such an appearance
as that described by Wager, and also instances in which the flagellum
arises from only one blepharoplast, it seems quite possible that such
a double condition is produced either by the splitting of an originally
single structure, or else by the beginning of outgrowth of the new
flagellum. Also, Dobell (1908), Berliner (1909), Bélar (1916),
Schiissler (1917), and Rhodes and Kirby (MSS) find only one
blepharoplast for each flagellum.

PARADESMOSE

As the blepharoplasts become further separated in the late meta-
phase and early anaphase, they are seen to be connected by a parades-
mose (pl. 41, figs. 17-19). This structure was first described by
Kofoid and Swezy (1915a) in Trichomonas as a ‘‘chromatie thread
which lies outside of the nuclear membrane.’’ Subsequent papers
(Kofoid and Christiansen, 1915a, b; Kofoid and Swezy, 1915b, 1919a,
b, c, 1920, 1922; Swezy, 1915a, b; Boeck, 1917; Cutler, 1919; Rhodes,
1919) have described such a structure in many different flagellates,
and it is probable that a paradesmose will prove to be a common
characteristic of mitosis in flagellates. It should be stated, however,
that in most cases the paradesmose is an exceedingly delicate structure
and is very difficult to stain and determine; hence, many observers
have failed to find it.

In Copromonas subtilis, Dobell (1908, pl. 5, figs. 44 and 45)
figures a fine strand connecting the two daughter ‘basal granules’
at division; this structure resembles very closely the paradesmose
found in Menoidium, so that it is probably permissible to assume that
Copromonas also possesses a paradesmose. Dobell, however, attributes
no significance to this structure, merely stating that ‘‘the basal granule
then divides, becoming dumb-bell shaped, and finally being constricted
into two daughter-granules.’’ Yet Dobell finds no rhizoplast in
Copromonas. It is probable, however, that a more adequate staining
technique would have diselosed such a structure, since Berliner (1909)
figures a rhizoplast in Copromonas major.

In Euglena agilis (fig. B) I have recently found a paradesmose
similar to that in Menoidium; here also this structure is spun out
between the two blepharoplasts after their division.

1923 | Hall: Binary Fission of Menoidium incurvum 459

In Menoidium, as stated above, the paradesmose is spun out
between the two daughter blepharoplasts. In Trichomonas Kofoid
and Swezy (1915a) describe in mitosis the division of each blepharo-
plast into two new structures, a basal granule (blepharoplast proper)
and a centrosome ; while the flagella and paradesmose remain attached
to basal granules, or blepharoplasts proper, the centrosomes become
located outside the nuclear membrane at opposite ends of the spindle
and connected to the blepharoplasts by the nuclear rhizoplasts. In
such a case as this the original blepharoplasts would be considered
centroblepharoplasts (Kofoid and Swezy, 1919a), since they divide
to form both centrosomes and blepharoplasts. In other flagellates,
such as Giardia (Kofoid and Swezy, 1922) the blepharoplast is
connected by a rhizoplast to a centrosome just outside the nuclear
membrane; during mitosis in Giardia the paradesmose is drawn out
between the two extranuclear centrosomes. Thus there are two types
of paradesmoses, differing in location; in Giardia, the paradesmose
connects the centrosomes, whereas in Trichomonas, in which the
centrosome originates from the centroblepharoplast in mitosis, the
paradesmose remains connected to the two blepharoplasts. It is quite
evident that the paradesmoses of Menoidium incurvum and Euglena
agilis are homologous with the type found in Trichomonas, in that both
of them are drawn out between the two daughter blepharoplasts,
rather than with the paradesmose seen in Giardia. The origin of
the extranuclear centrosome from the blepharoplast (centroblepharo-
plast) at the beginning of mitosis has not yet been demonstrated in
Menoidium, but from the essential similarity in structure to that of
Trichomonas it might be expected that the blepharoplast of this
flagellate may be shown eventually to be a centroblepharoplast.

GULLET AND RESERVOIR

With the progress of mitosis, the gullet and reservoir begin to
prepare for fission; the first step is the general widening of both
structures (pl. 41, fig. 17). Then, as the flagella move farther apart
the reservoir becomes divided into two lateral portions (pl. 41, figs. 16,
18, and 19), each of which is to give rise to a new reservoir. At a
later stage (pl. 41, figs. 22-25), the median plane of fission has con-
stricted the gullet and reservoir into two daughter structures, one
in each new flagellate.

460 University of California Publications in Zoology [Vou 20

After the separation of the two daughter nuclei in the early
telophase, binary fission is carried to completion and two daughter
flagellates are formed. The flagellates appear to swim about actively
throughout the whole process of mitosis. Since eysts could not
be identified, there is no evidence for division during encystment,
although it is quite possible that, as in Euglena (Tschenzoff, 1916;
Zumstein, 1899), such a process may occur.

DISCUSSION

As Hartmann and Chagas (1910) have suggested, the euglenoid
nucleus is probably more easily studied than that of any other group
of flagellates; yet the literature contains very few good accounts
such as that of Tschenzoff (1916), for example, and the different
authors hold conflicting viewpoints with regard to the structure of
the nucleus and the nature of nuclear division. Mitosis of one sort
or another has been deseribed by Klebs (1892), Blochmann (1894),
Keuten (1895), Dangeard (1902), Prowazek (1903), Berliner (1909),
Hartmann and Chagas (1910), Haase (1910), Bélar (1916), Tschen-
zoff (1916), Schiissler (1917), Dehorne (1920), and Gard (1920).

Amitosis has been described in a few forms, for example, Hutreptia
(Steuer, 1904) and Copromonas [Scytomonas?] (Dobell, 1908). As
for Copromonas, Berliner (1909) finds chromosome formation in well
stained preparations of Copromonas major; also, Schiissler (1917)
describes in Scytomonas chromosome formation in properly destained
haematoxylin preparations, and states that an appearance of amitosis
is Seen in overstained preparations; however, these chromosomes were
interpreted by Schiissler as being in the karyosome. Hence, it is quite
possible that Dobell’s account of amitosis in Copromonas subtilis 1s
the result of inadequate staining technique. Dobell (Dobell and
O’Connor, 1921) has recently modified his original designation of
nuclear division in this form to ‘‘amitosis or a simple form of
mitosis.”

Sexual phenomena have been deseribed in a few instances (Weisse,
1856: Dobell, 1908; Berliner, 1909; Haase, 1910), but such deserip-
tions do not rest upon critical cytological evidence and are not to
be accepted without confirmation. In Copromonas, Dobell asserts
that two flagellates become fused at their anterior ends, the union

1923 | Hall: Binary Fission of Menoidium incurvum 461

extending gradually to complete fusion. Two ‘‘reduction divisions’’
oeeur, producing in each conjugant a ‘‘reduced’’ nucleus and two

?

“‘reduction’’ nuclei, the latter comparable with the polar bodies of

oogenesis; the “‘reduced’’ nuclei unite to form a ‘‘zygote nucleus.’’
The flagellum of one flagellate is drawn in, while that of the other
persists, serving to keep the precocious pair in locomotion. After
fusion, this ‘zygote’ animal is said to have its choice of remaining
a single large flagellate which will later divide, or of eneysting. While
it is probable that Dobell really observed an apparent fusion, the
question remains as to whether or not such behavior is to be interpreted
as isogamy. Occurring as it does under such conditions that ‘‘Hven
in my most healthy cultures a number of monads always underwent
degenerative changes and died’’ (Dobell, 1908, p. 102), and that ‘‘one
can often observe degenerating monads side by side with perfectly
healthy individuals in active division or conjugation’’ (Dobell, 1908,
p. 103), the interpretation of this process as conjugation must remain
subject to criticism. Berliner’s (1909) deseription of isogamy in
Copromonas major is quite similar, so that to both of these reports
Dobell’s criticism (Dobell and O’Connor, 1921) of Woodeock’s de-
scription of conjugation in Cercomonas might appropriately be
applied:

. .. believes he has observed conjugation in... I have not done so, and
consider—from the account published—that there is little or no evidence that

conjugation oceurs. The phenomenon observed... appears rather to be an
abortive or regressive fission. .. .

Weisse (1856) describes small individuals of Huglena, or ‘‘Sperm-
atozoids,’’ which penetrate the cysts of larger individuals of Euglena,
thus effecting fertilization. Haase (1910) describes the formation of
gametes in Huglena sanguinea by an intrakaryosomal mitosis, and
the union of free gametes to form a zygote; the proof offered by her
figures is entirely inadequate, however, and her interpretation is
evidently at fault. With such little evidence for sexual behavior,
the occurrence of sexual reproduction in the euglenoids must, for the
present at least, remain doubtful. Other points of controversy have
been the nature of the outer chromatin and its behavior in division,
and the nature of the endosome—its function in division, and whether
or not it contains a centriole.

462 University of California Publications in Zoology [Vou 20

Tue Resting Nucieus

Tschenzoff (1916) describes the resting nucleus of Euglena viridis
as containing a ‘‘ Binnenkdrper’’ around which is found the
chromatische Substanz in Form von feinsten Bréckehen und Fadechen. Manchmal
tritt mehr brockliche Beschaffenheit des Chromatins, manchmals feinfaidige zutage.
Manchmal die Chromatinkérnen auf einer Netwerk aufgereiht zu sein. ... Ein

geschlossenes Webenwerk konnte ich nicht feststellen, wenn auch das Vorhandsein
eines solches zu vermuten ist.

His description agrees with that of Bélat (1916) for Astasia, Hart-
mann and Chagas (1910) for Peranema, Steuer (1904) for Hutreptia,
and Biitschli (1883-1889) for euglenoids in general. Klebs (1883)
seems to have mistaken a nucleus in the early prophase for the typical
resting nucleus, since he describes a spireme structure in the nucleus
of Euglena. Keuten (1895) has also described a prophase, since he
mentions distinct chromosomes in the resting nucleus of Huglena.
Dangeard (1902) considers the nucleus of Huglena and other
euglenoids to contain a karyosome, around which is wound a long
chromatin thread, the coils of which present the appearance of
chromosomes. This concept of the euglenoid nucleus has recently
been revived by Dehorne (1920) in his work on Euglena.

The structure of the nucleus in Menoidium is similar to that
described for Huglena by Tschenzoff. In Menoidiwm, however, the
nuclear membrane appears to persist throughout mitosis, while
Tschenzoff states that in Euglena
eine echte doppelkonturierte Membran ist indessen auscheinend nicht vorhanden,

sie wird nur manchmal durch ein Kunstprodukt vorgetauscht. Wahrend des ganzen
Prozesses der Kernteilung wird sie regelmassig vermisst.

Yet in his figures the outline of the nucleus stands out sharply from
the cytoplasm, so that a membrane of some sort may well nave been
present. Dehorne (1920) finds a nuclear membrane persisting in rare
eases only. Dobell (1908) describes the nucleus of Copromonas as
bounded by a nuclear membrane and containing a karyosome, around

‘ ”

which there is a clear zone containing ‘‘achromatie granules’’ and
erossed by radial ‘‘linin threads’’; his ‘‘achromatie granules’’ evi-
dently correspond to the chromatin granules which give rise to the

chromosomes in the mitosis of other euglenoids.

1923 ] Hall: Binary Fission of Menoidium incurvum 463

THE ENDOSOME

The endosome, or ‘‘ Binnenkdrper’’ (Doflein, Tschenzoff), is one
of the least understood structures in the euglenoid nucleus. Some
authors ascribe to it an inactive réle in mitosis; others believe it to
be active in initiating division; while a few have deseribed it as con-
taining a centriole. With a few exceptions, such as Berliner (1909),
Haase (1910), Schiissler (1917), all are agreed that the endosome
has nothing to do with the formation of chromosomes. Since Haase,
however, describes also the formation of gametes by an intrakaryo-
somal mitosis and the union of gametes to form a zygote, her interpre-
tation is open to suspicion.

The question of the presence or absence of a centriole in the
endosome of euglenoids is not yet definitely settled. A centriole or
centrosome has been described by Berliner (1909), Hartmann and
Chagas (1910), Haase (1910), Bélar (1916), Schiissler (1917).
Schiissler describes ‘‘Centren’’ in the middle of the. ‘‘ Pseudopol-
korper,’’ which are formed at the poles of the endosome; he reverts to
the theory of chromatic dualism when he states that in

allen genauer untersuchten einfacher Protozoenkernen sich stets zwei gesonderte
Komponenten nachweisen lassen, eine lokomotorische und eine generative.

He considers that in the phylogeny of the euglenoids the more primi-
tive types, represented by Scytomonas, have both nuclear components
localized in the endosome, while in the higher forms such as Euglena
the generative function is invested in the outer chromatin. Berliner
(1909) believes that in the division of Copromonas major the old
basal granule degenerates or is drawn back into the nucleus, and that
new basal granules arise from the centrioles of the daughter nuclei;
from these “newly produced granules the flagella of the daughter
flagellates grow out. This is denied by Schiissler, and Dobell (1908)
also fails to find any indications of such a process. The cytological
evidence of Haase (1910) for the presence of centrioles is quite as
inadequate as that for her other fanciful interpretations, so that her
account is of little value. Béla? (1916), however, has deseribed and
figured a structure resembling a centriole in the endosome of Astasia
levis, and has traced it from the prophase through the anaphase.
Hartmann and Chagas (1910) figure a divided centriole in the endo-
some of Peranema during the prophase, but they have not followed
this structure through mitosis, as Béla? has done in Astasia, and their

464 University of Califorma Publications in Zoology [Vou. 20

cytological evidence seems insufficient; Alexeieff (1913) even accuses
Hartmann and Chagas of arbitrarily selecting a pair of granules and
““centrioles,’’ as other centriolists are said to have done.
The structure of the endosome in Menoidium supports the views

calling them

of Tschenzoff and others, since there is no evidence at all of a centriole
in the endosome. Since the only account worthy of careful consider-
ation is that of Bélar (1916), the probabilities seem rather against
the presence of a centriole in the endosome of all euglenoids.

Fragmentation of the endosome occurs in the mitosis of some
euglenoids, such as Euglena (Haase, 1910; Dangeard, 1902) and
Heteronema (Rhodes and Kirby, MSS) but such a phenomenon has
not been discovered in Menoidium. The significance of such a process
is not known.

Another interpretation of the endosome—the theory of the
nucleolo-centrosome—was advanced by Keuten (1895). He com-
pared the endosome (‘‘ Nucleolo-centrosom’’) with the centrosome and
central spindle of the diatoms, and with the intranuclear centrosome
of Ascaris (Brauer, 1893). This theory was supported by Schaudinn
(1896), who cited an interesting experiment on Oxyrrhis marina as
evidence of the nature of the endosome. In the nucleus of this form
there is normally a large endosome; when the flagellates were placed
in diluted sea-water the endosome left the nucleus and behaved as an
extranuclear centrosome. Steuer (1904) accepts the term nucleolo-
centrosome, but the more modern investigators have discarded this
conception, employing such terms as karyosome, ‘‘ Binnenkérper’’
(Tschenzoff, Doflem), and endosome (Minchin).

It has been suggested to me by Dr. Kofoid that a solution of the
endosome problem is to be sought in connection with the intranuclear
rhizoplast (Kofoid and Christiansen, 1915); Boeck, 1917). In many
flagellates activity within the nucleus is apparently correlated with
the activity of the extranuclear centrosome ; the physical bond between
these two structures is the intranuclear rhizoplast, connecting the
centrosome with the karyosome. There is a possibility that the endo-
some of the euglenoids may, in some way, be comparable with this
intranuclear rhizoplast ; future investigation may perhaps throw some
light upon this phase of the question.

1923 | Hall: Binary Fission of Menoidium incurvum 465

THE PROPHASE

Tschenzoff (1916) describes the prophase as beginning in Huglena
viridis when the chromatic substance begins to form distinct granules,
which later become arranged in rows to form chromatic threads. He
notes the appearance of an increasing number of chromatic granules
which lie at the nuclear surface and appear to crop out into the
eytoplasm, producing wrinkles in the nuclear membrane. These
mysterious granules are believed either to break up and pass out into
the cytoplasm, or else to take part in the formation of chromosomes.
Later the chromosomes become arranged more or less radially around
the endosome; with the elongation of this structure, the chromosomes
come to form a eylindrical ring parallel to its long axis. Dehorne
(1920), in a comparative study of the euglenoid and other protozoan
nuclei, comes to the conclusion that, as Dangeard (1902) believed,
the nucleus of Huglena contains in the prophase a continuous
chromatin thread; this ‘spireme’ is said to be composed of granules.
He bases his statement on the appearance of loops in the chromosomes
and the facet that he is unable to find free ends; however, his figures
do not aftord sufficient evidence to convince one of the accuracy of
such an interpretation.

In Menoidium these early processes are similar to those described
by Tschenzoff, with the exception that no granules, such as he men-
tions, have been observed. In many eases structures resembling these
eranules of Tschenzoff have been seen, but they can always be resolved
into optical cross-sections of chromatin threads. It is thus possible
that Tschenzoff has mistaken optical sections of chromatin threads for
actual granules; such an explanation might account for most of these
struetures shown in his figures.

In connection with the formation of chromosomes in the prophase,
Sands (1922) presents some rather startling conclusions drawn from
his work on Tradescantia virginica. He states that in the so-called
resting stage, the genes attract to themselves “‘from the protoplasm,
materials of a similar kind, thus molding next to the original gene
another structure of similar parts, identically arranged, which then
become bound together to form another gene, a replica of the first,”’
and that these genes ‘‘after the genesis of their like, reassemble in such
a fashion that they form a continuous spireme thread.’’ He also states
that the chromosomes are formed in the prophase by segmentation of
a continuous spireme, and that no longitudinal splitting of the chromo-

466 University of California Publications in Zoology [Vou. 20

somes occurs. His views are such a radical departure from the com-
monly accepted conception of the status of the chromosomes that
they must be considered at present with some degree of skepticism,
but they are, to say the least, very interesting and should provide
an impetus to further critical investigation. His account, however,
does not seem to be supported by the mitotic phenomena described for
euglenoids.

Tur MerrarHAsE

In Euglena viridis, according to Tschenzoff, the chromosomes,
after being parallel to the endosome, come together in a thick equa-
torial zone, in which they are arranged at random with respect to
the axis of the endosome. Separation of the double chromosomes
observed at this stage begins at one end of the pairs, while the free
ends begin to migrate toward opposite ends of the endosome. Dehorne
(1920) finds in Euglena, at a stage approximating an early metaphase,
distinet longitudinal cleavage of separate chromosomes—‘‘ Le clivage
est trés surprenant chez un tel noyau, mais indubitable.’? At this
stage he is unable to find a continuous spireme, but he believes that
the free ends of the chromosomes later fuse again to form a single
thread. This formation of separate chromosomes, their longitudinal
cleavage, and the re-formation of the spireme occur prior to the
anaphase, so that the separation of the equatorial belt imto two
daughter groups is due to a “‘division transversale’’ of the spireme.
This interpretation of Dehorne is in marked contrast to that of
Tschenzoff (1916), who finds in Huglena viridis longitudinal division
of the chromosomes in a true mitosis, and no indications of a con-
tinuous spireme. When one considers that Dehorne, in the paper
under consideration, is bent upon tracing similarities in the nuclei
of ciliates, euglenoids, and Cyanophyeceae, his interpretations may
be suspected to have been influenced somewhat by his underlying
hypothesis.

From the behavior of the chromosomes in Menoidium, it is con-
eluded that the procedure in the metaphase is shghtly different from
that deseribed by Tschenzoff for Huglena. The thickened chromo-
somes of the late prophase appear to remain radially arranged with
respect to the axis of the elongating endosome. In the early meta-
phase, splitting apparently begins at one pole of these thick chromo-
somes and proceeds toward the other, while the free ends of the
daughter chromosomes begin to move toward opposite ends of the
endosome. Although no evidence for such a condition has been

1923 | Hall: Binary Fission of Menoidium incurvum 467

found, these chromosomes of the late prophase might, in reality, be
paired chromosomes which have already split at an earlier stage, as
described by Tschenzoff in Euglena viridis; if such were the case,
the metaphase ‘‘splitting’ would be merely the separation of pre-
viously formed daughter chromosomes. ‘Tschenzoff finds that in
Euglena the chromosomes split in the telophase or late anaphase,
while the daughter chromosomes so formed pair in the prophase of
the next mitosis and are permanently separated in the metaphase.
Such a process could not be confirmed in Menoidiwm; however, it
should be stated that the nucleus of this flagellate is much smaller
than that of Huglena, so that it may be that such a process occurs
and has not yet been detected.

The migration of the free ends of the V-shaped chromosome pairs
during the metaphase in Menoidium results in the formation of the
ring of chromosomes parallel to the endosome (‘‘equatorial plate
stage’’) ; in such a ease the ring is formed by the paired daughter
chromosomes which have unfolded but still remain attached at the
equator. This is supported by the condition shown in plate 41,
figures 16 and 17, in which a split has not yet appeared in the plane
of the equator through the end-to-end union of the daughter chromo-
somes. In slightly later stages (pl. 41, figs. 18 and 19) a split appears,
while the chromosomes are still parallel to the endosome. <A similar
process of equatorial plate formation is described by Kofoid and
Swezy (1919b) in Trichonympha campanula:

. with the beginning of the formation of the spindle fibres, or somewhat
earlier, another change takes place in the chromosomes, the loops straightening
out so that the chromosomes come to lie parallel to the paradesmose. . . . The com-
pletion of this gives the equatorial plate phase, with the chromosomes still joined
by an end to end union in the equatorial plane.

ANAPHASE AND TELOPHASE

Tschenzoft’s description of the later stages of mitosis in Euglena is
in accord with the observed phenomena in Menoidium, except for the
fact that a splitting of the chromosomes in the telophase or late
anaphase could not be determined in the latter. Dehorne (1920)
states that, in the late anaphase, the separate segments of the spireme
produced by the transverse division of the metaphase stage reunite
to form the continuous spireme characteristic of the prophase; in such
flagellates as Euglena, however, where the chromosomes are closely
massed together in the anaphase, it is difficult to understand how one
could be certain that such a structure exists.

468 University of California Publications in Zoology [Vou. 20

SUMMARY

Menoidium incurvum is a small saprozoie euglenoid characterized
by a rigid body marked on the surface with from ten to fifteen widely
separated longitudinal striations; the body contains a number of
nearly colorless plastids, or paramylum bodies, located usually at
the anterior end, although occasionally at both ends. The gullet and
reservoir are similar in structure to those of other euglenoids, but
are not used for ingestion of solid food.

The neuromotor apparatus is a simple system consisting of a
flagellum ending in a blepharoplast, from which a rhizoplast extends
to the nucleus. The granule, or centrosome, at the base of the rhizo-
plast in Menoidiwm, and also in Euglena agilis, is similar to the extra-
nuclear centrosome of such forms as Giardia and Trichomonas. Dur-
ing mitosis the daughter blepharoplasts remain connected for a time
by a paradesmose; this is similar in both Menoidiwm and Euglena
agilis to that of Trichomonas. There is some indication of a splitting
of the old flagellum instead of the expected outgrowth in mitosis,
but it is more probable that there is an outgrowth of the new flagellum
in close conjunction with the old.

The resting nucleus contains an endosome surrounded by chro-
matin granules in the nodes of a linin network; during mitosis these
granules become organized into distinct chromosomes, whose number
is tentatively determined to be twelve. The equatorial ring or plate
of the late metaphase is apparently produced by an unfolding of the
V-shaped chromosome pairs formed by the metaphase split; the free
ends of the V’s migrate to opposite poles of the endosome, leaving
their other ends attached in the plane of the equator. At this stage
the chromosomes are parallel to the long axis of the endosome, and
they remain so until the late anaphase. Splitting of the chromosomes
occurs in the metaphase; a precocious splitting in the preceding telo-
phase is possible, but there is no evidence whatever that this occurs.
The endosome contains no centriole at any stage of mitosis. The
nuclear membrane persists throughout nuclear division.

Eneysted forms of Menoidiwm have not been identified in either
living or stained material.

1923 | Hall: Binary Fission of Menoidium incurvum 469

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Cuarton, E.

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DANGEARD, P. A.

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pls. 1-4.
DEHORNE, A.

1920. Contribution 4 1’étude comparée de |’appareil nucléaire des Infusiores
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DoBELL, C. C.

1908. The structure and life-history of Copromonas subtilis n.g., n.sp.: A
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470 Unwersity of California Publications in Zoology [Vou. 20

*DOBELL, C., and O’Connor, F. W.
1921. The intestinal Protozoa of man (London, J. Bales, Son and Danielsson),
ix+211 pp., 8 pls.
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HAMBURGER, C.
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KHAWKINE, W.
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1923 ] Hall: Binary Fission of Menoidiwm incurvum 471

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EXPLANATION OF PLATES

Figures 6-12, 14, 16-19, 22, and 25 are drawn from material killed in Schau-
dinn’s fluid and stained in 0.1 per cent Bordeaux red preceding iron-alum
haematoxylin. Magnification X 2100 (except fig. 2).

PLATE 40

Fig. 1. Camera lucida sketch of unstained flagellate, showing longitudinal
striations, plastids, gullet, and flagellum.

Fig. 2. Diagrammatic enlargement of a camera lucida sketch. Note increase
in number of striations with the preparation for binary fission.

Fig. 3. Posterior end of flagellate showing fourteen striations converging to
a point.

Fig. 4. Anterior end; striations extend to edge of cytostome.

Fig. 5. Optical section through posterior end; the striations appear to be
elevated ridges in the pellicle.

Fig. 6. Unstained flagellate, showing plastids at both anterior and posterior
ends.

Fig. 7. Resting nucleus, showing aggregation of chromatin particles as the
prophase approaches.

Fig. 8. Very early prophase; chromatin in form of fine threads irregularly
coiled around the endosome. The ends of several threads can be seen.

Fig. 9. Nucleus in prophase; chromosomes show radial grouping around
endosome. Flagellum, blepharoplast, and rhizoplast are also shown.

Fig. 10. Surface view of nucleus at about same stage as that shown in figure 9.
Note appearance of two blepharoplasts; only one rhizoplast could be traced.

Fig. 11. Nueleus shows thicker chromosomes of later prophase.

Fig. 12. Note the two blepharoplasts and rhizoplasts and the apparent split-
ting of the proximal part of the flagellum; this probably represents the beginning
of outgrowth of the new flagellum. Chromosomes are still radially arranged, but
appear to have increased in number with the beginning of metaphase splitting.

Fig. 13. Later stage than figure 12. Nucleus shown in end view at about same
stage shown in figures 14 and 15. Several V-shaped chromosome structures are
seen.

[474]

UNIV. CALIF. PUBL. ZOOL. VOL. 20

[HALL] PLATE

40

PLATE 41

Fig. 14. Semi-composite view of nucleus in metaphase; the endosome has
elongated, and its upper end is at a deeper focal plane than the lower. The
rhizoplasts could not be traced from the blepharoplasts.

Fig. 15. Nucleus elongated in later metaphase; endosome now extends from
one end of the nucleus to the other, and the free ends of the chromosome pairs
have begun to migrate.

Fig. 16. Nucleus in late metaphase, or ‘equatorial plate’ stage. The free
ends of the daughter chromosomes have now reached the poles of the endosome,
but the other ends are still attached at the equator. The reservoir and gullet have
widened with the migration of blepharoplasts and flagella.

Fig. 17. Nucleus in ‘equatorial plate’ stage. The blepharoplasts are con-
nected by a paradesmose, and from each a rhizoplast extends to the nucleus.

Fig. 18. The ends of the daughter chromosomes are beginning to separate
at the equator in the early anaphase. Blepharoplasts still connected by the
paradesmose.

Fig. 19. Nucleus in early anaphase, showing complete separation of the
daughter chromosomes; the daughter groups are connected by the middle portion
of the endosome. Blepharoplasts still connected by the paradesmose.

Fig. 20. End view of nucleus at approximately the stage shown in figures 17
and 18.

Fig. 21. Optical section of the nucleus shown in figure 20, at a plane about
one-third the distance from the upper end. Note, with figure 20, the parallel
arrangement of the chromosomes.

Fig. 22. Later anaphase; the daughter nuclei are distinctly constricted; the
chromosomes are still parallel to the endosome.

Fig. 23. Later anaphase, showing further separation of the daughter nuclei.

Fig. 24. Early telophase. Constriction is just completed. The chromosomes
of the daughter nuclei are still parallel to the endosomes.

Fig. 25. Later telophase. The daughter nuclei have rounded up and regene-
ration has begun. Cell division is nearly completed.

[476]

UNIV. CALIF. PUBL. ZOOL, VOL. 20 [HALL] PLATE

41

MW
16 : Le Nene

UNIVERSITY OF CALIFORNIA PUBLICA TIONS— (Continued)
11. The Life Cycle of Echinostoma revolutum (Froelich), by John C. Johnson.

if : 18. Demonstration of the Function of the Neuromotor Apparatus in Euplotes

_ Vol. 20. 1. Studies on the Parasites of the Termites. I. On Streblomastix striz, a Poly-

ree mastigote Flagellate with a Linear Plasmodial Phase, by Charles Atwood
oe Kofoid and Olive Swezy. Pp. 1-20, plates 1-2, 1 figure in text. July, 1919 .25

am os 2. Studies on the Parasites of the Termites. II. On Yrichomitus termitidis, a

; Polymastigote Flagellate with a Highly Developed Neuromotor System,

a by Charles Atwood Kofoid and Olive Swezy. Pp. 21-40, plates 3-4, 2
PAROS MN COK Gs NY 1 ORO ce cee NR Ne Sah SI pee ON 25

» Studies on the Parasites of the Termites. III, On Trichonympha campanula

iJ)

Bole VAtieures AN CeXt.. veh; LOLO 2 ce ee ee Se Bi Ree
4, Studies on the Parasites of the Termites. IV. On Leidyopsis sphaerica gen.

5,

6. A Critical Review of the Nomenclature of Human Intestinal Flagellates,
Cercomonas, Chilomastiz, T'richomonas, and Giardia, by Charles. A. Kofoid.
Pp. 145-168, 9 figcures’in text... June, 1920). Se ee RAS 365
7. On the Free, Encysted, and Budding Stages of Councilmania lafleuri, a Para-
sitic Amoeba of the Human Intestine, by Charles Atwood Kofoid and
Olive Swezy. Pp. 169-198, plates 18-22, 3 figures in text. June, 1921 .._. -60
8. Mitosis and Fission in the Active and Encysted Phases of Giardia enterica
(Grassi) of Man, with a Discussion of the Method of Origin of Bilateral
Symmetry in the Polymastigote Flagellates, by Charles A. Kofoid and
Olive Swezy. Pp: 199-234, plates 23-26, 11 figures in text. March,

fn 12 eee ARS aC FO oer Ra ag « toe A Ae SO BO) ie a fi 50
9, The Micro-Injection of Paramaecium, by Chas: Wm. Rees. Pp. 235-242,

91 0) ar Pom AS (2) 7a eae aac ra Oe OR Seen eee C08 CL aI SE Ad 20
10. On Balantidiwm coli (Malmsten) and Balantidium suis (sp. nov.), with
~ an account of their neuromotor apparatus, by J. Daley McDonald.

3 Pp. 243-300, plates 27, 28, 15 figures in text. May, 1922 200. 1,00
? 11. Mitosis in Endamoeba dysenteriae in the Bone Marrow in Arthritis de-
oe formans, by Charles Atwood Kofoid and Olive Swezy. Pp. 301-307,

wo Be : 7 figures in text.

Bayt 12. Endamoeba dusenteriae in the Lymph Glands of Man in Hodgkin’s Disease,

by Charles A, Kofoid, Luther M. Boyers, M.D., and Olive Swezy. ‘Pp. 309-
$12, 4 figures in text.

Nos. 11 and 12 in one cover. April, 1922 oon... eece cece eeeeeereeee 26
®; 13. Mitosis in the Encysted Stages of Endamoeba coli (Loesch), by Olive
1 Swezy..)-Pp.-313-332; plates 29-31. “May, 1922-2... eee 30
: 14, The Neuromotor Apparatus of Paramaecium, by Charles William Rees.
Pp. 333-364; plates 32-36, 5 figures in text. November, 1922. 000.0.0 0. 45

15. A Comparison of the Cysts-of Hndamoeba coli and Councilmania lafleuri in
Congo Red, by Nathaniel Bercovitz, M.D. Pp. 365-371, February, 1923. .25
16. On the Morphology and Behavior of Pentatrichomonas ardin delteili. (Der-
rieu and Raynaud), by Charles Atwood Kofoid and Olive Swezy. Pp.
873-890, plate 37, 1 figure in text. August, 1923.20 cece 25
17. The Pseudopodial Method of Feeding by Trichonymphid Flagellates Para-
sitic in Wood-eating Termites, by Olive Swezy. Pp. 391-400, 14 figs. in
Oaths AT OTIS bee LOLS ce etal e Sac ae ta pat carol on eceoupueatte deca asbeusvmsectcochacees 25
18. Methods of Obtaining Amoeba-free Rats for Experimental Infection with
Intestinal Amoebae, by John F. Kessel. Pp. 401-408.
19. Experimental Infection of Rats and Mice with the Common Intestinal
Amoebae of Man, by John F. Kessel. Pp. 409-430, plates 38-39.
Nos. 18 and 19 in one cover, October, 1923-0222 2...22et eee eene eee 50
20. On the Genus Councilmania, Budding Intestinal Amoebae Parasitic in Man
and Rodents, by Charles A. Kofoid, Olive Swezy, and John F. Kessel.

ee Pp. 431-445, 19 figures in text. October, 1923 0 tec eee eee 25
i 21. Morphology and Binary Fission of Menoidiwm incurvum (Fres.) Klebs,
Pp. 447-476, plates 40-41, 2 figures in text. October, 1923 __...02-..-..... 50

21. Morphology and Binary Fission of Menoidiwm incurvum (Fres.) Klebs,
“Ye by Richard P. Hall. Pp. 447-476, plates 40-41, 2 figures in text. Novem-
a8 PR agli OR eT BRC ADS SP Se SO oa ORL ise URES pee BP Re eS 50

UNIVERSITY OF CALIFORNIA PUBLICATIONS— (Continued)

Vol. 21. 1. A Revision of the Microtus californicus Group of Meadow Mice, by Reming-
ton Kellogg. Pp. 1-42, 1 figure in text. December, 1918 -.2.o2

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

Py. 43-47. 5 iarch 191 Oh eo rs a) tn ee ae
8. Notes on the Natural History of the Bushy-tailed Wood Rats of California,

by Joseph Dixon. Pp. 49-74, plates 1-3, 3 figures in text. December, 1919 ~

4, Revision of the Avian Genus Passerella, with Special Reference to the Dis-
tribution and Migration of the Races in California, by H. S. Swarth. Pp.
75-224, plates 4-7, 30 figures in text. September, 1920

5. A Study of the California Jumping Mice of the Genus Zapus, by A. Brazier

Howell, Pp. 225-238, 1 figure in text. May, 1920). 2 wna.
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Wovember, 1921 hes a a ee ee
7. A Study of the Californian Forms of the Microtus montanus Group_ of
Meadow Mice, by Remington Kellogg. Pp. 245-274, 25 figures in text.

8. A Synopsis of the Microtus mordax Group of Meadow Mice in California,

by Remington Kellogg. Pp. 275-302, plate 8, 29 figures in text.
Nos.. 7~ard-8>in- one cover... April, 1922 25 oro aac eccoeseee

9. Behavior of the Leaf-nosed Snake, Phyllorhunchus decurtatus, by Sarah

Rogers. Atsatt.. Pp. 303-312. February, 1923 202.0. ne
10. A Systematic List of the Mammals of Fon ao by Joseph Grinnell.
Pp. 313-324. January, 1923.
11. The Systematic Status of the Mountain Hon of California, by Joseph

Grinnell and Joseph Dixon. Pp. 325-332, plates 9-10. April, 1923.02..

12. New Subspecies of Birds from Patagonia, by Alexander ‘Wetmore: Pp.
935-337. <JSUNO, 1923 SS Sa eee

Vol. 22.1. A Quantitative and Statistical Study of the Plankton of the San Joaquin

River and Its Tributaries in and near Stockton, California, in 1913, by

Winfred Emory Allen. Pp. 1-292, plates 1-12, 1 figure in text. June, 1920.
2. Variations in the Shell of Teredo navalis in San Francisco Bay, by Robert

Cunningham Miller. Pp. 293-328, plates 13-17, 6 figures in text. Novem- sak

TROT 5 AGA a ee a a a Sa a ra ee

8. Quantitative Studies on Marine Phytoplankton at La Jolla in 1919, by

Winfred Emory Allen. Pp. 329-347, 2 figures in text. November, 1922
4. On the Effect of Low Salinity on Teredo navalis, by Harold Francis Blum.
Pp. 349-368, 4 figures in text. December, 1922 22.20 cece eset

5. Quantitative Studies on Inshore Marine Diatoms and Dinoflagellates of ve

Southern California in 1920, by -Winfred Emory Allen. Pp. 369-378,
1 figure in text. : ;

by Christine E. Essenberg. Pp. 379-381, 8 figures in text.

Nos. 5 and 6 in one cover. December, 1922 2.02.2

1

. The Digestion of Wood by Teredo navalis, by Walter H. Dore and Robert
C. Miller. Pp. 383-400, plate 18. February, W923 Lae

. Variations in the Pallets of Teredo navalis in San Francisco Bay, by Robert
Cunningham Miller. Pp. 401-414, plates 19-20. February, 1923 2.220.

9. Some Tide-Water Collections of Marine Diatoms taken at Half-Hour Inter-

vals near San Diego, California, by Winfred E. Allen. Pp. 413-416, -

1 -figure:in text. dune, 923 ee

10. Preliminary Statistical Report on the Occurrence of Marine Copepoda in Z
the Plankton at La Jolla, California, by Calvin O. Esterly. Pp, 417-483.

Fh (= Mage 82.73 Salle ar eee AU I Pe mse SEN nah ae De EE pa ES
11. Studies on Marine Diatoms and Dinoflagellates Caught by Aid of the
Kofoid Bucket in 1922, by Winfred Emory Allen. Pp. 435-445, 5 figures
in text; - October: 1929°). 23 SS 5 niga x
12. Statistical Studies of Marine Diatoms of the San Diego Region Collected
by U.S.S. Pioneer in Midwinter of 1923, by Winfred Emory Allen. Pp.

Aa5-258..Octeber, 1923). 2 se ee Bes

18. Life Cycle of Oithona nana Reared Experimentally, by Helen E. Murphy.

Pp. 449-454, 5 figures in text. November, 1928 o-oo. eee ieceectteeeatee
Vol. 23. The Marine Decapod Crustacea of California, by Waldo L. Schmitt. Pp.
1-470, plates 1-50, 165 figures in text. May, 1921 ~... ee rs

Vol 24. 1. A Geographical Study of the Kangaroo Rats of California, by Joseph
Grinnell, Pp. 1-124, plates 1-7, 24 figures in text. June, 1922

2. Birds and Mammals of the Stikine River Region of Northern British ©

Columbia and Southeastern Alaska, by H. 8. Swarth. Pp. 125-314, plate

8, 34 figures in texts. Dume, 1922 anna conc cceaecetncententerncsnennecoennmereace

. Stylarioides papillosa, sp. nov., a New Annelid from the San Diego Region,

6 |

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SKIN" REACTION TO EXTRACTS OF LEISHMANIA
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UNIVERSITY OF CALIFORNIA PUBLICATIONS
1N

ZOOLOGY

Vol. 20, No. 22, pp. 477-488, plate 42 December 31, 1923

A SKIN REACTION TO EXTRACTS OF LEISHMANIA
TROPICA AND LEISHMANIA INFANTUM

BY

EDNA HANNIBAL WAGENER

INTRODUCTION

The cutaneous reaction has been so largely and suecessfully used
im demonstrating hypersensitiveness to protein derivatives of both
fungi and bacteria that it seemed possible that a similar reaction
might be obtained with protein derivatives of protozoans. Some work
has been done in this field of investigation, but thus far no conclusive
results have been published. (See discussion at close of this paper.)

The selection of a favorable protozoan for experimentation offered
several difficulties as apparently none of the intestinal forms has
been grown in pure culture. Finally, however, Leishmania was found
to fulfil the requirement; also to grow readily in quantity sufficient
for the essential experimental tests with derived proteins.

ACKNOWLEDGMENTS

This investigation has been carried on under the direction of
Professors J. N. Foree and Charles A. Kofoid, to whom thanks are
due for kindly suggestions and eriticism. Acknowledgments are also
made to Professor F. E. Novy, of the University of Michigan, for
cultures of Leishmania tropica, to Professor EH. E. Tyzzer, of Harvard
University, for cultures of Leishmania infantum, to Professor T. D.
Beckwith for valuable suggestions during the course of the work,
and to Dr. Olive Swezy, for the excellent illustrations accompanying
the paper.

478 University of California Publications in Zoology (Vow. 20
gy

MATERIAL AND TECHNIQUE

Cultures of Leishmania tropica and Leishmania infantum were
grown on N.N.N. media made up in the proportions of 1.4 per cent
agar and 0.5 per cent sodium chloride in distilled water. This was
adjusted to pH 7.8, was tubed, autoclaved, and stored until needed.
One day before being used 30 per cent of defibrinated rabbit blood
was added and the tubes incubated overnight, to test them for
sterility of the culture media. The tubes were then inoculated and
the cotton plugs covered with rubber caps to prevent evaporation.
After 10 to 12 days’ incubation at room temperature, the surface of
the media was found to be covered with minute, dewdrop colonies
of the flagellates. This growth was rubbed off with a platinum loop
into 1 ¢.c. of normal saline and decanted into a centrifuge tube. The
washings from 15 to 20 cultures of each species were centrifuged
separately for half an hour at 1800 revolutions a minute and the
supernatant fluid decanted off. The organisms in the bottoms of
the tubes were then diluted with normal saline to which 0.25 per cent
phenol had been added until each ecubie centimeter contained
1,500,000 organisms by direct count in a blood-counting chamber.

With the suspension thus prepared, two rabbits were immunized
to each of the two species of Leishmania by intravenous injections
of 2 ¢.c. each at three day intervals. Seven days after the last injec-
tion the rabbits were bled from the ear and macroscopic tests made
for the-presence of agglutinins. All four rabbits showed complete
agglutination of the specific flagellate at a 1 to 980 dilution of the
serum. These results were considerably higher than those of Bandi
(1913), who found that immunized rabbits would agglutinate the
specific strains of Leishmania infantum at a 1 to 160 dilution of the
serum.

A leishmaniosin was prepared, following the technique used by
Force and Stevens (1917) in the preparation of typhoidin. This
consisted in washing off the growth from about 50 cultures of each
species with 1 ¢.c. of saline, precipitating the protein with 95 per cent
alcohol, washing with ether, and drying the residue over concentrated
sulphurie acid in vacuum. As a small amount of haemoglobin from
the culture media was dissolved in the saline, a control was prepared
from washings from sterile culture media. About 0.1 gm. of dry

1923] Wagener: Leishmania tropica and Leishmania infantum 479

powder was obtained from each species and from the control. Unlike
the typhoidin, however, these dry powders would not go into solution
in saline, and no reaction occurred in the rabbits from the intra-
dermal injection of 0.2 ¢.c. of the suspension of 1 part of powder in
100 parts of saline. It was therefore decided to grind each of the
powders in a mortar, while adding slowly a weakly alkaline solution
such as that recommended by Coca (1922) for the extraction of
proteins from pollens and danders. So 100 parts of the extracting
fluid of Coca, consisting of sodium chloride 0.5 per cent, sodium
bicarbonate 0.05 per cent, and earbolic acid 0.4 per cent, in sterile
distilled water were added to one part of dry powder. The suspen-
sions were then placed in sterile containers, covered with toluol, and
allowed to stand at room temperature for three days. Unfortunately,
there was not a sufficient quantity of the extracted proteins to enable
us to follow Coca’s technique farther and determine the nitrogen
content of the extract before using it. These aleoholie extracts will
be designated alcoholic extracts of L. tropica and L. infantum.

At the same time a number of cultures of each species of Leish-
mania were washed off in saline, the washings centrifuged, and the
protozoans in the bottom of the tube diluted with Coca’s extracting
fluid until 1 ¢.c. contained 2,000,000 organisms. These suspensions,
spoken of hereafter as alkaline extracts of L. tropica and L. infantum,
were also covered with toluol and allowed to stand at room tem-
perature for three days. The alcoholic and alkaline extracts were
then centrifuged at high speed for half an hour, and the super-
natant fluid decanted into sterile tubes.

One normal and four immune rabbits which had been shaved on
the back were inoculated intradermally with 0.2 ¢.c. of each extract
and with a control of the extracting fluid to which a little toluol had
been added. All extracts were tested for their sterility before being
used for the inoculations.

The extracts prepared from the protein precipitated by means
of alcohol gave unsatisfactory results. A reddened and occasionally
a slightly indurated papule, indistinguishable from the control of
that series, appeared within 24 hours and then disappeared after 48
hours. The alkaline extracts of L. tropica and L. infantum, on the
other hand, produced at the end of 24 hours a small reddened papule
which reached its height in 48 hours and persisted for five days. The
center of this papule in all the immune animals consisted of an
irregular ivory-white area 1 to 2 mm. in diameter, surrounded by an

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1923] Wagener: Leishmania tropica and Leishmania infantum 481

irregularly shaped red halo, which faded to a flesh color beyond the
edge of the indurated area (pl. 42, fig. 4). The areas of induration
varied from 5 to 7 mm. in diameter on the different immune rabbits.
At about the fifth day these white centers formed a hard, yellowish
brown crust, which sloughed off three or four days later without
leaving a sear.

Neither the white center nor the area of induration was present
in the control tests made on each rabbit with the extracting fluid
(pl. 42, fig. 4b) or in any of the tests on the normal rabbit.

This reaction is comparable to that produced in tubercular animals
by the intradermal injection of Old Tubereulin, which results in a
red and indurated papule with a central necrotic area which sloughs
off several days after the injection.

Krause and Peters (1920) demonstrated a local reaction to
tuberculosis re-infection when living tubercle bacilli were injected
intracutaneously into guinea pigs. This reaction was characterized
by a reddened papule, rapid ulceration, and slough followed by
healing. These authors considered this an immune reaction in the
euinea pigs. As this reaction corresponds very closely to that
obtained in the rabbits immunized to Leishmania, an attempt was
made to further correlate the results by injecting imto an immune
and a normal rabbit cultures of the living flagellates. These injections
were made intradermally on the testicle, this site being chosen on
account of the thinness of the epidermis and also because Laveran
(1917) had found it possible to produce Bagdad boils in white mice
by injecting cultures of Leishmania tropica into the testicular sac.
The rabbits were observed daily for a week and frequently there-
after. No reaction occurred. The reason, probably, was because,
as suggested by Delanoe (1911), the living flagellates were being
rapidly phagocytized.

One week after the first series of skin tests had been applied to
the immune rabbits, they were retested in order to standardize more
definitely the alkaline extracts of L. tropica and L. infantum. This
was considered necessary because throughout the first tests the extract
from L. infantum had produced in general a less marked reaction
than that from LD. tropica. This difference might have been due to
an unequal concentration of protein because of the slightly larger
size of L. tropica as compared with L. infantum. Hence extracts
made by the addition of a definite number of flagellates per c.c. would
not necessarily represent equivalent amounts of protein. Flagellate

[ Von. 20

University of California Publications in Zoology

48

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1923] Wagener: Leishmania tropica and Leishmania infantum 483

growths from a number of cultures were therefore washed off in
saline and packed by rapid centrifugalization for one half-hour.
One part of the packed organisms was then diluted with four parts
of the alkaline extracting fluid, treated as before, and stored in the
ice box. It was then possible to make up any dilution desired from
these stock alkaline extracts of L. tropica and L. infantum.

Tests made by the intradermal injection of 0.2 c.c. of these stock
alkaline extracts into the immune rabbits showed both of them to
be unduly toxie (pl. 42, figs. 3b and d). When diluted 50 per cent
with extracting fluid, however, the toxicity of both was greatly
reduced, though a red and indurated papule more than 5 mm. in
diameter, easily distinguishable from the control test and from the
tests on the normal rabbit, was still produced (pl. 42, figs. 1 and 2).
The difference in the size of the papule produced by L. tropica and
by L. infantum persisted. The cause of this difference is, as yet,
obscure.

The results of these tests are as shown in table 2.

DISCUSSION

It is probable that the immunization of rabbits to Leishmania
produces in them the condition classified by Coca (1923) as hyper-
sensitiveness of infection. Hypersensitiveness to derivatives of bac-
teria has been demonstrated by a number of investigators through
the use of the intradermal test. Von Pirquet (1911) showed that
the intradermal injection of vaccine lymph produees, in previously
vaccinated persons, marked papules after 48 hours. Force and
Stevens (1917) have described in detail the papule produced by the
intradermal injection of typhoidin in typhoid immune rabbits. The
intradermal tuberculin reaction in both man and animals is so gen-
erally known that a resumé of the literature is scarcely necessary.
Recently Fairley (1923) found that the Casoni intradermal reaction
gave positive results in 57.9 per cent of patients with proved hydatid
infestation. This intradermal reaction is not constant in all cases
giving positive complement fixation or precipitin reactions; in cases
of ruptured eysts, this is due to the desensitization of the patient.
Lanfranchi and Sani (1921) produced, with antigens prepared from
Trypanosoma brucci, an ophthalmic reaction which persisted 36 hours

484 University of California Publications in Zoology (Vou. 20

in horses infected with dourine. Normal horses showed a reaction
persisting from 20 to 24 hours. The authors considered the reactions
in the infected animals to be similar to those obtained in positive
mallein reactions. Nusshag (1921) described inconclusive results
obtained in dourine by the intradermal injection of .25 ec. of antigen
prepared from trypanosomes. <A faintly reddish papule appeared
in 5 hours, reached its height in 18 hours, and disappeared within
24 hours. In the light of the tuberculin and typhoidin reactions,
these results could scarcely be considered positive, but rather as a
non-specific protein reaction. Force and Stevens (1917) consider
specific only those intradermal reactions which reach their height
at 48 hours and persist for at least 72 hours. The work of De Gaspari
(1921) on dourine has been inaccessible. The reactions obtained by
the intradermal injection of the alkaline extracts of L. tropica and
L. infantum in immune rabbits are without doubt comparable to those
obtained with tuberculin, typhoidin, and vaccine lymph.

It is of importance to note that rabbits immunized to one species
of Leishmania are sensitive to the protein of both species, L. tropica
producing the most pronounced reaction even in animals immunized
to L. infantum. Hamman and Wolman (1912) have found that both
human and bovine Old Tuberculin produced approximately the same
sized papule in 127 out of 150 cases of tuberculosis tested. Meyer
and Christiansen (1917) stated that some typhoid immune rabbits
show non-specific reactions with paratyphoid and B. coli extracts
which can be considered as group reactions.

These group reactions do not impair the diagnostic value of the
skin reaction as a means of detecting hypersensitiveness to protozoan
proteins. At the present time insufficient work has been done to
determine the value of these tests in man or in animals infected with
protozoa. It is hoped, however, that the results of this investigation
may be made the basis for similar tests on cases or convalescents from
leishmaniosis.

1923] Wagener: Leishmania tropica and Leishmania infantum 485

SUMMARY

The intradermal injection of alkaline extracts of Leishmania
tropica or Leishmania infantum will produce a local reaction in the
skin of a sensitized rabbit. This reaction is characterized by an
erythematous papule which reaches its height in 48 hours and persists
from 72 hours to 5 days.

The injection of a concentrated extract produces necrosis and
sloughing. Normal rabbits show no reaction.

The reaction in immune or sensitized rabbits is not specifie for
the homologous species of Leishmania.

Transmitted October 8, 1928.

FROM THE DEPARTMENTS OF ZOOLOGY AND OF HYGIENE,
UNIVERSITY OF CALIFORNIA.

LITERATURE CITED
Banpt, I.
1913. Preliminary note on the identity of certain leishmanioses based on bio-
logical reactions. Jour. Trop. Med. and Hyg., 16, 50.
Coca, A. F.
1922. Studies on specific hypersensitiveness. Jour. Immunol., 7, 163-178.

Coca, A. F., and Coox, R. A.
1923. On the classification of the phenomena of hypersensitiveness. Jour.
Immunol., 8, 163-182.
DELANOE, P.
1911. L’immunité naturelle de la souris 4 1’égard des cultures de Kala-Azaz
et de bouton d’Orient tunisiens. C. R. Soc. Biol., Paris, 70, 387-388.

Fairey, K. D.
1923. The investigation of the immunity reactions in hydatid disease.
Med. Jour. Australia, 10, II, 27-37.

Force, J. N., and Stevens, I. M.
1917. Further studies on typhoidin. Arch. Int. Med., 19, 444-456.

GASPARI, F. DE.
1921. Morbo coitale maligno e anafilassi. Aleune recerche sperimentali sulla
sensibilizzazione dell’organismo animale con i prodotti tossici du
trypanisomi e con le cue lorazione. Clin. Vet. Milano, 44, 671-687.

Hamman, L., and Wotan, 8.
1912. Tubereulin in diagnosis and treatment (New York, Appleton), xiv +381
pp-, 30 figs. in text.

486 University of California Publications in Zoology (Vou. 20

Krause, A. K., and Prerrrs, D.
1920. Immunity in tuberculosis. Am. Rev. Tuberce., 4, 551-556, 2 pls.

LANFRANCHI, A., and Sani, L.
1921. L’intrapalpebroreazione nella diagnosi del morbo coitale maligno. Clin.
Vet. Milano, 44, 647-650, 1 pl. in text. (Trop. Dis. Bull., 19, 150.)
LAVERAN, A.
1917. Leishmanioses (Paris, Masson), iii +515 pp., 4 pls., 40 figs. in text.

Meyer, K. F., and CHRISTIANSEN, O. R.
1917. The intracutaneous ‘‘typhoidin’’ reaction. Jour. Inf. Dis., 20, 357-441.

NussHac, W.
1921. Allergische Reactionen bei der Beschalseuche. Berl. tierarztl. Wochen-
sehr., 37, 567-569.
Von PirqQueEt, C.
1911. Ueber die verschiedenen Formen des allergischen Reaktion bei der

Revaccination. Ztschr. f. Immunitatsforsch. u. exper. Therap., Orig.,
10, 1-23, 13 figs. in text.

EXPLANATION OF PLATE 42

All reactions shown forty-eight hours after the intradermal injection of alka-
line extracts of Leishmania tropica and Leishmania infantum. Figures natural
size and color. Drawings by Dr. Olive Swezy.

Fig. 1. Normal rabbit No. 21 showing the results of the intradermal injection
of a 50 per cent dilution of stock solution of the alkaline extracts of L. tropica
and L. infantum. a. Alkaline extract of L. tropica. b. Alkaline extract of L.
infantum. c. Extracting fluid control. Drawn September 24, 1923.

Fig. 2. The same as fig. 1 on immune rabbit No. 7. a. 50 per cent dilution
of the alkaline extract of L. tropica. b. 50 per cent dilution of the alkaline
extract of L. infantum. c. Extracting fluid control. Drawn September 24, 1923.

Fig. 3. Immune rabbit No. 7 showing the effect of the intradermal injection
of the stock alkaline extracts of L. tropica and L. infantum as compared to a
50 per cent dilution of the stock alkaline extract of L. tropica. a. Extracting
fluid control. b. Stock alkaline extract of L. tropica. c. 50 per cent dilution of
the stock alkaline extract of L. tropica. d. Stock alkaline extract of L. infantum.
Drawn September 17, 1923.

Fig. 4. Immune rabbit No. 127 injected intradermally with alkaline extracts
of L. tropica and L. infantum containing 2,000,000 flagellates per cubic centi-
meter. a. Alkaline extract of L. infantum. b. Extracting fluid control. c. Alka-
line extract of L. tropica. Drawn September 10, 1923.

[488]

. PUBL. ZOOL. VOL. 20 (WAGENER] PLATE 42

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UNIVERSITY OF CALIFORNIA PUBLICATIONS—(Continued)
yy Vol 20. 1. Studies on the Parasites of the Termites. I. On Streblomastizx striz, a Poly-

vi ye ' mastigote Flagellate with a Linear Plasmodial Phase, by Charles Atwood
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Ce 2. Studies on the Parasites of the Termites. II. On Yrichomitus termitidis, a

Le? Polymastigote Flagellate with a Highly Developed Neuromotor System,
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sp. nov., by Charles Atwood Kofoid and Olive Swezy. Pp. 41-98, plates
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plates IBLE, AL eure In Text. OuUlyy LITO): tes Ak Si ee es
ye! 5, On the Morphology and Mitosis of Chilomastiz mesnili (Wenyon), a Common
‘ Flagellate of the Human Intestine, by Charles A. Kofoid and Olive Swezy.
BRON’ Pp. 117-144, plates 15-17, 2 figures in text. April, 1920 oo
TR, - 6. A Critical Review of the Nomenclature of Human Intestinal Flagellates,
PAS Cercomonas, Chilomastiz, Trichomonas, and Giardia, by Charles A. Kofoid.
Pp. 145-168,-9 figures in text: Jume,,1920 2022s eee lee cecceeee
7. On the Free, Encysted, and Budding Stages of Councilmania lafleuri, a Para-
sitic Amoeba of the Human Intestine, by Charles Atwood Kofoid and
Olive Swezy. Pp. 169-198, plates 18-22, 3 figures in text. June, 1921 .._.
8. Mitosis and Fission in the Active and Encysted Phases of Giardia enterica
(Grassi) of Man, with a Discussion of the Method of Origin of Bilateral
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rae Swezy. Pp. 199-234, plates 23-26, 11 figures in text. a ahi
922
9. The Micro-
PATI BODE ss Ee ea OI SN as AES RRA ne 30 AGEN Ba BEE PRR
10. On Balantidium coli (Malmsten) and Balantidium suis (sp. nov.), with
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11. Mitosis in Endamoeba dysenteriae in the Bone Marrow in Arthritis de-
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12. Endamoeba dysenteriae in the Lymph Glands of Man in Hodgkin’s Disease,
by Charles A. Kofoid, Luther M. Boyers, M.D., and Olive Swezy. Pp. 309-
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Nos,.11 and:12 in one cover, April, 1922 ooo... cece ccleeecenceeeetenterneseeeee
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14. The Neuromotor Apparatus of Paramaecium, by Charles William Rees.
Pp. 333-364; plates 32-36, 5 figures in text. November, 1922.2... 04.
15. A Comparison of the Cysts of Hndamoeba coli and Councilmania lafleuri in
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873-890, plate 37, 1 figure in text. August, 1923 20000. ee
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hay Map Cad Ga fe bog oo Maal bs ll ATA Ce Rt NS a ae ed een Alls RAN LR FE Els
18. Methods of Obtaining Amoeba-free Rats for Experimental Infection with
Intestinal Amoebae, by John F. Kessel. Pp. 401-408.
19. Experimental Infection of Rats and Mice with the Common Intestinal
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‘ t Nos. 18 and 19 in one cover. October, 1923_............---2-------e-- eee
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and Rodents, by Charles A. Kofoid, Olive Swezy, and John F. Kessel,
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i i 21. Morphology and Binary Fission of Menoidiwm incurvum (Fres.) Klebs,
i by Richard P. Hall. Pp. 447-476, plates 40-41, 2 figures in text. Novem-

[ss PiU RS ys Ra ASS SAAT OCA ca RU NAL SRA SSS RS a Se SRA A
he 22, A Skin Reaction to Extracts of Leishmania tropica and Leishmania in-
i fame) by Edna Hannibal Wagener. Pp. 477-488, plate 42. December,

923

* Yoli22. 1, ne Bevision of the Microtus californicus Group of Meadow Mice, by Reming-
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2, Five New Five-toed Kangaroo Rats from California, by Joseph Grinnell.

Py 48-47; Mearchy, 1919 12-1 ca een ern mn tneemmetnn cnn rennnicene

Vol. 22.

Vol. 23.
VoL 24.

Vol. 25.
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3. Notes on the Natural History of the Bushy-tailed Wood Rats of California,
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9. Behavior of the Leaf-nosed Snake, Phyllorhunchus decurtatus, by Sarah ?

Rogers Atsatt.. Pp. 303°312. . February, 1923 =e
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Pp, 318-824) Jaruary,; 1928 eo See ee oe :

11. The Systematic Status of the Mountain Lion of California, by Joseph
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1. A Quantitative and Statistical Study of the Plankton of the San Joaquin
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2. Variations in the Shell of Teredo navalis in San Francisco Bay, by Robert.

Cunningham Miller. Pp. 293-328, plates 13-17, 6 figures in text. Novem-
Ber}, AGAZh Sch a a Seek RE ee ST Toe ee Re
8. Quantitative Studies on Marine Phytoplankton at La Jolla in 1919, by

Winfred Emory Allen. Pp. 329-347, 2 figures in text. November, 1922 —
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9. Some Tide-Water Collections of Marine Diatoms taken at Half-Hour Inter-

vals near San Diego, California, by Winfred E. Allen. Pp. 413-416,

A. figure in‘ text-s June, 2923 so et eR ae Sg ek See
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the Plankton at La Jolla, California, by Calvin O. Esterly. Pp. 417-433.
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445-4487, ‘November,-8923 7 e ee eae ee Oe nS
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The Marine Decapod Crustacea of California, by Waldo L. Schmitt. Pp. —

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ae ee ee eee ee ee ee ee ee

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COMDET; 191 eee eee ee ATS abn rata ats Aw ces alee usecase 3} EN
10: A New Morphological Interpretation of the Structure of Noctiluca, and Its
Bearing on the Status of the Cystoflagellata (Haeckel), by Charles A. ‘
Kofoid. Pp. 317-334, plate 18, 2 figures in text. February, 1920............... 25 -
11. The Life Cycle of Hchinostoma revolutum (Froelich), by John O. Johnson. :
Pp. 338-888, plates 19-25, 1 figure in text. May, 1920 —..2...21...c...--ccsceee 60 |
12. On Some New Myriopods Collected in India in 1916 by C. A. Kofoid, by
Ralph V. Chamberlin. Pp. 389-402, plates 26-28. August, 1920 ............... 20
18. Demonstration of the Function of the Neuromotor Apparatus in Huplotes
by the Method ef Microdissection, by Charles V. Taylor. Pp. 403-470,
plates 29-33, 2 figures in text. October, 1920 22.2... jeeeeccn ete eteneeneee 85
Index, pp. 471-480,
Vol. 20. 1. Studies on the Parasites of the Termites. I. On Streblomastix strix, a Poly-
mastigote 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

. 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 taxty July, 101 ee ee ee ee eee UA Jeol deo ceatiainsecseeets

a ag peal ey

UNIVERSITY OF CALIFORNIA PUBLICATIONS

IN

ZOOLOGY

Vol. 20, No. 23, pp. 489-544, plates 43-48, 3 figures in text

February 20, 1924

THE DISTINGUISHING CHARACTERISTICS OF
THE PARASITIC AMOEBAE OF CULTURE

RATS AND MICE

BY

JOHN F. KESSEL

CONTENTS

Introduction...
Eistoricall. 2... ..cccc:
Acknowledgments...
Material and methods
Incidence of infection of amoebae in rats and mice
Councilmania muris........
The motile amoeba.
Encystment...... ;

IMUIGOSIS;sesotesccee ec
Councilmania decumani

The motile amoeba...

Encystment....

Budding......

Mitosis. .......:. si
Endamoeba ratti......

The motile amoeba.

Encystment....
Experiments in cross-infection......
Discussion....................
UMM ST Va eee cies.
Literature cited.
Explanation of plates........

PAGE
490
490
491
492
493
495
495

. 503

507
509
511
512
512

. 514

514
515

515

. 516

517

. 522

529
Del
534

490 University of California Publications in Zoology [VoL 20

INTRODUCTION

The present investigation was begun in order that, later, we might
attempt the experimental infection of rats and mice with the common
intestinal amoebae found in man. For it was early realized that, before
such critical experimental infections could be carried out satisfactorily,
a detailed study of the amoebae normal to the rat and mouse was
essential.

Our attempts to culture in rats and mice most of the common intes-
tinal amoebae of man have met with success (Kessel, 19236), and it is
our purpose in the present paper to describe the distinguishing char-
acteristics of the amoebae normally occurring in culture rats and mice.

HISTORICAL

Grassi (1881) found in both the rat and the mouse an amoeba which
he named Amoeba muris. He published no figures and described no
cysts but stated that the motile forms resembled Amoeba coli (Losch)
inman. He distinguished the amoeba of the mouse from £. coli largely
on the basis of size, and stated that the maximum size of the species
found in the mouse slightly exceeds the minimum size of the species
found in man.

Wenyon (1907) in connection with his work on the intestinal
Protozoa of mice made a careful study of an amoeba found in the
mouse, and concluded that the free forms and cysts bore a very striking
resemblance to Endamoeba coli. When the genus Councilmania was
later described (Kofoid and Swezy, 1921), resemblances were at once
apparent between this new genus and the amoeba described by
Wenyon. These are also very well shown in Wenyon’s figures.

Wenyon’s work is excellent as a pioneer investigation, but certain
structures, such as the chromatoidal body, were not then regarded as
important diagnostic characters and were described (his pl. 10, figs.
13, 20) as being remains of food products that had been thrown out of
the cyst. It is now generally accepted that solid food products are
extruded in the precystic stage. His account of autogamy in the
amoeba of the mouse, as previously described by Schaudinn for Enda-
moeba coli, cannot be upheld in the light of our present knowledge of
nuclear phenomena in cysts.

1924] Kessel: Amocbae of Culture Rats and Mice 491

Brug (1919) coneluded that the amoebae of the rats, identified
as Mus rattus, and of the mice examined by him were identical, and
that their resemblances to Endamoeba coli were close, the greatest
difference being in the size. His figures indicate that he was dealing, at
least for the most part, with the species Councilmania decumani.
Rudovsky (1921) deseribed and figured an amoeba found in five rats of
the species Mus decumanus Pall., which is a synonym for Rattus nor-
vegicus (Erxleben). Their figures show that Rudovsky and Brug have
deseribed an identical species. Rudovsky, however, drew a morpho-
logical distinction between Hndamoeba coli and the amoeba of the
mouse, which he described as having a heavy nuclear membrane, a
large karyosome, and little outer chromatin; and also, between these
two amoebae and the amoeba of the rat, which he deseribed as having a
thin nuclear membrane, small granules of peripheral chromatin, and
a small karyosome. He speaks of this amoeba of the rat as Endamoeba
muris decumant.

In our own work, following investigations, first of an amoeba of the
mouse and then of an amoeba of the rat, it was thought that there was
a difference between the morphological characteristics of the two
amoebae. But a second lot of rats produced amoebae identical in all
respects with the amoeba found in the mouse. For a time it seemed
that the differences noted in the first rats examined represented differ-
ent phases of the same life cyele, but further investigation has led to
the conclusion that there are at least three species of amoebae parasitic
in the intestine of culture rats and mice.

For reasons to be stated later, these three species of amoebae should
be classified as Councilmania muris (Grassi, 1881, emend. Wenyon,
1907) Kessel emend.; the species Councilmania decumani (Rudovsky,
1921) Kessel, emend.; and Endamoeba ratti, sp. nov., a species having
granular pseudopodia and found, to date, only in the rat.

ACKNOWLEDGMENTS

The writer is greatly indebted to Professor C. A. Kofoid, under
whose supervision the work here presented has been completed; to
Dr. E. L. Walker of the Hooper Foundation, who has offered many
helpful suggestions; and to Dr. Olive Swezy, who has been generous
in extending many courtesies. The work of the past year has been
made possible by the writer’s appointment as University Fellow in
Zoology.

492 University of California Publications in Zoology [VoL 20

MATERIAL AND METHODS

Culture albino mice, Mus musculus Linnaeus, obtained from four
different colonies, and culture rats, Rattus norvegicus (Erxleben),
were used. The colony of rats from which our stock came was started
in 1911, when Professor J. A. Long crossed three albino females,
obtained from the Wistar Institute, with a wild brown male, pre-
sumably Rattus norvegicus, caught in Berkeley. New commercial
stock of albinos has been added once to this colony. Huber (1915),
quoting Donaldson, concluded that the albino rats from the rat colony
of the Wistar Institute of Anatomy and Biology belong to the species,
Mus norvegicus albinus (Donaldson). Dr. Joseph Grinnell, of the
California Museum of Vertebrate Zoology, has identified an adult
brown rat taken this year from our stock colony as Rattus norvegicus.

To determine the presence of amoebic infection in the culture
animals, the normal faeces from the rats and mice were at first
examined daily. This method proved to be slow, tedious, and imac-
curate (see Brug, 1919, and Kessel, 1923a). Suecessful attempts were
then made to evacuate the amoebae in the faeces in greater quantities
by the administration of a purgative, epsom salt, as described by Kessel
(1923a), and this method was used throughout the investigation.

Preliminary examinations were made of all material in fresh
smears, one-half of the cover-glass preparation being smeared in
normal salt solution, the other half in Donaldson’s iodine-eosin stain.
The active forms are easily detected in the normal saline solution,
while the cysts are more readily seen in the stained portion. Per-
manent slides were made by fixing the material for two minutes in
Schaudinn’s fluid heated to 60° C. The slides were stained in iron-
haematoxylin.

The activity of the motile forms has been observed in normal saline
on an electric warming stage at 37° C.

1924] Kessel: Amoebae of Culture Rats and Mice 493

INCIDENCE OF INFECTION OF AMOEBAE IN
RATS AND MICE

During the investigation 80 mice and 288 rats have been examined
for amoebae. As stained slides have been made in only 45 of the
positive cases found, the exact species of amoebae has been determined
only in these 45 rats and mice. The other examinations were made for
the purpose of procuring animals for infection experiments and were
studied only in iodine-eosin. The data in the first table serve merely
to differentiate between amoebae of the genus Councilmania and the
genus Endamoeba.

TABLE 1

TABLE SHOWING INCIDENCE OF INFECTION OF RATS AND MICE WITH AMOEBAE
BELONGING TO THE GENERA Councilmania AND Endamoeba

Mice

Positive
Lot Wh A ; - Nega- Total | P t
ese Obtatied i CS | eee || ees es leas
A |e rabon eee. Over 10 months.. 3 7 10 | 30.0%
2 | Dr. Morgan ....| 2 to 4 months...... 4 5 9 | 44.5%
3 | Dr. Hagedoorn| 2 to 4 months... 7 8 15 | 46.6%
4 | Dr. Long.......... About 10 months.. 7 11 18 | 39.0%
5 | Dr. Morgan ....| Five weeks.......... 2 7 9 | 22.0%
6 | Dr. Long..........| About 10 months 2 10 12 | 16.6%
7 | Dr. Gaylord* ..| 2 to 4 months... 5 2 Ch ANAS)
30 50 80 | 37.5%

Rats

Positive
; Nega- Total | P t
Reel) (Ovtained ia Couneil-| Enda- | Nees | Total | positive
Tae prongs. _| 4 to 6 months... 6 1 13 20 35.0%
2 | Dr. Evans....... Over 10 months 1 19 20 5.0%

3 | Dr. Long and

Dr. Evans....| Mixed.................. 29 1 77 107 | 28.0%
2 to 4 months 17 8 25 68.0%
2 to 4 months... 23 2 25 92.0%
3 to 5 months... 8 1 9 18 | 50.0%
2 months. ss 6 14 20 | 30.0%
5 and 6 weeks....... 0 15 5 0.0%
5 and 6 weeks....... 3 15 18 16.6%
Over 10 months 2 18 20 10.0%
95 3 190 288 | 34.0%

*New York State Institute for the Study of Malignant Diseases.

494 University of California Publications in Zoology [Vou 20

The following conclusions may be drawn from a study of the data
in the accompanying tables.

1. The incidence of infection of amoebae among culture mice in
this laboratory is 37.5 per cent while among rats it is 34 per cent (see
table 1).

2. The incidence of infection is much lower in very old and in very
young mice and rats than it is in middle-aged animals (table 2).

TABLE 2

Mice
Per cent
Age Positive Negative Total positive
Unders2imonthsyaee see ee 2 7 9 22.0%
2ito 4 months)............... Perce - 16 15 31 51.6%
About Oimonths yes ee 12 28 40 30.0%

Rats
Per cent
Age Positive Negative Total positive
Under2imonthss eee 9 44 53 17.0%
2 to 10 months............ Re eee 54 42 96 56.3%
OverslOimonthshee eee : 3 40 43 7.0%

3. The incidence of infection is lower in wild mice and rats than
in culture mice and rats (see table 3). It is assumed that the mice
used by Wenyon were culture mice, and while he gives no figures of
the number he examined he states that he found amoebae in about half
of the cases. The culture mice and rats examined in this laboratory
show an average infection of 35.7 per cent, while all the wild rats and
mice recorded in the literature and the 20 wild mice examined in this
laboratory show an average infection of 7 per cent.

Balfour (1922) made his examinations from autopsied rats and
it is assumed that Rudovsky worked with dead animals. The examina-
tions of the wild mice in this laboratory were made after death.
Examination after autopsy should be just as certain a method of
determining infection as the epsom salt method. Brug does not state
how his 50 rats were examined, but, as his rate of infection is higher
than Rudovsky’s and only slightly lower than the lower percentage
recorded by Balfour, it would seem fair to include his results in the
table. These results indicate that animals living in the crowded con-
ditions of a laboratory colony are more liable to acquire amoebic
infection than those living in the open.

1924 | Kessel: Amoebae of Culture Rats and Mice 495

COUNCILMANIA MURIS (Grasst, 1881), emEND. Kessen

Occurrence.—Councilmania muris has been found in 14 of the 25
rats and in 4 of the 17 mice from which stained slides of the faecal
material have been prepared in this laboratory (see table 4). An exam-
ination of the stained slides has been our main and most conclusive
method of differentiating this species from Councilmania decumani;
the positive cases diagnosed in fresh smears only have not been con-
sidered in the percentages cited above.

TABLE 3

TABLE SHOWING INCIDENCE OF AMOEBIC INFECTION IN RATS AND MICE
RECORDED IN LITERATURE

‘ ; . . Per cent
Investigator Species of Animal Positive Negative Total positive
Wenyon.........| Mus musculus........ Not stated] Not stated} Not stated] 50.0%
SEU Peeerecerees IVEUIS Cat GUUS se see eeeere eee reese 7 43 50 14.0%
Rudovsky....| Mus musculus........ : 0 37 37 0.0%
Rudoysky....|. Mus musculus............. 5 159 164 3.0%
Balfour........ Blackspratseeete sscr Not stated} Not stated 34 25.0%
IBrOWwnens tensor sees ses Not stated] Not stated 444 15.7%

Mus norvegicus.......... 96 172 268 3.6%

Mus musculus (albino).. 30 00 80 37.5%

Mus musculus (wild) .... 2 PAL 23 9.0%

TABLE 4

INCIDENCE OF INFECTION OF AMOEBAE OF RatTS AND MIcE DETERMINED FROM
EXAMINATION OF SLIDES STAINED WITH IRON-HAEMATOXYLIN

: F é - Mixed per cent
Councilmania Councilmania Endamoeba C. eae and
muris decumani ratti C. deecumani

Number | Per cent | Number | Per cent | Number | Per cent | Number | Per cent

Tue Morite AMOEBA

Habitat——The normal habitat of this amoeba is the caecum. Unless
examination is made following a purgative, the motile forms are sel-
dom found in the colon at autopsy. Just how the active amoebae are
restricted in habitat almost entirely to the caecum, while the contents
of the caecum within which the amoebae have been mixed are dis-
charged into the colon, affords an interesting problem. According to
Cannon and MecNease (1923) the pH of the caecal contents is more

496 University of California Publications in Zoology [VoL 20

acid than the contents of the ileum or the colon. Determinations of
the pH by us in this experiment verify this conclusion and it would
seem that a definite chemotropism restricts the active amoebae to this
region.

The degree of infection varies greatly ; in some instances as many
as three or four amoebae have been found in a single field of a freshly
prepared smear, while in other cases only a single amoeba has been
found in an entire smear. There is no evidence that the degree of
infection varies in different parts of the caecum, for if amoebae are
present in a smear from one region they are usually present in other
parts of the caecum. They may be scraped with caecal contents from
the surface of the epithelium, but no visible pathological conditions
have been observed in the wall of the caecum.

Food and pathogenicity—The amoebae live normally upon food
procured from the contents of the caecum. They may ingest bacteria
(pl. 48, figs. 1, 2), yeasts, and flagellates (pl. 43, fig. 4), as shown by
the presence of a partly digested Chilomastix in the largest vacuole.
At the suggestion of Dr. I. C. Hall of the Department of Bacteriology
in the University of California, gram stains were made of the free
amoebae from the caecum in order to determine the type of bacteria
ingested. After a number of attempts, successful results were obtained.
Bismark brown was found to be the most successful counterstain. The
cytoplasm is gram-negative while the nucleus retains the gentian violet.
The amoebae ingest both gram-positive and gram-negative bacteria,
the cases examined giving 75 per cent gram-negative rods and 25 per
cent gram-positive cocci. It is significant that no gram-positive rods
have been found ingested, though many are present on the slides in the
material surrounding the amoebae. Whether or not a change in the
stainability of the bacteria is produced by their partial assimilation
by the amoebae which causes them to stain gram-negative, it is as yet
impossible to say. Sphaerita (Dobell, 1921) has been found as a
parasite in the amoebae (pl. 43, fig. 1).

Because no evidence has been encountered which indicates that
the free amoebae ingest normal tissue cells—the reason being that no
caecal ulcers have been found in any of the rats—and because 34 per
cent of normal rats harbor amoebae in the caecum, it is thought that
no pathogenic results accompany the presence of these amoebae in
the rodent hosts.

Pseudopodia and amoeboid movement.—As examination in normal
saline reveals no marked specific differences between Couwncilmania

1924] Kessel: Amocbae of Culture Rats and Mice 497

muris and Councilmania decumani, the pseudopodia and the amoeboid
movement of the two species will be discussed together.

Grassi (1881) in describing the amoebae of the rat and mouse said
that they are slightly motile and that two or more large and blunt
pseudopodia can be demonstrated. Ordinarily, only one is evident
at a single instant and this is retracted before the second is formed.
He described the pseudopodia as ‘‘hyaline,’’ i.e., ectoplasmic, and
stated that the endoplasm flowed into the ectoplasm, which he inter-
preted as the method by which the amoeba moves.

- Movement, according to Wenyon (1907), may be rapid on a warm
stage. He wrote, ‘‘As a rule only a single pseudopodium is formed at
one time. This consists at first only of eetoplasm [his pl. 10, fig. 3]
into which the endoplasm suddenly streams, carrying the nucleus
with it.”’ Brug (1919) stated that the amoebae creep about more
actively than he ever saw Endamoeba coli creep and that in the pseu-
dopodia only ectoplasm is found.

Rudovsky (1921) found that the protruding pseudopodia are
plainly divisible into a transparent and a finely granular zone and he
differentiates between ectoplasm and endoplasm (his pl. 7, fig. 1). It
is evident from these descriptions that the amoebae already described
from rats and mice possess hyaline pseudopodia.

Endamoeba coli (Losch) from man, on the other hand, differs
remarkably from these amoebae in this regard. Dobell and O’Connor
(1921) stated that in the change of shape of Endamoeba coli, without
evident progression (fig. on their pl. 1),‘‘no sharp line of demarcation
separates the ectoplasm and endoplasm, and the formation of large,
clear, blade-like pseudopodia—so characteristic of Endamoeba histo-
lytica—is never seen.”’

One of the distinguishing characteristics between Endamoeba coli
and Councilmania lafleuri (Kofoid and Swezy, 1921) is this difference
in formation of pseudopodia, hyaline pseudopodia being the type
possessed by Councilmania lafleuri. It therefore seems quite evident
that in this one respect alone there is sufficient differentiation to war-
rant drawing a distinction between Endamoeba coli and the amoebae
of the rat and mouse.

The formation of pseudopodia of Couwncilmania muris and of
Councilmania decumani can be described most satisfactorily by divi-
sion of their movements into two types: first, that during progressive
movement and second, that during attachment to the substrate. In
both types of movement the pseudopodia are charaeteristically hyaline.

498 University of California Publications in Zoology [Vou 20

It is not always possible to procure amoebae, even from the caecum,
that will remain active for more than a few minutes on the warm
stage. At times, however, amoebae have been secured which have
exhibited typical movement for thirty minutes. The reasons for this
variation in activity are undetermined.

18

Fig. A

Free-hand sketches, showing progressive movement, of Councilmania muris
from rat.

In progressive movement an amoeba may move as far as 500
microns in five minutes. Usually, however, the movement is less rapid.

1924 | Kessel: Amoebae of Culture Rats and Mice 499

This forward movement may be described as an ectoplasmic looping.
As represented in figure A, 1, the rounded amoeba shows at the most
only a narrow ectoplasmic margin. This rounded amoeba protrudes,
with almost explosive suddenness, a hyaline pseudopodium (fig. A, 2)
into which the endoplasm may later flow (fig. A, 3). In the progressive
movement, the pseudopodia appear blade-like in optical section, but
in reality are blunt lobes. They are inclined to be more pointed than
the pseudopodia formed when the amoeba is attached. In the forward
movement, the hyaline pseudopodium is fully formed before the vacuo-
lated and granular endoplasm flows into it. At the posterior, or
attached end, a conical projection or attachment cone is often in evi-
dence (fig. A, 1). This appears to be used as a point of adhesion to
the substrate and as a point of leverage. As the endoplasm flows for-
ward, this root-like projection persists for a short time as a hyaline
structure (pl. 43, fig. 3). The amoeba then resumes a spherical or
ovoidal shape before it thrusts out another pseudopodium. The whole
process is then repeated.

In progressive movement the pseudopodia are not all thrust out
in a constant forward direction, but lateral pseudopodia (figs. A, 8, 11)
or even posterior pseudopodia (fig. A, 14) may be formed. In the
observations made, however, a permanency of movement in a given
direction was maintained. The formation of these lateral and posterior
pseudopodia may indicate a reversing motor reaction such as is found
in ciliates, or it may indicate that the path, if observed for a sufficient
time, might represent a ‘‘flattened spiral’’ as described by Schaeffer
(1920). As in these amoebae there appears to be no constancy in the
formation of left or right-handed pseudopodia, it is not certain that
either a right or left-handed spiral would be retained. In progressive
movement, it is not common to see two pseudopodia (fig. A, 14) in
action at the same time, and this probably oceurs only when the amoeba
changes its direction, as perhaps in a motor reaction.

Pseudopodial formation during attachment.— In a cover-class
preparation, the amoeba often appear to be attached either to the slide
or to the cover-glass, as they may also adhere to a clump of bacteria
from which they are apparently making a continuous effort to become
free. Pseudopodia are then thrust out rapidly in all directions, ocea-
sionally as many as three or four being evident at one time (fig. B, 11;
pl. 43, figs. 1, 2). These pseudopodia are always hyaline and while
some present the pointed, blade-like appearance of the one formed
by amoebae in progressive movement (fig. B, 12), the broad, blunt,

500 University of California Publications in Zoology (Vou. 20

balloon-like pseudopodia (pl. 48, fig. 2; fig. B, 1) are more common.
The endoplasm may move into and fill the pseudopodia formed when
the amoeba is thus attached, but more commonly the pseudopodia are
retracted or a pseudopodium may be partly filled with endoplasm and
the remaining portion retracted.

In accordance with the gel-sol theory of pseudopodial formation
(Hyman, 1917), this hyaline ectoplasm after protrusion represents
a gel and the more vacuolated endoplasm a sol, each of which is
reversible, one into the other, as protrusion, advancement, or retraction
oceurs.

10
Fig. B

Free-hand sketch, showing formation, while attached, of pseudopodia of
Councilmania muris from rat.

In the formation of a pseudopodium, solution of the ectoplasm
occurs at the initial point of formation. This liquid sol spreads out-
ward and forward, its outer surfaces forming a gel, as contact occurs
with the medium of the environment. The liquid endoplasm then flows
in and oceupies the space between these gelated layers. In retracting
a pseudopodium, the gelated ectoplasm is gradually reversed to a sol
state.

In this discussion it is preferred to describe the protoplasm as
separated into two layers, the ectoplasm and endoplasm, rather than to

1924 | Kessel: Amoebae of Culture Rats and Mice 501

describe a third outer layer, the hyaloplasm, as is found in some
amoebae (see Schaeffer, 1920). While it appears at times that there
are two layers outside the endoplasm, a thin, clear outer layer and an
alveolar middle layer, these two layers blend so, one into the other,
that, as a rule, the line of demarcation is indistinguishable. These two
layers will therefore be described as constituting the ectoplasm. In
stained slides the ectoplasm appears to be finely alveolar in structure.

The great activity with which the amoebae move forward, the
remarkable coordination during the progressive movement, the sudden-
ness with which pseudopodia are formed, the contractility exhibited
by the protoplasm, and the indescribable contortions indulged in by
the active amoebae, lead one to disregard the earlier theories, which
endeavored to explain amoeboid movement as the result of surface
tension, and to conelude with Schaeffer (1921) that the mechanism
controlling locomotion and movement is primarily internal rather than
that the pseudopodia move the amoeba.

The endoplasm contains the nucleus and the numerous vacuoles,
some containing undigested food and some being filled with liquid.
The endoplasm which surrounds these structures presents a flaky
appearance similar to that of the cytoplasm which is found in the
encysted stages. No contractile vacuole has been observed at any time.

Size—Grassi (1881) described the average size of the motile
amoebae as 13.2u. Wenyon (1907), however, recorded the motile forms
as measuring from 30-40u. Rudoysky (1921) figured motile forms
measuring 30 in diameter. The rounded motile forms, measured on
permanently stained slides in this investigation, average 194. With
extended pseudopodia, the length is greatly increased, forms 40 in
length and 30 in breadth having been encountered. An attempt to
determine racial differences in size within the species, from the motile
forms, such as is very evident among the cysts, indicates that there
is a corresponding racial difference in the motile forms, the average
diameter of the largest race in this phase being 21.54 while the
average of the smallest is 18.14. The cysts of these races average
respectively 17.74 and 14.54. This affords an approximate ratio of
the volume of rounded unencysted amoeba to the eyst as 1.2 is to 1; 1.e.,
the volume of the rounded motile amoeba is 0.2 greater than the
corresponding eyst which it forms.

Nuclei—The nuclei of free forms of Councilmania muris are dis-
tinguishable from the food vacuoles in normal saline solution by a
more opaque appearance and by a darker gray color. In the iodine-

502 University of California Publications in Zoology (Vou. 20

eosin stain the nuclei take the iodine stain first and for a brief period it
is possible to definitely distinguish the structure; but as the amoeba
gradually assumes the red color of the eosin and disintegrates, the
well-defined nuclear structure disappears. In the slides stained with
iron-haematoxylin, the same distinguishing characteristics are shown
that are found in the nuclei of eneysted stages. The nuclear mem-
brane is very thin and stains very faintly (pl. 43, fig. 3). A vacuole
surrounding the nucleus has been figured by Wenyon (1907, pl. 10,
fig. 3). This condition has been seen in my own investigations, but I
have considered it to be an abnormality resulting from staining rather
than as a normal occurrence.

Little or no chromatin is encrusted on the nuclear membrane (pl.
43, figs. 1 and 3). This condition, in which small chromatin granules
are found lining the internal margin of the nuclear membrane, is
characteristic of a very early prophase. The bulk of the chromatin
material within the nucleus occurs in a ‘central’ karyosome of the
dispersed type, in the form of clustered spherical granules which
always present a more or less dispersed appearance. At times the
granules may be congregated in a dispersed central mass (pl. 43,
fig. 4). This dispersed type of karyosome is figured by Wenyon (his
pl. 10, figs. 1-3) and while he notes a distinct nucleolus (his pl. 10,
fig. 1) this is shown as being more dispersed than is the case in
C. decuman. A network of faint linin fibers connects the chromatin
granules with the nuclear membrane. The average diameter of the
nuclei in motile forms is 5.6p.

The nuclei do not retain a constant spherical form but are elastic
and may be elongated at times. In one instance the nucleus in a living
amoeba elongated and constricted so definitely in the middle that it
was thought division was in progress. The nucleus, however, soon
resumed its normal spherical shape and did not divide. It was kept
under careful observation until the amoeba had again assumed a
rounded condition.

Asexual reproduction.—It is assumed that this species of amoeba
multiplies by two methods, by binary fission of the motile amoeba and
by budding, a modification of multiple fission following encystment.
No occurrences of binary fission have been witnessed in the motile
stages of this species though motile stages of C. decumani have been
found in which two nuclei were present (pl. 46, fig. 31) and the last
stages of a division process were seen in a case of C. laflewri which had
been recovered from a ease of infection established in a rat (see
Kofoid, Swezy, and Kessel, 19230).

1924] Kessel: Amoebae of Culture Rats and Mice 503

Precystic stage—tIn the motile stages of this amoeba, a certain
number of the vacuoles usually contain solid food material. As the
amoeba prepares for encystment, all solid material is either digested
or egested, leaving the vacuoles filled with liquid substance only.
These vacuoles tend to coalesce, and a few large vacuoles result from
a number of small ones (pl. 43, fig. 5).

The amoeba assumes a spherical form and in time secretes a cyst
wall (pl. 43, fig. 7). There is a further coalescing of the vacuoles at
about this stage (fig. 6), until a single large vacuole is formed in the
early phase of encystment (fig. 7). This vacuole has the subeentral
position of the glycogen mass, the glycogen having been dissolved out
in the process of staining. It seems certain, however, that this gly-
cogen material is derived, at least in part, from the smaller vacuoles
found in the precystic stages that contain liquid. There is an analogy
between this method of formation of vacuoles and the method of the
formation of contractile vacuoles in ciliates as described by Taylor
(1923).

ENCYSTMENT

Occurrence—Until recently it was generally considered that
encystment was for the purpose of protecting the organism while it
was being transferred from host to host. There seems, however, to be
growing evidence that encystment may occur periodically, following
intervals of the accumulation of excess food material (Kofoid, 1923).
It has been noted that, in cases where the cysts appear in the faecal
material, they also usually occur in the caecum, indicating that encyst-
ment, for the most part, takes place in the caecum. It is further signi-
ficant to note that following the administration of epsom salt there
is no regular response in the formation of cysts. One would think
that, as a result of exposure to this deleterious environment, the forms
would encyst immediately. This does happen occasionally and a few
hours after the extrusion of the amoebae has begun, cysts may be
found in the faeces. It happens just as often, however, that no cysts
appear in the faeces, although motile amoebae may appear for a period
of two or three days or until the final effects of the purgative have dis-
appeared. Numerous eysts are often encountered on the first examin-
ation after the purge is administered, indicating that the cysts were
already formed in the caecum, and that the caecal contents, whether
they contain free amoebae, encysted forms, or a mixture of the two,
are emptied to the outside.

504 University of California Publications in Zoology [Vou 20

A daily examination of the normal faeces, as they may be pressed
from the rectum of the rat, shows that there is no regular periodicity
in the formation of cysts (Brug, 1919, and Kessel, 1923a). At times
as long an interval as three weeks may elapse between the appearance
of cysts in the faeces, while at other times they may appear daily for
three consecutive days.

It is possible to detect the cysts in normal saline solution by their
light gray color and by the inner darker layer of the peripheral wall
which surrounds the cyst. It is, however, extremely difficult to dis-
tinguish the nuclei, and differentiation of species should not be based
on this type of superficial examination alone. In this preparation the
glycogen mass appears as a large fluid-filled vacuole.

In the iodine-eosin stain the cysts assume a greenish-yellow color,
presenting a shade which is about midway between the pale yellowish
green cysts of Endamoeba dysenteriae, and the more yellow cysts of
Endamoeba coli. In this preparation the nuclei stain definitely, and
the type of karyosome often may be clearly demonstrated. The
elyeogen masses which are present in a certain number of the eysts
are always stained dark brown by the iodine.

Shape.—The majority of the cysts are spherical or ellipsoidal in
shape, in optical section appearing round (pl. 45, fig. 23), perfectly
elliptical (pl. 45, fig. 20), or presenting varying degrees of elliptical
outline (pl. 48, fig. 7; pl. 44, fig. 10). No extremely irregular eysts,
like those often encountered in C. decumani (pl. 47, fig. 49), have ever
been observed by us in C. muris.

Cyst wall——A thin wall surrounds the eyst. In the iron-haema-
toxylin stain this appears quite transparent and can be defined only
by the inner and outer margins. In the iodine-eosin preparations,
however, it appears as a circle composed of three layers, in optical
section consisting of a thin outer line, a middle clear zone, and an
inner dark line. This resembles the cyst wall of C. laflewri (Kofoid
and Swezy, 1921). The thickness of the cyst wall is relatively con-
stant in all eysts, no matter what their dimensions may be. Measure-
ments show only a slight variation from 0.50u in the newly-formed
eysts to 0.75y in the larger and older cysts.

Sizes and races of Councilmania muris—Wenyon and O’Connor
(1917) and Dobell and Jepps (1918) have shown that EF. dysenteriae
and E. coli are divided into a number of distinet races, the determina-
tion being based on the size of the cysts. The five races of HE. dysen-
teriae have mean diameters of 6.6, 8.3, 11.6, 13.3, and 15y respectively,

1924] Kessel: Amocbae of Culture Rats and Mice 505

while in £. coli, the four races distinguished have mean diameters of
15, 16.5, 18.7, and 21.7 respectively.

In C. muris the eysts vary in diameter from 13 (pl. 44, fig. 16)
to 19 (pl. 45, fig. 23). Measurements of eysts have been made from
the material stained in iron-haematoxylin and three distinet races
have been determined, one with an average diameter of 14.5, a second
with an average of 15.7, and a third with an average of 17.74. The
accompanying table gives the range, average diameter, standard devia-
tion, coefficient of dispersion, and variability in percent of the differ-
ent races of C. muris. Cross-infection experiments of Cowneilmania
muris to non-infected rats have produced no racial change in the size
of the eysts.

TABLE 5
Races,
Range Average Standard Coéfficient Variability
diameter deviation of dispersion in per cent
C. muris. 13 to 16. 14.5 92 0634 44%
15 to 17.. 15.7 68 0511 33%
16 to 21. : le, ee 1.51 0856 43%

Cytoplasm and inclusions.—The cytoplasm in the cysts of C. muris
in structure looks like an accumulation of fine flakes. Stained with
iron-haematoxylin it assumes a gray appearance and the darker inclu-
sions within the eyst may be distinguished easily. This flaky structure
of the eytoplasm can be distinguished definitely from the alveolar type
of eytoplasm found in EF. dysenteriae and from the more coarsely
granular cytoplasm of EF. coli.

Three types of structures within the cytoplasm of the cyst are to
be distinguished, namely, the nuclei, and two types of inclusions, the
elyecogen mass and the chromatoidal bodies.

The glycogen mass, though present in the cysts at the time of
fixation, is dissolved out by water during the process of staining and
leaves merely a large clear vacuole. This vacuole is predominantly
present in the mononucleate and binucleate cysts (pl. 43, fig. 7; pl. 44,
fig. 11). An occasional mononucleate cyst is found without a glycogen
vacuole (pl. 44, fig. 9), but it is significant to note that no binucleate
cysts without glycogen vacuoles have been discovered. As the glycogen
is probably utilized in the metabolic processes attendant upon mitosis,
it seems likely that a mononucleate cyst without glycogen cannot com-
plete development. Around the margin of the glycogen vacuole chro-
matoidal bodies often appear (pl. 44, fig. 15). As the chromatoidal

506 University of Californa Publications in Zoology [Vou. 20

bodies often appear coincidently with the disappearance of the gly-
cogen mass, it seems likely that the latter may function in the forma-
tion of the former (Kofoid, 1923). These may be used up early in
the developmental process as is evidenced by the fact that many of the
four-nucleate, five-nucleate, and eight-nucleate cysts do not possess
chromatoidals (pl. 44, figs. 17-19) or they may persist to the eight-
nucleate stage (pl. 45, fig. 21). These chromatoidal bodies in the latter
stages have the form of massed bundles of splinter-like processes as is
shown by the irregular ends of the masses (figs. 24, 27). It is likely
that these massed chromatin bodies are formed by a union of the
smaller bodies that first appear around the margin of the glycogen
mass as is deseribed for C. laflewrt (Kofoid and Swezy, 1921). The
seattered acicular and filamentous type of chromatoidal body so often
seen in F. coli has not been found in this species.

Four-nucleate cysts may be found either with or without the glyco-
gen vacuole (pl. 44, figs. 16-17) though it is quite unusual to find the
vacuole persisting longer than to the four-nucleate stage.

Nuclear phases.—As the nuclei within the cysts are frequently in
a state of mitotic change, it is necessary to regard these changes in
making a diagnosis. Nuclei in cysts, prior to the eight-nucleate stage,
may show figures representing any phase of mitotic development. The
typical resting nucleus is more often encountered in the eight-nucleate
cysts and on this account they are more commonly used as a basis of
diagnosis.

The nuclear membrane of C. muris is characterized by its very
thin and transparent appearance. Small chromatin granules may lie
around the inner margin of the membrane, but this is common only
in the early prophase. As a rule the nucleus is characteristically clear,
and in the middle is the dispersed karyosome (pl. 45, figs. 21, 24, 25).
Linin fibers connecting the karyosome with the nuclear membrane are
more common in the earlier than in the later divisions. The nuclear
membrane is so faint that it is frequently difficult to detect, and its
position can often be determined only by the limits of the cytoplasm
which surrounds the nucleus. This nucleus presents an entirely differ-
ent picture from the nucleus of #. coli, which has a thick membrane
heavily encrusted with chromatin granules, and from the nucleus of
C. decumani the more distinct membrane of which is usually encrusted
with several heavy chromatin blobs.

The nuclei in the mononucleate eysts (pl. 43, fig. 7) are the largest,
and a corresponding diminution in the size of the nuclei is seen as the

1924 | - Kessel: Amoebae of Culture Rats and Mice 507

number of nuclei increases. For a ratio of the size of the nuclei in this
species anid in C. decumani see Kofoid, Swezy, and Kessel (1923a).
In material obtained after the administration of a purgative and
from autopsies, the eight-nucleate cysts are the most numerous while
the binucleate cysts are next in number. The binucleate cysts usually
show some form of mitotic spindle and it is from this type of eyst
that the majority of the chromosome counts have been made.

No sixteen-nucleate cysts have been found from this species, though
nuclei have been found in the eight-nucleate cysts which indicate that
a further division is in progress.

Chromophile ridge—In a majority of the eight-nucleate cysts, a
dark-staining ridge is found, which is located in the cytoplasm along
the inner margin of the cyst wall. It may often extend a distance
ranging from one-quarter to one-half of the circumference of the cyst
(pl. 45, figs. 20, 25). This ridge is not to be confused with the chro-
matoidal bodies, which are usually not curved and as a rule le
embedded in the cytoplasm. Similar ridges, which they have termed
chromophile ridges, have previously been described in Councilmania
lafleuri by Kofoid and Swezy (1921). It is significant to note that
Wenyon (1907, pl. 10, fig. 1) has figured a cyst which presents this
typical structure.

BuppING

Although it is a fact that the motile amoebae divide by simple
fission, it is to be noted that this process is comparatively rare, few
mitotic spindles ever having been recorded in motile forms and only
a few instances of binucleate motile forms ever having been seen.
The present investigation verifies this rarity of dividing motile forms,
even in material collected from the caecum of autopsied animals. How-
ever, in these same caeca numerous cysts have been found, a fact which
indicates that multiplication by encystment in this species of amoeba
may be much more common than multiplication by binary fission of the
motile phase.

The determination of the manner by which the amoebae escape
from the cysts has presented numerous difficulties and has called forth
several interesting theories. In EF. dysenteriae it is generally thought
that in some manner the four-nucleate amoeba escapes from the cyst,
and that subsequent division into four amoebulae occurs (Chatton,
1917; Dobell and O’Connor, 1921). Again, E. coli is supposed to
eseape from the eyst in a similar manner as an eight-nucleate amoeba

508 Unwersity of California Publications in Zoology [Vou. 20

(Yoshida, 1918). Wilson (1916) described pores in the cyst wall of
the soil amoeba and observed the process of an amoeba emerging from
the eyst through one of these pores. Kofoid and Swezy (1921)
recorded the process of budding for Cowncilmania laflewri. By this
process a small amount of cytoplasm, together with a single nucleus,
emerges through a pore in the cyst wall, and the cytoplasm containing
the nucleus breaks off forming a small amoebula. This process is
repeated until the cyst is emptied of its contents. If pores are easily
demonstrable in cysts of soil amoebae, it seems likely that they may
occur in other amoebae.

The present investigation of the amoebae of rats and mice was just
being started at the time the observations on budding in C. lafleuri
were recorded. Many similarities were noticed between C. lafleuri and
the amoebae of the rat and mouse. These, together with certain
figures by Wenyon (1907, pl. 10, figs. 12, 13, 18, 20, 21), though
explained by him in a different manner, led to the thought that bud-
ding would probably be found in Councilmania of rats and mice. The
assumption was easily verified by observation in the first few animals
examined and numerous instances of the process have since been
recorded. In C. muris, chromophile ridges and also chromatoidal
bodies are common, and observations to date indicate that the budding
process occurs in a manner similar to that in which it occurs in
C. laflewri (Kofoid and Swezy, 1921).

Evidence for this process is based on the following observations:

1. Buds have been seen in fresh smears prepared in iodine-eosin
and normal saline, without pressure or other mechanical disturbance
having been brought to bear on the cysts.

2. Buds have been seen in slides permanently stained with iron-
haematoxylin. These cysts contain varying numbers of nuclei, indi-
cating that some of the nuclei have passed out in previously formed
buds. In plate 45, figure 26, the whole eight nuclei are present, one
having passed into the bud; plate 44, figure 28, shows a eyst with six
nuclei, one in the bud, and figure 22 of the same plate shows four
nuclei in the cyst, and one in the bud. It further shows light patches
in the cytoplasm, indicating spaces from which the nuelei and their
surrounding protoplasm have emerged.

3. Small amoebulae about the size of buds are often found in
proximity to the cysts (pl. 45, fig. 22), indicating that they have
previously emerged from the cyst.

1924 | Kessel: Amoebae of Culture Rats and Mice 509

Mirosis

Accurate cytological interpretation of cell phenomena in parasitic
amoebae is difficult at best and requires the careful and detailed study
of a great number of specimens. In the present investigation, 170
drawings have been made with the aid of the camera-lucida and
large numbers of other forms have been studied in detail with the
best magnification available. The 62 figures on the plates have been
chosen as the most representative of the various phases of cytological
development.

The developmental process in this species of amoeba is similar in
method to that recorded for other parasitic amoebae, and except in
specific differences the mitotic stages simulate those already described
for C. lafleurt (Kofoid and Swezy, 1921) and for Endamoeba coli
(Swezy, 1922). The nucleus in the mononucleate cyst divides mito-
tically to form two nuclei, a second division then occurs to form four
nuclei, and finally a third division, which results in a typical eight-
nucleate cyst which is regarded as the mature cyst of this species.

The typical resting stage of the nucleus has already been described.
This persists for a longer or a shorter time between each division, is
always typical, and is found most commonly in the eight-nucleate
eysts. The second division seems to require a longer time than either
of the following divisions, judging from the fact that mitotie figures
are most commonly encountered in binucleate cysts. Owing to the
presence of the glycogen vacuoles, the nuclei are usually somewhat
flattened and pressed to one side of the cyst (pl. 44, fig. 11).

Prophase.—The fact that more prophase stages were found than
stages of any other phase indicates that this period occupies a longer
time than any other phase of mitosis. During this stage there is a
complete reorganization and transformation of the contents of the
nucleus. The first indication of this phase is the arrangement of a
few small granules of chromatin on the nuclear membrane (pl. 43,
fig. 4). The granules of the karyosome become somewhat more dis-
persed than usual and numerous linin fibers connect the granules of
the karyosome with the chromatin granules on the nuclear membrane.
The granules on the membrane disappear and a darkly staining region
appears around the granules arranged in the middle of the cyst (pl. 43,
fig. 7; pl. 44, fig. 11). About this time a distinct line, the intradesmose
(Kofoid and Swezy, 1921) appears between two of these granules
(pl. 48, fig. 6). These granules then pull further and further apart

510 University of California Publications in Zoology {Vo- 20

(fig. 7) and finally take their positions at the ends of the spindle, form-
ing the polar caps or centrosomes (pl. 44, fig. 9). They are still con-
nected by the darkly staining line which appears to lie in close prox-
imity to the inner face of the nuclear membrane. During the time
that the centrosomes are taking their position at either margin of the
nuclear membrane, the granules of the dispersed karyosome and the
dark gray region around them undergo a complete readjustment, out
of which emerges a characteristic spindle upon which are arranged
the chromosomes in a late prophase (fig. 8). During this process the
linin network disappears and the nucleus becomes elongated (fig. 9).
Wenyon (1907) stated that there did not appear to be a formation of
definite chromosomes, but from the present study it appears that six
chromatin masses are arranged on the spindle (pl. 44, fig. 8), and the
conclusion is that these are the chromosomes. C. muris thus differs
from C. laflewri, which has eight chromosomes (Kofoid and Swezy,
1921), but agrees with H. coli, which also has six (Swezy, 1922).

Metaphase——tThe separation of the chromosomes on the spindle to
form the metaphase is not always synchronous. In plate 44, figure 9,
the two upper chromosomes appear in the metaphase, while the lower
chromosomes have already divided and the daughter chromosomes are
beginning to separate. This non-synchronous division of chromosomes
is also illustrated in the lower nucleus (pl. 44, fig. 14) in which some of
the daughter chromosomes have nearly reached the polar caps, while
two are still in the middle of the spindle.

The chromosomes appear as small granules, some representing
spheres, while the others are somewhat elongated. There is evidence
that of the six chromosomes in this species, two are large ellipsoids,
two are small spheres, and the other two are slightly elongated and
midway between the other pair in size (pl. 44, figs. 12, 17).

Anaphase——After the division of the chromosomes in the meta-
phase there is an immediate pulling apart of the daughter chromo-
somes toward either end of the spindle as is shown in the lower nucleus
(pl. 44, fig. 18) and the upper nucleus (fig. 14). This separation is
accompanied by a disappearance of the spindle fibers between the
separating daughter chromosomes and by a further elongation of the
nucleus (pl. 48, fig. 13, lower nucleus). The intradesmose also becomes
fainter in the median region and a complete separation of the polar
caps ensues as the intradesmose retracts or fades away.

Telophase.—As good figures of this phase have been particularly
rare, it is concluded that the final separation into two nuclei is com-

1924 | Kessel: Amoebae of Culture Rats and Mice 511

pleted rather suddenly. The middle nucleus of figure 18, plate 44,
represents the polar cap and chromosomes arranged at either end of
the nucleus, only traces of the extreme ends of the spindle fibers
remaining, and shows a very marked constriction of the nuclear mem-
brane in the middle region. This constriction of the nuclear mem-
brane is also shown in figure 10, plate 44, but the nuclear contents
have already become arranged in an early prophase of the second
division. The upper nucleus (pl. 44, fig. 16), shows the final dividing
stage of the nucleus into the two daughter nuclei, the nuclear contents
representing a typical resting stage while the lower nucleus of the
same figure shows the final separation as having oceurred and the
nuclear membrane fully formed around each daughter nucleus.

The process of nuclear division in parasitic amoebae differs from
that in the metazoan cell in that the nuclear membrane remains
intact throughout the whole process. It is for this reason that the
polar caps of the spindle and the intradesmose connecting them must
be formed from material within the nucleus.

The divisions of the nuclei within the same cyst do not always
take place concurrently (pl. 44, fig. 15). Here the larger nucleus
represents the first division while the two smaller nuclei have already
completed the second division. The nuclei in figure 18 exhibit the same
phenomenon: the two larger nuclei have resulted from the second
division, the middle nueleus is just completing the third division, and
the two small nuclei have already completed the third division.

COUNCILMANIA DECUMANI (Rupovsky, 1921) EMEND. KESSEL

Councilmania decumani has been found in 14 of the 20 mice and
in 9 of the 24 rats examined by us. We believe this is the species of
amoeba from rats dealt with, for the most part, at least, in the accounts
by Brug (1919) and by Rudovysky (1921). Rudovsky (1921) noted
a difference between the amoeba of the mouse and the amoeba of the
rat. He called the latter Endamoeba muris decumant. On account
of the characteristics to be mentioned in the following account, it is
necessary to transfer this species from the genus Endamoeba to which
it was assigned by Rudovsky to the genus Cowncilmania (Kofoid and
Swezy, 1921).

eile University of California Publications in Zoology [Vou 20

Tue Morire AMOEBA

When examined in smears prepared in normal saline solution,
there is little or no apparent difference between the motile forms of
C. muris and C. decwumani. The same type of hyaline pseudopodial
formation (pl. 46, fig. 32) and of amoeboid movement are exhibited
and the amoebae inhabit the same parts of the digestive tract. The
food is similar and there is no evidence of a pathogenic effect upon
the rodent host. Cases of apparent cannibalism, however, have been
encountered in this species (pl. 46, fig. 30). A typical small amoeba
of this species is contained in a food vacuole within the larger amoeba.
The nucleus and food vacuoles of the ingested form are intact. Enda-
moeba coli has been shown to ingest cysts of H. dysenteriae (Wenyon
and O’Connor, 1917) and Lepage (1922) recorded a very interesting
instance of cannibalism in Amoeba vespertilio (Penard). In C. decu-
mani instances of binucleate motile forms have also been encountered
(pl. 46, fig. 31) by us in which the nuclei appear quite normal.

When stained in iron-haematoxylin the nuelei of C. decwmani
present an entirely different appearance from those of C. muris. The
nuclear membrane is heavier and more distinct and much more chro-
matin material is encrusted thereon in C. decwmani than in C. muris.
In the former species, a distinct, more or less massed karyosome is
found in an exeentrice position. This may assume the form of a sphere
(pl. 46, fig. 30) or it may appear in the form of a crescent (fig. 32).
The karyosome may be surrounded by a light gray halo (figs. 30, 32),
or it may appear as an outstanding black sphere (fig. 31, upper
nucleus). Linin fibers connect the karyosome with the chromatin
material encrusted on the nuclear membrane, and small dots often
appear where these fibers cross.

ENCYSTMENT

The process of eneystment is a generalized one for parasitic
amoebae and nothing of importance differentiates the methods in C.
decumant and C. muris. Of great importance, however, is the differ-
ence in appearance of the contents of the eysts. The cytoplasm in
the eysts of C. decwmani is more coarsely flaked than in C. mwris, and
the chromatoidal bodies, while often arranged in sheaves of splinter-
like processes, as in C. muris (pl. 47, fig. 48), also commonly oeeur with
rounded ends, curved surfaces and as small spheres (fig. 50). Chro-

1924] Kessel: Amoebae of Culture Rats and Mice 513

matoidal bodies are not found as commonly in the eyst of C. decumani
as in those of C. muris. Chromophile ridges (pl. 48, fig. 51) are not
common in this species, traces of small ones having been found only
three times.

The structure of the nuclei is the important feature differentiating
the cysts of C. decumani from those of C. muris. As already noted
in the motile forms, the nuclear membrane is more distinet in C. decu-
mani than in C. muris, and small granules or large blobs of chromatin
material are encrusted on the inner surface. This presence of char-
acteristic chromatin material on the nuclear membrane is a constant
feature throughout all the stages of mitosis. The typical resting
nucleus is characterized by the presence of an excentric karyosome
which is much more massed than the karyosome of C. muris (pl. 48,

TABLE 6
Races,
Range Average Standard Coéfficient Variability
diameter deviation of dispersion in per cent
C. decumani 12 to 15...... 14.0 1.00 0714 51%
14 to 16.5. 15.5 98 .06382 A1%
eutorZ0 ee Wie 1.09 0641 388%

fig. 52), and yet, in certain instances, presents a tendency toward
diffusion (pl. 46, fig. 37). Linin fibers connect the karyosome and
nuclear membrane, and these can usually be easily detected, even in
the eight-nucleate eysts. The nucleus, seldom, if ever, presents the
transparent appearance of the nuclei so characteristic of C. muris.

The majority of the cysts are spherical, though ellipsoidal (pl. 46,
fig. 35), and irregularly-shaped forms (pl. 47, fig. 49) are found. The
irregular shape of the cyst in figure 49, simulates the shape of a motile
amoebae. One wonders if Schaudinn (1903) did not confuse irregu-
larly-shaped cysts of this type with motile amoebae when he elaborated
his theory of ‘‘schizogony’’ for E. coli.

As in CO. muris, three races of amoebae have been determined in
OC. decumani, the smallest averaging 14, the next 15.5 and the largest
17 (see table 6). The eight-nucleate cyst is regarded as the typical
mature cyst, but instances of sixteen-nucleate cysts have been found
(pl. 48, fig. 52). In this ease the eyst is abnormally large, being 22y

in diameter.

514 University of California Publications in Zoology (Vou. 20

BUDDING

Instances of budding have been found in C. decumani in material
from the mouse, from the rat, and in induced cross-infections from
each of these animals to the other.

One important feature, however, differentiates the budding process
in C. decumani from that in C. muris. This is the absence of its
association with an excessive amount of chromatoidal material or with
the general presence of chromophile ridges. A comparison of the
figures at the top of plate 48 with those on plate 45 brings out a most
striking difference. This feature affords important evidence in favor
of the theory that a pore is normally present in the cyst wall and that
at the proper juncture in the life history, this opens and permits the
contents of the cyst to pass to the outside. Figure 54, plate 48, shows
a first bud in the early stages of formation. Figure 55 shows a
nucleus at the pore or point of emergence of the bud. It is somewhat
pointed and has begun to elongate. Three other nuclei remain in the
cyst and there are empty patches in the cytoplasm, the material from
which has gone to form buds that have previously been discharged.

Mitosis

The general process of mitosis is similar to that of C. muris, but
certain important differences should be noted.

Prophase.—The first indication of this phase is the enlargement
of the halo or gray area around the karyosome (pl. 46, figs. 33, 34)
in which chromatin granules, much smaller than the chromosomes, may
appear. About this time the large, massed karyosome begins to divide
into two parts (fig. 834) which move away from each other. As the
portions separate, they are connected by the intradesmose (fig. 38).
These two masses take up their position at either end of the somewhat
elongated nucleus (pl. 47, fig. 39) and form the centrosomes. As the
amount of chromatin material encrusted on the nuclear membrane
becomes less during these processes of development, it is probable that
a portion of this material functions in the formation and organization
of the spindle and chromosomes. In the gray-staining area between
the centrosomes, the spindle emerges with the chromosomes arranged
on its fibers. As in C. muris, no linin fibers are seen in nuclei in the
late prophase.

Metaphase.—Figures of the metaphase (pl. 47, figs. 41, 45) in this
species exhibit four chromosomes. They are spheroidal in shape, one

1924 | Kessel: Amocbae of Culture Rats and Mice 515

being large, two medium in size, and the fourth so small that it does
not stain as darkly as the others.

The processes, resulting in the formation of the anaphase and telo-
phase, are carried out in a manner similar to those already described
for C. muris.

ENDAMOEBA RATTI sp. nov.

This species of amoeba has been encountered in only 3 of the 288
rats examined to date and in none of the mice. As its characteristics
warrant its classification with the genus Endamoeba, this species has
been named Endamoeba ratti sp. nov. Permanent slides, on which
are to be found stained specimens of both the cysts and free amoebae,
have been available from two of these three rats.

THe Morine AMOEBA

Early in this investigation, a motile amoeba was observed in a
normal saline preparation which presented characteristics entirely
different from those exhibited by the other amoebae found in rats
and mice. In comparison with amoebae of the genus Councilmania,
the general appearance of this amoeba, when rounded, is more
opaque; the structure of the endoplasm is more vacuolated and granu-
lar; and the pseudopodia never present the characteristic hyaline
appearance of the pseudopodia formed by amoeba belonging to the
genus Councilman.

In progressive movement, the pseudopodia formed are always
granular and the endoplasm constantly extends to the outermost
margin of the pseudopodium (fig. C). The amoeba does not progress
by a looping method, as described for Cowncilmania, but rather flows
forward by a streaming movement, rounding up less often than
Councilmania. The nucleus is often located in the extreme anterior
end of the amoeba during this forward movement. The posterior,
conical projection, already described in Councilmania, is more
prominent and more constant in this species. The pseudopodia are
characteristically blunt and granular.

When attached, the amoeba occasionally produces a narrow ecto-
plasmic margin when the pseudopodium first begins to form. This
lasts only for an instant and the endoplasm immediately flows into
it before its completion. A completely formed hyaline pseudopodium
has never been seen, and the conical pseudopodia, common in Cowncil-
mania, are not formed. On the whole, the amoeboid movement of this

516 University of Califorma Publications in Zoology [Vou 20

species of amoeba simulates very closely the movement of Endamoeba
coli as described by Dobell (1921), and Kofoid, Swezy, and Kessel
(19230).

Endamoeba ratti, although possessing many of the characteristics
of EF. coli, is smaller in size, the average diameter of rounded, unen-
eysted forms being about 19, while that of HZ. coli ranges from 20p
to 30” (Dobell and O’Connor, 1921).

10 11
Fig. C
Free-hand sketch of Endamoeba ratti while in progressive movement.

The nuclear membrane is definite and is encrusted with chromatin
which may present a beaded appearance or a darkly staining ring
(pl. 48, fig. 57). The karyosome is excentrie and may be surrounded
by a clear or a gray halo (fig. 57).

The food appears to be the same as that ingested by the other non-
pathogenic amoebae of the intestinal tract of rats and mice.

ENCYSTMENT

As permanent slides have been procured from only two cases of
infection of Endamoeba ratti, it has been impossible to secure a com-
plete series of the developmental stages of the cysts. A sufficient num-
ber of the eight-nucleate cysts have been found to give a typical picture
of the ripe eysts, and a few of the intervening stages have been seen.

1924] Kessel: Amocbae of Culture Rats and Mice 517

The nuclei in the resting stage simulate very closely the typical
nuelei of E. coli (pl. 48, figs. 58-62). The nuclear membrane is dis-
tinet and may or may not be encrusted with chromatin granules. The
karyosome is in the form of a small but distinct excentrie sphere. The
diffuse, excentric karyosome and the heavy, peripheral chromatin blobs
found in C. decumani have not been seen in this species. The binu-
cleate cysts (pl. 48, fig. 59) have a large glycogen vacuole similar to
the type found in £. coli, but the chromatoidal bodies (fig. 60) are
more massed than in the type usually found in FL. coli.

No evidence of budding has been found in the eysts thus far
encountered.

The average diameter of the cysts in this species is 14.64. This
is much smaller than the average diameter of the cysts of FE. coli which
is 17.3p.

The conclusions to be drawn at the present time from the study of
this species is that it differs from E. coli with respect to size, and in
the massing of the chromatoidal bodies.

Endamoeba coli that have passed through rats during this investi-
gation maintain the structural and size differences mentioned above
and it is on this basis that #. ratti is differentiated from £. colt.

EXPERIMENTS IN CROSS-INFECTION

While careful study of the stained material at hand indicates
decisively that Cowncilmania muris and C. decumani are distinet
species, in the light of the successful infection of rats with the amoebae
of the human intestinal tract (Kessel, 1923b) in which transfer the
amoebae underwent no visible morphological change during the period
of the experiment, cross-infection of C. muwris and C. decumani was
attempted from rats to rats, from rats to mice, from mice to mice, and
from mice to rats. The results are tabulated in the accompanying
tables.

In this experimental work the amoeba-free rats and mice were
chosen by the epsom salt method as in the other infection experiments
carried on in this investigation. As very young amoeba-free mice
were not always on hand, it was necessary in most cases to use old mice.
It has been found throughout this investigation that old rats and mice
are less susceptible to infection than young stock. This suggests that
the older animals are able to establish an active immunity against
infection. In the series of experiments in attempting to transfer C.

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1924 | Kessel: Amoebae of Culture Rats and Mice 519

decumani from mouse to mouse and from mouse to rat, faeces were
fed from the same mouse to both the mice and the rats. As the result
was positive in rats and negative in the mice, the indication is that
the reason for the negative results in the mice is to be traced to some
condition within the mice rather than to a non-viable condition of the
eysts. In transferring C. muris from mouse to mouse and from mouse
to rat, faeces from the same mouse were used to feed both the rats and
mice. As similar results were obtained in this experiment as in the
previous one with reference to non-infeetion of the mice, similar
conclusions are applicable.

TABLE 7

TABLE SHOWING RESULTS OF EXPERIMENTS IN CROSS-INFECTION OF C. muris AND
C. decumani FROM Rats TO MICE AND FROM MICE To Rats

C. muris
Period of |Number of Amoeba
feeding animals Positive Negative found Conclusion

Mouse to mouse | 3 weeks 2 0 2 0 Immunity
Mouse to rat........| 4 weeks t 4 0 C. decumani| No change
Rat to rat 3 months 5 3 2 C. decumani| No change

3 weeks 3 2 1 C. decumani| No change
Rat to mouse.....| 2 months 2 0 2 0 Immunity

3 weeks 2 0 2 0 Immunity

C. ratti
Period of |Number of Amoeba
feeding animals Positive | Negative found Conclusion

Mouse to mouse | 3 weeks 2 0 2 0 Immunity
Mouse to rat........| 3 weeks 3 2 1 C, muris No change
Rat to rat 1 feeding 3 3 0 C. muris No change
Rat to mouse.....| 2 months 4 2 2 C. muris No change

2 months 2 0 2 0 Immunity

1 feeding 2 2 0 C. decumani

and C.
muris Mixed*

* This case of mixed infection is thought to be due to a positive infection of
C. decumani which was overlooked in the examination with epsom salt.

The facts that C. decwmani has been successfully transferred from
rats to rats and from mice to rats without any perceptible morpho-
logical change, and that C. muris has also been transferred from rats
to rats, from mice to rats, and from rats to mice without a morpho-
logical change, are further evidence in favor of the constancy of the
characters upon which both of these species are established.

520 University of California Publications in Zoology [Vou 20

It is much more difficult to force mice to take epsom salt than it
is to induce rats to take it. Because of this fact, one is not always
certain that the experimental animal is amoeba-free, as is the case
with the experimental rats. Further, the salt method of obtaining
amoeba-free animals has not been conclusively tested with mice, as it
has with rats. Attempts to infect mice with the amoebae of the human
digestive tract have not met with the success that attended similar
infection experiments with rats. It is thus easily understood that
mice are much more difficult to work with than rats. The mixed
infection of the third attempt to transfer C. muris from rat to mouse
may be therefore attributable to a probable previous infection of
C. decumani that had been overlooked in determining the amoeba-free
mice.

Culture——No success has attended our attempts to culture para-
sitic amoebae in artificial media. Although instances have been
recorded in the earlier works in which E. coli and EF. dysenteriae are
supposed to have been cultured, it is now generally recognized that
the workers were dealing in their culture amoebae with non-parasitie
species (Dobell, 1919). In this investigation it was impossible to
engage in extensive attempts to culture amoebae from the rat, but
several brief endeavors were made.

The first medium used was that recommended by Musgrave and
Clegg (1904), for E#. coli. Attempts were made with an alkaline
medium in an anaerobie environment only and were unsuccessful.
Wenyon (1907) had previously tried this medium both aerobically
and anaerobically, but without success.

In order to simulate more closely the environmental conditions of
the caecum in which the amoebae live in greatest numbers, at the sug-
gestion of Dr. T. D. Beckwith of the Department of Bacteriology of
this University, the hydrogen ion concentration of the caeca of several
rats was taken. The caeca of eight rats non-infected with amoebae,
and of eight rats that showed amoebic infection, were tested. The
average pH of the caecal contents of both amoebae-free rats and of
rats with positive amoebic infection was 6.35.

Rettger and Cheplin (1921) showed that the pH of the faecal
material from the caecum and colon ranged from 5.5 to 6.7 in the
various rats they tested. They failed to differentiate between the pH
of the caecum and other parts of the digestive tract. Furthermore,
their work was carried on in connection with the intestinal flora of
rats and no record was given of the presence or absence of amoebae.

1924 | Kessel: Amocbae of Culture Rats and Mice 521

At the suggestion of Dr. I. C. Hall of the Department of Bacteri-
ology of this University, one series of rats of known amoebic infection
with C. muris or C. decumani was fed on a meat diet, only, for two
weeks, and another series of rats known to be infected with amoebae
was fed only skimmed milk for two weeks. Infections were determined
by the epsom salt method (Kessel, 1923a). After the feeding had
been continued for two weeks, the experimental animals were autopsied
and examined for amoebae. Of the 15 rats fed on milk diet only, the
whole number had been cleared of amoebie infection. Of the 6 rats
fed on meat diet for the two weeks, 4 had been cleared of amoebic
infection. Nineteen amoebae-positive rats were fed on the regular diet
of table scraps and were used as a control. Of the previously infected
control rats 7 were found to have been cleared of amoebic infection
on terminal examination, indicating that the purgative may, at times,
clear some of the rats of infection, or that an active immunity may
arise.

As all the rats fed on milk diet, while only two-thirds of the rats
fed on a meat diet, had been cleared of infection, it was considered
that a culture medium of high animal protein content was more suit-
able for the amoebae than one of higher carbohydrate content such as
milk. Cannon and MecNease (1923) have shown that when meat is
fed to white rats the reaction of the contents of the caecum is prac-
tically neutral. Consequently, beef broth and brain media were pre-
pared and titrated to a pH of 6.35. Motile amoebae from the caecum
of newly autopsied animals and cysts were planted in these media, both
in the broth and in a 0.5 per cent agar. These were incubated at 37° C.
under both aerobic and anaerobic conditions. No success attended
these’ attempts, apparently because of the putrefaction produced by
the rapid growth of bacteria.

One important result derived from this phase of the work should
not be overlooked and that is the fact that all the rats fed on a milk
diet, only, for two weeks were amoeba-negative at the end of that time,
while of the control rats only 27 per cent were negative. It is interest-
ing to note that Hegner (1923) obtained reductions in the number of
flagellates in rats fed on a milk diet though his rats were not entirely
rid of the infection.

It has been shown by Rettger and Cheplin (1921) and Cannon and
McNease (1923) and others preceding them that feeding milk only to
rats produces a change in the flora of the digestive tract from a
majority of gram-negative rods, presumably Bacillus coli, to a pre-

522 University of California Publications in Zoology [Vou 20

dominance of gram-positive rods, Bacillus acidophilus. Cannon and
McNease have further shown that the pH of the caeca of rats, fed on
meat only, averages 7.1, while the caecal contents assume an acidity
of pH 4.5 when the animals are fed on milk only.

It would seem that one or both of two main factors are responsible
for the disappearance of the amoebae from the digestive tract of the
rats. First, it has previously been noted in this paper that amoebae
ingest gram-negative bacilli rather than gram-positive bacilli. The
change in the flora from gram-negative to gram-positive rods may
result in the starvation of the amoebae. Second, the great increase
in the acidity of the caecal contents as the result of the lactie acid
produced from the milk may have an antagonistic effect on the
amoebae.

DISCUSSION

Classification and systematic relationships.—Differentiation into
species of animals so minute as amoebae and with such a limited num-
ber of characteristics has always been a difficult problem. In non-
parasitic amoebae, species characters are generally based on the type
of pseudopodia and the nuclear structure (Schaeffer, 1920). Schaef-
fer said that these characteristics are hereditary and are due to
“‘fundamental chemical structure of the protoplasm which is specific
for the species.’’ In speaking of parasitic amoebae, Dobell (1921)
states that the important structural characters distinguishing the
various genera and species are supplied by the nuclear apparatus and
by the cysts. Differences in the types of pseudopodia formed and
structural differences in the protoplasm are also generally regarded
as bases for differentiation of species. It is recognized by the writer
that much unwarranted differentiation into species has oceurred in
the past, involution forms of the same species having been regarded
as showing specifie differences. The mechanism of mitosis is similar
in all species of parasitic amoeba and in certain phases of mitosis it
is often impossible to distinguish different species from each other.
The tendency to confuse these different phases should be carefully
euarded against and only those characteristics which are permanent
at some definite phase of the life cycle should be considered in differ-
entiation of species; e.g., the resting stage of the nucleus.

The further facts that it is possible for the same species of para-
sitie amoeba to be transferred from host to host without any recogniz-
able morphological change (Kessel, 1923)) and that transfer of

1924 | Kessel: Amocbae of Culture Rats and Mice 523

Councilmania muris and C. decumani recorded in this investigation
has been successful indicate that the environment in which a parasitic
amoeba may be found is not necessarily indicative of its species. This
possibility should always be taken into consideration before new
species are named and described.

In this investigation, the different species of amoebae found in
rats and mice have been separated mainly on differences in pseudo-
podial formation and protoplasmic structure, on differences in the
nuclei found in the resting stage, for in the cysts a condition exists
primarily in which environmental factors are least influential, on the
differences in chromosome count, and on differences in the structure
of the chromatoidal bodies.

Councilmana muris belongs to the genus Couwncilmania because it
possesses chromophile ridges, reproduces by budding from the eyst in
the caecum, produces hyaline pseudopodia, and possesses a dispersed
karyosome. It differs from C. laflewri in size, and in the possession of a
very thin nuclear membrane upon which little or no chromatin is ever
encrusted. The granules in the karyosome are smaller and are more
dispersed in motile amoebae of this species than in those of C. laflewri.
It has six chromosomes, while C. laflewri has eight.

Councilmania decumant belongs to the genus Cowncilmania because
of its reproduction by budding from the cyst, because it produces
hyaline pseudopodia, and because of its dispersed karyosome. It
differs from C. laflewri and from C. muris in that the karyosome is
excentric and is more massed than in either of these species, and in that
the nuclear membrane is more distinct and is encrusted with large
chromatin blobs. There is less chromatoidal substance in this species
than in either C. lafleuri or C. muris, and there is not so pronounced a
chromophile ridge. This species has four chromosomes.

Up to the present time no amoeba possessing granular and vacuo-
lated pseudopodia has been described from the rat or mouse. Balfour
(1922), however, did mention that on two occasions during his investi-
gation cysts of the EF. coli type were noted from black rats. As he
also suggested that they were possibly the same as those found by
Brug (1919), we conelude that Balfour had no opportunity to differ-
entiate between E. coli of man and the coli-like and other amoebae cf
the rat.

If differences in the structure of pseudopodia have specific values,
as the extensive work of Penard (1904) and Schaeffer (1921) indi-
cates, it then follows that an amoeba which possesses hyaline pseudo-

524 University of California Publications in Zoology [Vou 20

podia cannot belong to the same species as an amoeba that forms granu-
lar pseudopodia. This difference alone should be sufficient evidence
for the establishment of a new species.

During this investigation, an amoeba has been encountered in the
intestinal tract of the rat which moves by the formation of granular
and coarsely vacuolated pseudopodia. Except in very rare instances,
when the amoeba is very sluggish in movement and for the most part
rounded, when a narrow hyaline margin of ectoplasm may oceur for
an instant only, this amoeba shows absolutely no differentiation
between ectoplasm and endoplasm. It has never been observed to
form a pseudopodium, completely hyaline, which is the only type of
pseudopodium ever observed in Councilmania. The type of pseudo-
podial formation observed in this amoeba resembles that described by
Dobell (1921) for E. coli.

The nuclear structure of this amoeba also differs from the nuclear
structure of Couwncilmania in that the karyosome is solid rather than
diffuse. It further: differs from species of amoebae found in the rat
that belong to the genus Councilmania in that reproduction by bud-
ding from the cyst in the caecum has not been observed in it. Because
of these differences, it is concluded that this species of amoeba cannot
be placed in the genus Councilmania.

Since the pseudopodia are of the granular type and since the
karyosome is massed and excentric, as in the type species E. blattae
(Biitschli) and also in E. coli (Lésch), we conelude that this amoeba
from the rat should be placed in the genus Endamoeba.

This amoeba found in the rat differs from EH. coli of man in size
and in the type of chromatoidal body, which simulates the chroma-
toidal body of C. decumani more than the chromatoidal body of F. coli.
There is also less tendency for the chromatin material encrusted on the
nuclear membrane to arrange itself in a bead-like structure. It is
usually encrusted in a solid ring, or in granules spaced widely apart.
‘This, however, may be physiological rather than specific. Because of
these differences between the amoeba possessing granular pseudopodia
that is found in the rat and EZ. coli of man, it is proposed to eall this
amoeba Endamoeba ratti sp. nov.

Pseudopodial formation—The structure of the protoplasm and the
type of pseudopodia formed by the various amoebae, both free-living
and parasitic, must be associated with the biochemical construction
of the protoplasm of which the specific amoebae are composed. It
has been suggested by some investigators that the difference in pseudo-

1924] Kessel: Amoebae of Culture Rats and Mice 525
podial formation may be merely a difference of the moment, dependent
upon the immediate environmental condition. It is, however, more
generally accepted that the structure of the protoplasm and the type
of pseudopodia formed are constant in the normal environment and
may be used as characters for differentiating species.

In the amoebae previously described as parasitic in rats and mice, it
is Significant to note that, without exception, they have been described
as possessing hyaline pseudopodia. It is mentioned by every investi-
gator that there is a sharp line of demarcation between the endoplasm
and ectoplasm in the formation of pseudopodia. It seems that the
work has been carried on over a sufficient period of time and by men
of sufficiently sound methods in this line of investigation to con-
elude that if there had been any possibility of change from a hyaline
to a granular type of pseudopodium in this species of amoeba, it would,
at some time, have been noticed. It therefore seems from the work of
the past and from the present investigation that the characteristic of
hyaline pseudopodial formation is a definite and constant general
character in these two amoebae, C. muris and C. decumani.

As already quoted from Dobell (1921), the growing impression
is that in Endamoeba coli ‘‘no sharp line of demarcation separates the
ectoplasm from the endoplasm.’’ With the exception of Rudovsky,
all the previous investigators mention size as the chief differentiating
character between E. coli and the amoebae of the rat and mouse that
have been described by them. It follows that, if pseudopodial forma-
tion is to be regarded as a specific character, #. coli and the amoebae
of the rat and mouse described as Councilmania muris and C. decumani
cannot be identical. ;

The possibility that one species of amoeba may form hyaline
pseudopodia at one time and granular pseudopodia at another time
has been constantly borne in mind during the present investigation,
but no evidence supporting this theory has been found in any of the
cases examined. One instance, which the writer considers to be a case
of a mixed infection, was found in which amoebae possessing hyaline
pseudopedia and other amoebae possessing granular pseudopodia were
found in the same field of the microscope. The actively motile forms
of the amoebae exhibiting hyaline pseudopodia in this particular
instance rounded up after being under observation about ten minutes,
and showed no further activity, while the amoebae with granular
pseudopodia remained active for an extended length of time. This
tendency of amoebae to become inactive after being in the unusual
environment of the normal saline smear for a short period is char-

526 University of California Publications in Zoology Vou. 20

acteristic of the species belonging to the genus Councilmania found
in the rat. On the other hand, Endamoeba coli is not so easily affected
by the change of environment and may continue its activity in normal
saline on the warm stage for several hours (Kofoid, Swezy, and Kessel,
19230).

Evolutionary source of infection of Councilmania of man.—The
source of the infection of the parasites of man is an important prob-
lem. While it is impossible to state in all instances the source from
which man originally acquired his parasitic infections, the evolution-
ary origin of certain protozoan infections is at least suspected.
Iodamoeba biitschlui has been described as occurring in the pig and
it is suspected (Kofoid, 1923) that man acquired his original infection
from that source. The wild game in Africa is thought to be the source
of infection to man of the causative organisms of African sleeping
sickness and Reichenow (1920) has recently claimed that monkeys
are a dangerous source of infection to man, of the organisms causing
malaria.

Rats and mice have long since been regarded as among the most
annoying pests of mankind. On account of their tendeney to inhabit
the dwelling places of the human race, they have from earliest times
doubtless been brought into very close proximity with the substances
used by man for food. It seems very likely that these rodents may
also have functioned in the evolutionary origin of man’s parasitic
Councilmania in the past (Kofoid, 1923).

The remarkable similarity between the species of amoebae belong-
ing to the genus Councilmania found in man and the two species of
that genus found in rats and mice leads one to suspect that these
animals may be the original source of infection by the genus Council-
mania in man; i.e., Man may have originally acquired his infection of
this species of amoeba from rodent hosts. This suspicion leads one to
the consideration of the question of the obligate specificity between
host and parasite which has been discussed by Kessel (1923). The
successful experimental transfer of C. muris and C. decumani recorded
in this paper affords valuable evidence against the specificity of
parasitic amoebae to a given host.

Immunity.—The question of the establishment of an active immun-
ity to amoebiasis is a new one. No experimental work with respect to
this question has been done with the amoebae of man. Yet, in the
examination of rats during this investigation, the question of the
possibility of certain animals being immune to infection has presented
itself on several occasions.

1924 | Kessel: Amoebae of Culture Rats and Mice 527

First, the incidence of infection among old rats is much lower than
among young rats or rats of middle age. Secondly, in 27 per cent of
the cases in this investigation rats and mice which had a previously
determined amoebic infection have been known to clear themselves
of the infection without the administration of curative measures.
Thirdly, among rats and mice subjected to precisely the same feeding
experiments, some animals became infected while others remained free
from amoebic infection. No data were kept for the rats, but among
the mice those that did not become infected as a result of the feeding
experiments were old mice that had been diagnosed as amoeba-free at
the beginning of the experiment.

These results have been obtained from experiments bearing on
subjects other than immunity, but they present striking suggestions
and open the way for further investigation along this line.

Reproduction.—In so far as is known, all reproduction in amoebae
is by the asexual method. There has been no recent evidence to sup-
port the earlier idea of autogamy and no sexual cycle or syngamy has
been determined. It is generally accepted that asexual reproduction
may occur either in the motile amoeba or in the encysted stages.

Although Schaudinn (1903) and Mathis and Mercier (1917)
described the process of multiple schizogony as occurring in Enda-
moeba coli, conclusive evidence has not been produced for this method
of reproduction. It is the opinion of the writer that irregularly
shaped eysts (pl. 47, fig. 49), which may simulate motile amoebae in
shape, may have been interpreted as motile amoebae and the theory
of schizogony based on this evidence.

Binary fission seems to be the usual method of reproduction in
motile amoebae; at any rate, motile amoebae with more than two nuclei
have never been seen in this investigation. Motile amoebae with two
nuclei have been observed and, while they are not common, the fact
that they have been found indicates that the process of binary fission
is quite normal.

Probably the more usual method of reproduction of parasitic
amoebae is by multiple nuclear formation within the cyst. A mono-
nucleate amoeba encysts and by nuclear divisions produces a cyst with
successively two, four, eight, or sixteen nuclei. In the three amoebae
here described from the rat and mouse, eight nuclei is the common
number for the mature cyst.

The question of periodicity of encystment has been discussed in an
earlier paper (Kessel, 1923a), but the growing impression among
investigators is that there is no regular periodicity of similar intervals

528 Unwersity of California Publications in Zoology \Vou. 20

in the formation of cysts and that no regular periodical life cycle is
apparent for parasitic amoebae. Kofoid (1923) has suggested that
encystment occurs as the result of the presence of reserve food rather
than for the purpose of protection to the protozoan. Protection is
afforded while passing from one animal to another, but this is a second-
ary function following from the formation of the cyst. As eysts are
found in the caecum as well as in the faeces, there are indications that
reproduction by multiple fission within the cyst in Councilmania may
occur within the host independent of discharge. The fact that budding
from the cyst apparently in the caecum of the host in which the eysts
were formed has been found in Councilmania muris is further evidence
in support of this theory.

Mitosis and the mechanism of heredity—Karly workers in proto-
zoology expressed the opinion that cell division among the Protozoa
was affected by amitosis. Perfection of cytological methods and
further detailed work have resulted in the description of cell division
by the process of mitosis for one after another of the Protozoa that
had been previously thought to divide amitotically. It is very prob-
able that mitotic cell division is the method of cell division in all
normal cells in the animal kingdom and that amitotie division must
be reserved merely to explan pathogenic or abnormal cell division.

If the mechanism of heredity is bound up in the complicated
structures recognizable in mitotic division, one would expect to find
a hereditary mechanism in the Protozoa similar to the one known
in the Metazoa. Among the amoebae parasitic in man, accounts of
mitosis have been given for Endamoeba dysenteriae by Dobell (1919),
though he did not record the complete mitotic cycle, by Kofoid and
Swezy (1921) for Councilmania lafleuri, and by Swezy (1922) for
Endamoeba coli.

These accounts show that a characteristic spindle is formed in the
amoebae with a centrosome at either pole, and that the centrosomes
are connected by an intradesmose (Kofoid and Swezy, 1921). The
centrosomes are formed from chromatin material within the karyo-
some. The nuclear membrane remains intact throughout the whole
process of division and merely lengthens and constricts in the middle,
finally dividing into two parts at the close of the telophase.

In the present investigation the process of mitosis has been recorded
for Councilmania muris and C. decumani. It is essentially the same
in these two species and similar to that in Endamoeba coli and C.
lafleuri, the chief difference being that the chromosomes are smaller

1924 | Kessel: Amoebae of Culture Rats and Mice 529

in size. Exact chromosome counts, however, have been made and
these are important in differentiation of species. The chromosomes
found in the parasitic amoebae are relatively small and simple when
compared with the chromosomes of the Metazoa. They are for the
most part small spheres or ellipsoids. In this investigation we have
found evidence of the precocious splitting of the chromosomes as
deseribed by Swezy (1922) for Endamoeba coli. The chromosomes
do not retain their constancy of form during the resting stage of the
nucleus, but are reformed at each mitotic division from the chromatin
material scattered within the nucleus and encrusted upon the nuclear
membrane. It is probably because of insufficient observation of mitotic
stages of division that Wenyon (1922) seems to hold to the opinion
that ‘‘no definite chromosomes are formed during the division of the
nucleus’’ of parasitic amoebae.

The formation of an intradesmose (Kofoid and Swezy, 1921) is
characteristic of the type of nuclear division in amoebae. This is
formed within the nuclear membrane and thus differs from the
paradesmose of the Mastigophora and from the centrodesmose of the
metazoan cell.

SUMMARY

1. The present investigation was begun in order to determine
whether or not rats may become infected with Endamoeba dysenteriae.
It was found necessary first to determine the amoebae that normally
inhabit the intestinal tract of rats and mice and this paper describes
the three species of amoebae found in the intestinal tract of culture
rats and mice. These three amoebae are:

(a) Councilmania muris, transferred from the genus Amoeba to
which it was assigned by Grassi (1881) and by Wenyon (1907).

(b) Councilmania decumani, transferred from the genus Enda-
moeba to which it was assigned by Rudovsky (1921).

(c) Endamoeba ratti sp. noy., found in only three of the rats dur-
ing the present investigation.

2. Specific differences of these amoebae are based on differences in
the structure of the protoplasm and pseudopodial formation, morpho-
logical differences in nuclear structure, in chromatoidal bodies, in the
formation of chromophile ridges, in the budding of cysts in the bowel
by which young amoebulae are discharged from the cyst, and on the
fact that the amoebae have been transferred by experiment from one
rodent to another without apparent racial or morphological change.

530 University of California Publications in Zoology [Vou 20

3. Councilmania muris is characterized by the formation of hyaline
pseudopodia; by a central, diffuse karyosome; by the occurrence of
numerous and massed chromatoidal bodies having irregular ends in
the mature cysts, by the frequency of chromophile ridges, by budding
cysts in stools, and by the presence of six chromosomes.

4. Councilmania decumani is characterized by the formation of
hyaline pseudopodia; by a fairly distinct nuclear membrane, heavily
encrusted with chromatin material ; by an excentrie, diffuse karyosome,
somewhat more massed than the karyosome of C. muris; by the pre-
sence of relatively few chromatoidal bodies which have a tendeney to
form rounded ends; by the relative rarity of well defined chromophile
ridges ; and by the process of budding of cysts in the stools.

5. Endamoeba ratti is characterized by the possession of granular
and vacuolated pseudopodia; by a distinet nuclear membrane upon
which the chromatin has a tendency to be encrusted in a regular ring
or small beads; by chromatoidal bodies which resemble those of C.
muris; and apparently by the absence of the budding process.

6. Distinet size races of Councilmania muris and Councilmania
decumani have been observed, those of C. muris being 14.5, 15.7u, and
17.7, respectively, and those of C. decwmani being 14, 15.5, and 17p,
respectively.

7. The process of mitosis has been worked out in detail for the
species C. muris and C. decumam. It resembles mitosis as already
deseribed for C. laflewri (Kofoid and Swezy, 1921) and for Enda-
moeba coli as described by Swezy (1922). C. muris possesses six
chromosomes while C. decwmani has only four. These chromosomes
present recognizable and constant differences in form.

8. Reproduction of parasitic amoebae may occur either by binary
fission of the motile amoebae, or by multiple fission following encyst-
ment, which is the most common method. Young amoebae are extruded
from the cysts of C. muris and C. decumani by a process of budding
through a pore in the cyst wall in the caecum and stools by a process
similar to that described by Kofoid and Swezy (1921) for Cowncil-
mania lafleurt. No evidence of buds has been found to date for Enda-
moeba ratti.

9. The possibility of the establishment of immunity to amoebic
infection by the rodent host is suggested by the facts that the incidence
of amoebic infection is higher in younger animals than in old ones,
that rats and mice may clear themselves of amoebic infection without
apparent treatment, and that it has been difficult to establish an

1924 | Kessel: Amoebae of Culture Rats and Mice 531
experimental infection in old animals that have previously been
determined to be amoeba-free.

10. The fact that the amoebae common in the digestive tract of
rats and mice may be transferred by experimental infection from one
species of rodent to another without immediate morphological or
racial change, and also that the common amoebae of the human diges-
tive tract may be transferred to rodents without noticeable morpho-
logical change affords valuable evidence:

(a) In favor of the morphological constancy of species of parasitic

forms.

(b) Against the theory of host specificity of parasites.

11. The fact that all rats of known positive amoebic infection that
have been fed on a milk diet for two weeks have cleared themselves of
infection while only 27 per cent of the control rats fed on table seraps
have been cleared of infection indicates that a diet of milk only is for
some reason antagonistic to the normal amoebic infection of the rat.

Permanent slides, representing paratypes of species Cowncilmania
muris, Councilmania decumani, and Endamoeba ratti are deposited
in the laboratory of the Department of Zoology, University of
California.

ZOOLOGICAL LABORATORY,
UNIVERSITY OF CALIFORNIA.

Transmitted September 4, 1923.

LITERATURE CITED

Ba.rour, A.
1922. Observations on wild rats in England, with an account of their ecto-
and endoparasites. Parasit., 14, 282-298, pls. 23-24, 2 figs. in text.
Brug, S. L.
1919. De entamoeben van de rat. Geneesk. Tijd. Nederlandsch-Indie, 59,
1-15, pl. 1-2.
Burscu i, O.
1878. Beitriige zur Kenntniss der Flagellaten und einiger verwandten
Organismen. Ztschr. wiss. Zool., 30, 206-281, pls. 11-15.

Cannon, P. R., and McNEASE, B. W.
1923. Factors controlling intestinal bacteria. The influence of hydrogen-ion
-eoncentration on bacterial types. Jour. Inf. Dis., 32, 175-180.
CHATTON, E.
1917. L’éclosion des kystes et les premiers stades de 1’évolution de 1’amibe
dysentérique humaine chez le chat. Bull. Soc. Path. Exot., 10,
834-841, 9 figs. in text.

532 University of California Publications in Zoology (Vou. 20

DOBELL, C.

1917. Reports upon investigations in the United Kingdom of dysentery
cases received from the Eastern Mediterranean. 1. Amoebie dys-
entery and the protozoological investigation of cases and carriers.
Brit. Med. Res. Com., 4 (London, Sir Joseph Caston & Sons), 85 pp.

1919. The amoebae living in man. (London, John Bale, Sons and Daniels-
son) vi-+ 155 pp., 5 pls.

DOBELL, C., and O’Connor, F. W.

1921. The intestinal Protozoa of man. (Ibid.), v + 211 pp., pls. 1-8, 2 figs.

in text.
GrRaASssI, B.

1881. Intorno ad aleuni Protisti endoparassitici ed appartinenti alle classi
dei flagellati, lobosi, sporozoi e ciliati. Atti Soe. Ital. Sci. Nat., 24,
135-224, pls. 1-4.

GRINNELL, J.

1923. A systematic list of the mammals of California. Univ. Calif. Publ.

Zool., 21, 313-324.
HEGNER, R. W.

1923. The effects of changes in diet on the incidence, distribution and
number of certain intestinal Protozoa of rats. Am. Jour. Hyg., 3,
180-200, pl. 6.

Hinton, M. A. C.

1920. Rats and mice as enemies of mankind (London, British Museum

Natural History), Ex. Se., 8, ix + 67 pp., pls. 1-2, 6 figs. in text.
HUBER, G. C.

1912. The development of the albino rat, Mus norvegicus albinus. Jour.

Morph., 26, 1-142, 42 figs. in text.
Hyman, L. H.

1917. Metabolic gradients in amoeba and their relation to the mechanism

of amoeboid movement. Jour. Exp. Zool., 24, 55-99, 14 figs. in text.
KESSEL, J. F.

1923a. Methods of securing amoebae-free rats for experimental infection
with intestinal amoebae. Univ. Calif. Publ. Zool., 20, 401-408.

1923b. The experimental infection of rats and mice with human intestinal

amoebae. Ibid., 20, 409-432, pls. 38, 39.
Koro, C. A.

1923a. Amoeba and man. Univ. Calif. Chron., 25, 149-174 and 291-312,
pls. 1-6.

1923b. The life cycle of the Protozoa. Science, 57, 397-408.

Korow, C. A., KoRNHAUSER, E. I., and Swezy, O.

1919. Criteria for distinguishing the Endamoeba of amoebiasis from other

organisms. Arch. Int. Med., 24, 35-50, 43 figs. in text.
Koro, C. A., and Swezy, O.

1920. On the prevalence of carriers of Endamoeba dysenteriae among
soldiers returning from overseas service. New Orleans Med. and
Surg. Jour., 73, 4-11.

1921. On the free, encysted, and budding stages of Councilmania lafleuri,
a parasitic amoeba of the human intestine. Univ. Calif. Publ.
Zool., 20, 169-198, pls. 18-22, 3 figs. in text.

Koro, C. A., Swezy, O., and KESSEL, J. F.

1923. The genus Councilmania, budding amoebae parasitic in man and

rodents. Ibid., 20, 431-445, 10 figs. in text.

1924 | Kessel: Amoebae of Culture Rats and Mice 933

LAPAGE, G.
1922. Cannibalism in Amoeba vespertilio (Penard). Quart. Jour. Mier. Sci.,
66, 669-710, pls. 28-29, 3 figs. in text.
Lyncu, K. M.
1915. The rat as a carrier of dysenteric amoeba. Jour. Am. Med. Assoc.,
65, 2232-2234.
Maruis, C., and Mercier, L.
1917. La schizogonie chez les entamibes intestinales de 1’homme. Bull. Soe.
Path. Exot., 10, 311-322, 15 figs. in text.
PENARD, E.
1904. Quelques nouveaux Rhizopodes d’eau douce. Arch. Prot., 3, 391-422,
11 figs. in text.
RerrTGcer, L. F., and CHEpuin, H. A.
1921. The intestinal flora with special reference to the implantation of
Bacillus acidophilus (New Haven, Yale University Press), pp. 135,
pls. 1-8.
RICHENOW, E.
1920. Uber das Vorkommen der Malariaparasiten des Menschen bei den
afrikanischen Menschenrassen. Centrbl. f. Bakt., Abt. I, 85,
2-7-2116, 1 pl., 3 figs. in text.
Rupovsky, F.
1921. Uber Rattenamodbiase. Wien. Tieriirzt. Monatsber., 8, 1-9, pl. 7.
SCHAEFFER, ASA A.
1920. Amoeboid movement (Princeton, University Press), v +156 pp.,
46 figs. in text.
ScHAUDINN, F.
1903. Untersuchungen uber die Fortpflanzung einiger Rhizopoden. Arb.
Kais. Gesundh., 19, 547-576.
Swezy, O.
1922. Mitosis in the eneysted stages of Endamoeba coli (Lésch). Univ.
Calif. Publ. Zool., 20, 313-332, pls. 29-31.
TayLor, C. V.
1923. The contractile vacuole, an,example of the sol-gel reversibility of
protoplasm. Jour. Exp. Zool., 37, 3 pls. 11 figs. in text.
WALKER, E. L.
1908. The parasitic amoebae of the intestinal tract of man and other
animals. Jour. Med. Res., 17, 379-460, pls. 21-24.
WENYON, C. M.
1907. Observations on the Protozoa in the intestine of mice. Arch. f. Prot.,
Suppl. 1, pp. 169-201, pls. 10-12.
1922. Supposed new endamoebae of man. Trop. Dis. Bull., 19, 19-22.
WENyoN, C. M., and O’Connor, F. W.
1917. Human intestinal Protozoa in the Near East (London, Wellcome
Bureau of Scientific Research), 218 pp., pls. 144.
WILSON, C. W.
1916. On the life-history of a soil amoeba. Univ. Calif. Publ. Zool., 16,
241-292, pls. 18-23.
YosHipa, K.
1918. The encystment of dysentery amoebae in vitro. Jour. Exp. Med., 28,
348-413, pls. 30-43.

EXPLANATION OF PLATES

All figures are made with camera lucida from smears fixed in hot Schaudinn’s
fluid and stained with iron-haematoxylin. XX 2500.

PLATE 43
Councilmania muris

Fig. 1. Motile amoeba, showing hyaline pseudopodia. Undigested food is
in some of the vacuoles. The large, dark-staining mass in the vacuole to the
right of the nucleus is probably Sphaerita.

Fig. 2. Attached motile amoebae forming balloon-like hyaline pseudopodia.

Fig. 3. Amoeba with typical hyaline pseudopodium formed as if in progressive
movement. Note the posterior, root-like projection. The nucleus shows the
thin membrane and diffuse karyosome, characteristic of this species.

Fig. 4. Rounded, motile amoeba, with partly digested Chilomastix in the
large food vacuole.

Fig. 5. Rounded, pre-cystic amoeba. Note the absence of food in the vacuoles
and a small number of large vacuoles, indicating that smaller ones have
coalesced to form the large ones.

Fig. 6. Rounded, pre-cystic amoeba with three large vacuoles. Karyosome
is composed of diffuse granules and has a gray area in center. Note the short
intradesmose.

Fig. 7. Mononucleate cyst with large glycogen vacuole and nucleus in
early prophase. Small chromatoidal bodies around the margin of the vacuole.

[KESSEL] PLATE 43

PUBL. ZOOL. VOL. 20

UNIV. CALIF.

PLATE 44
Councilmania muris

Fig. 8. Mononucleate cyst with glycogen vacuole showing nucleus in late
prophase.

Fig. 9. Mononucleate cyst without glycogen vacuole. The upper chromo-
somes represent a metaphase and lower ones are in early anaphase.

Fig. 10. Large mononucleate cyst with glycogen vacuole. Nucleus in
telophase.

Fig. 11. Binucleate cyst showing glycogen vacuole. The upper nucleus

represents a resting stage with the lower nucleus in prophase, showing intra-
desmose connecting centrosomes.

Fig. 12. Binucleate cyst with glycogen vacuole. Upper nucleus is in ana-
phase, showing six divided chromosomes and the centrosomes at either end of
the spindle. The lower nucleus in very early prophase.

Fig. 13. Binucleate cyst with upper nucleus in early anaphase and lower
nucleus in late anaphase.

Fig. 14. Binuecleate cyst with the nuclei in anaphase showing massed chro-
matoidal body formed between margin of glycogen vacuole and cytoplasm.

Fig. 15. Cyst with three nuclei showing diffuse karyosome. Large nucleus
is the result of the first division, the two small nuclei the result of the second
division. Chromatoidal bodies are arranged between the glycogen vacuole and
cytoplasm.

Fig. 16. Cyst, showing the upper nucleus in very late telophase, the lower
nuclei having just separated. The diffuse condition of the karyosome is apparent
in each nucleus,

Fig. 17. Four-nucleate cyst without glycogen vacuole. Three spherical
nuclei are in prophase while the elongated nucleus showing chromosomes is in
metaphase. Centrosomes are shown at either end of the spindle and are econ-
nected by the intradesmose.

Fig. 18. Five-nucleate eyst, without glycogen vacuole. The two larger
nuclei on the left are the result of the second nuclear division, the two smaller
nuclei on the right the result of the third nuclear division. The middle nucleus
is in a late telophase.

Fig. 19. Eight-nucleate cyst without chromatoidal body. Nuclei show
characteristic thin nuclear membrane and diffuse karyosome.

[536]

UNIV. CALIF. PUBL. ZOOL. VOL. 20 [KESSEL] PLATE 44

PLATE 45
Councilmania muris

Fig. 20. Ellipsoidal cyst with eight nuclei showing chromophile ridge
extending the full length of the cyst.

Fig. 21. Spherical eight-nucleate cyst +with chromophile ridges on either
side and chromatoidal body in the center.

Fig. 22. Ellipsoidal eyst with bud protruding from the region of the ehro-
mophile ridge. One nucleus is located in the bud, four nuclei are left in the
eyst, and a small amoebula is in close proximity to the cyst, having just pre-
viously budded off. Note the empty space in the cytoplasm and the general
thin appearance of the cytoplasm, indicating that part of the cytoplasm has
previously passed out of the cyst in forming the earlier amoebulae.

Fig. 23. Large, eight-nucleate cyst without chromatoidal bodies, bud, or
chromophile ridge.

Fig. 24. Smaller, eight-nucleate cyst with long chromatoidal body in middle
of cyst. The karyosomes are widely dispersed.

Fig. 25. Eight-nucleate cyst, showing chromophile ridge.

Fig. 26. Eight-nucleate cyst with bud showing chromatoidal body and
typical dispersion of the karyosome toward the region of the pore, and remnants
of chromophile ridges. One nucleus is in the bud.

Fig. 27. Seven-nucleate cyst showing bud, large, massed chromatoidal body,
and chromophile ridges in the region of the pore.

Fig. 28. Six-nucleate cyst with one nucleus in the bud. Chromatoidal body
and chromophile ridge are present.

Fig. 29. Eight-nucleate cyst with bud and showing peculiarly curved ehro-
matoidal body in middle of cyst. Chromophile ridge is in the region of the pore.

[538]

UNIV, CALIF, PUBL. ZOOL. VOL, 20 [KESSEL] PLATE 45

PLATE 46
Councilmania decumani

Fig. 30. Large, rounded motile amoeba with partly digested Chilomastix in
the upper large vacuole and a small amoeba in the lower vacuole. The nucleus
shows an excentric karyosome which is surrounded by a halo.

Fig. 31. Large, rounded motile amoeba with two nuclei.

Fig. 32. Medium size, motile amoeba, showing hyaline pseudopodia extended.
The nucleus shows a crescent-shaped, excentrie karyosome.

Fig. 33. Large mononucleate cyst without glycogen vacuole, and small
spherical chromatoidal bodies in the cytoplasm.

Fig. 34. Mononucleate cyst with glycogen vacuole. The karyosome has
begun to separate and is connected by the intradesmose. Heavy peripheral
chromatin is encrusted on the nuclear membrane.

Fig. 35. Ellipsoidal mononucleate cyst with glycogen vacuole. The spindle
shows most of the chromosomes in early anaphase and the centrosomes are con-
nected by the intradesmose. Note the blobs of chromatin encrusted on the
nuclear membrane.

Fig. 36. Small, binucleate cyst with glycogen vacuole. The spherical nucleus
is in the resting stage. The ellipsoidal nucleus is in the early prophase.

Fig. 37. Binucleate cyst showing nuclei slightly slougeted in very early
prophase prior to the division of the karyosome.

Fig. 38. Binucleate cyst with chromatoidal bodies near the margin of the
glycogen vacuole. The centrosomes are beginning to pull apart and are con-
nected by the intradesmose. '

[540]

UNIV. CALIF. PUBL. ZOOL. VOL. 20 [KESSEL] PLATE 46

1
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=
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PLATE 47
Councilmania decumani

Fig. 39. Binucleate cyst with nuclei in prophase. Centrosomes are arranged
at either end of the spindle and are connected by the intradesmose. Chroma-
toidal bodies are arranged around margin of the glycogen vacuole.

Fig. 40. Binucleate cyst with the nuclei in prophase. Note the area in the
region of the spindle in which are located four chromatin granules. The centro-
somes are connected by the intradesmose.

Fig. 41. Binucleate cyst with chromosomes arranged on spindles in meta-
phase. Heavy peripheral chromatin is formed on the nuclear membrane.

Fig. 42. Binucleate cyst with small glycogen vacuole. Chromatoidal bodies
of varying shapes are in the cytoplasm. Chromosomes on spindle are in anaphase.

Fig. 43. Cyst without glycogen vacuole and with three nuclei. The large
nucleus is the result of the first division, and the small nuclei the result of the
second nuclear division. Chromatoidal bodies are in the shape of small spheres
and splinters.

Fig. 44. Cyst with three nuclei and large glycogen vacuole. The nuclei
represent different phases of mitosis.

Fig. 45. Four-nucleate cyst without glycogen vacuole. Two lower nuclei
show the chromosomes in metaphase. One large chromosome, two medium-sized
chromosomes, and one small chromosome are evident.

Fig. 46. Cyst with five nuclei. The three large nuclei have not undergone
second division.

Fig. 47. Irregularly shaped cyst with eight nuclei and no chromatoidal
body. The nuclei show the typical resting stage with excentric karyosome and
large blobs of chromatin encrusted on the nuclear membrane.

Fig. 48. Spherical, eight-nucleate cyst with typical chromatoidal bodies
having irregular ends.

Fig. 49. Irregularly shaped eight-nucleate cyst with small chromophile
ridge.

Fig. 50. Spherical eight-nucleate cyst containing small, spherical chroma-
toidal body and large massed chromatoidal bodies with rounded ends.

[542]

UNIV. CALIF, PUBL. ZOOL. VOL. 20

[KESSEL] PLATE 47

PLATE 48
Councilmania decumani, figs. 51-56

Fig. 51. Spherical, eight-nucleate cyst showing small chromophile ridge.
Fig. 52. Very large, sixteen-nucleate cyst showing no chromatoidal bodies.

Fig. 53. Eight-nucleate cyst showing bud. One nucleus is in the bud and
the second nucleus is in the region of the pore.

Fig. 54. Eight-nucleate cyst showing bud just beginning to protrude.

Fig. 55. Cyst containing four nuclei. One nucleus is in the region of pore
and shows elongation prior to its entrance into the bud. The cytoplasm in the
cyst is thin and shows regions from which other buds have formed.

Fig. 56. Cyst with bud showing eight nuclei.

Endamoeba ratti, figs. 57-62

Fig. 57. Motile amoeba showing vacuolated and granular pseudopodia.
The karyosome is excentric and is surrounded by a halo.

Fig. 58. Eight-nucleate cyst showing no chromatoidal body. The karyo-
some is massed and excentric.

Fig. 59. Binucleate cyst showing glycogen vacuole and two small, chro-
matoidal bodies.

Fig. 60. Eight-nucleate cyst showing large, massed chromatoidal body with
splintered ends.

Fig. 61. Eight-nucleate cyst showing massed chromatoidal body, the margins
of which are rounded. Small chromatin beads are arranged on the nuclear
membrane.

Fig. 62. Eight-nucleate cyst showing typical massed, excentric karyosomes
in the nuclei. The peripheral chromatin on nuclear membranes is heavy.

[544]

SEL] PLATE 4

[KES

UNIV. CALIF. PUBL. ZOOL. VOL. 20

ae

yale isstyp,

x x30!

INDEX

Univ. Calif. Publ. Zool., vol. 20.

Titles of papers and names of new systematic units in boldface. Acknowledg-
ment of authors cited is made for each paper under the heading ‘‘ Literature

Cited.’’

Alveolar layer, in Trichonympha, 54;
in Leidyopsis, 105, 111.

Amitosis, 460.

Amoeba coli, 490.

dysenteriae, 170.
hyalina, 323.
muris, 433, 434, 490.

Amoeba, intestinal, of man, 169-198,
313-332, 431-445; in bone mar-
row, 301-307; difficulty of cul-
ture, 313; sexual process in, 324;
discharge of, in rats, 402; evacu-
ation through purgative, 403; in-
eidence of infection in rodents,
405, 406, 494, 496, 527; rate of
infection and age of hosts, 407,
408, 423, 494, 527; inoculation in
guinea pigs, 410, in rabbits, 410;
commonly in caecum of rodents,
421; parasitic in rats and mice,
489-544; reproduction, 527-528.

Arthritis deformans, 301-307.

Asearis toxin, 235-242.

Askaron, proteose toxin from Ascaris,
°

Astasia levis, 463.
proteus, 448.

Astasiopsis distorta, 448.

Autogamy, in Endamoeba coli, 324.

Axostyle, in Giardia enterica, 208; in
Pentatrichomonas, 380, 384; in
Trichomonas, 373.

Balantidium, systematie position of
genus and 22 known species, 248;
comparative measurements, 252—
254.

coli, 243-300.
entozoon, 244.
suis, 243-300.

Banks, Nathan, acknowledgment, 2.

Basal apparatus, in Balantidium, 271.

Beckwith, T. D., acknowledgment,
477.

Berecovitz, N., 365-371.

Bilateral symmetry, in polymastigote
flagellates, origin of, 215.

Binary fission, see Fission, binary.

Biochemical changes, susceptibility
of flagellates to, 29, 43.

Blastocystis, 324, 376.

Blepharoplast, in Chilomastixr, 124,
primary, 124, secondary, 125,
and tertiary, 125; in Giardia,

206; in Menoidiuwm, 451, 459; in
Pentatrichomonas, 378; in Streb-
lomastiz, 8, 14; in Trichomitus,
23, 31, 32; in Trichonympha, 70-
71.

Board of Research, University of
California, acknowledgment, 375,
432.

Bodo intestinalis, 163.

Bone marrow, in Arthritis deformans,
Endamoeba dysenteriae in, 301-
307.

Boyers, L. M., 309-312; acknowledg-
ment, 375.

Budding, in Councilmania, 181, 431-
445, 508, 514.

Bursaria, 270.

California State Board of Health,
Division of Parasitology of the
Bureau of Communicable Dis-
eases, 117, 146, 201, 370, 432.

Carnegie Institution of Washington,
acknowledgment, 375, 432.

Centroblepharoplast, in Chilomastiz,
123, 124, 137; in Leidyopsis, 104,
111; in Menoidium and Tricho-
monas, 123, 124, 459; in Tricho-
mitus, 27, 31, 37; in Trichonym-
pha, 47, 48, 53, 60, 62, 70, 71;
connection of with neuromotor
fibers in flagellates, 345.

Centrosome, in Chilomastixz, 118, 122,
124; in Endamoeba coli, 323; in
Giardia, 206; in Pentatricho-
monas, 379; in Streblomastix, 7,
16; in Trichomitus, 23 31, 32; in
Trichonympha, 47 60, 71.

Cerearia tenax, 157, 158.

Cercomonas, 147-150.

erassicauda, 147, 148.
davainei, 152, passim.
hominis, 147, 148, passim.
intestinalis, 160, 163.
longicauda, 148, 149, 150.
obliqua, 152.
parva, 147, 148, passim.
Chemotropism, 496.

>

Ut,

Index

Chilomastix, 148, 151, 205, 210, 216,
406, 496. See also under Cysts,
Cytoplasm, Mitosis, ete.

davainei, 147, 152, 155, 208, 366,
376.
mesnili, 117-144, 147, 151.

China Medical Board, acknowledg-
ment, 370.

Chromatoidal bodies, 130, 176, 178,
316, 506, 508, 517.

Chromomeres, 67.

Chromophile ridge,
508, 513.

Chromosomes, in Cowncilmania decu-
mani, 441; in C. laflewri, 177, 306,
435; in C. muris, 439, 510; in Chilo-
mastiz, 134; in EBndamoeba coli,
177, 319, 320; in Luglena viridis,
466-467; in Leidyopsis, 108, 109,
111; in man, 305; in Menoidium,
455, 466, 467; in Streblomastia,
11; in Trichomitus, 32, 33, 34; in
Trichonympha, 67, 72-77, 81;

continuity of, 76;

7

179, 187, 507,

differentia-
tion of species by, 529.

Cilia, in Balantidium, 265, 266, 270,
273, 284, 289; in Paramaecium,
movement of isolated pieces of,
350; in Streblomastiz, 14; in
Trichonympha, 48, 50; in Tricho-
nympha and Paramaecium, 345;
adoral, 284, 287, 291, 293; adoral
ciliary rootlets, 286.

Cockroach, host for Endamoeba, 432.

Comparison of the Cysts of Enda-
moeba coli and Councilmania
lafieuri in Congo Red, 365-371.

Congo red, reactions to, of cysts,
365-371.

Cooper, H. N., acknowledgment, 334.

Copromonas major, 458, 463.

subtilis, 458.

Cort, W. W., acknowledgment, 245.

Councilmania, generic diagnosis, 187,
431-445, 493; key to species of,
443; comparison of species, 523,
530.

decumani, 411, 433, 438, 439-443,

491, 495, passim.

lafieuri, 169-198; activities, 171;
free stage, 173, 186; encysted,
175, 186; dimensions, 180; bud-
ding and escape of amoebulae,
181; occurrence, 183; culture and
inoculation, 185; eysts, viability
of, 185; distinctions, 186; com-
parison of cysts in, with Enda-
moeba coli, 365-371; infection of
rodents with, 409-430, 497, 502,
506, passim.

muris, 411, 438-439, 491, 495,
passim.

Cross-infection, experiments in, with
parasitic amoeba, 517.

[546]

Craigia, 148, 149, 150, 431.
hominis, 150.
Craspedomonadina, direction of tor-
sion in, 9.
Critical Review of the Nomenclature
of Human Intestinal Flagellates,
Cercomonas, Chilomastix, Tricho-

monas, Tetratrichomonas, and
Giardia, 145-168.
Culture of Councilmania, 185; of

Leishmania, 447 ; of Paramaecium,

336.

Cummings, W. J., acknowledgment,
334,

Cutaneous reaction, 477-488; table,
480.

Cysts, in Chilomastixz, 129, 130; in
Councilmania decumani, 443, 513;
in C. lafleuri, 179, 185; in C.
muris, 439, 503-507; in Enda-
moeba coli, 179, 313-332; in En-
damoeba coli, and Councilmania
laflewri, 365-371, 432,435,437; in
Endamoeba dysenteriae-infected
rats, 414; in #. ratti, 516, 517;
in Giardia enterica, 217, 218, 219.
degenerative changes in, 314.
feeding experiments with, 413-421.
difference in viability of, 422, 425.
Cytolysis, 182.
Cytopharynx, in Paramaecium, 342.
Cytoplasm, in Balantidium, 263; in
Chilomastixz, 121; in Councilmania
lafleuri, 176, 181, 182; in C. muris,
505; in Giardia, 204; in Pentatri-
chomonas, 381; in Streblomastia,
7; in Trichomitus, 24.
Cytopyge, 280.
Cytostome, in Balantidium, 260, 267,

276; in Chilomastiz, 126; in
Giardia, 204; in Paramaecium,
342, 344; in Pentatrichomonas,

380; in Trichomitus, 26.

Devescovina, 78, 373.

Dexiotropism in polymastigotes, 9.

Dientamoeba fragilis, 170.

Diet, milk only, antagonistic to nor-
mal amoebic infection, 521, 522;
meat only, suitable, 521, 522.

Dimorphus muris, 160, 163.

Dinenympha, 9, 78.

gracilis, 2, 14, 216.
Dinoflagellata, direction of torsion in, 9.
Diplodinium, 21, 60, 267.
ecaudatum, 273, 279, 280, passim,
335.

Diplozoie organization, in flagellates,

29

Distinguishing Characteristics of the
Parasitic Amoeba of Culture
Rats and Mice, 489-544.

Domonoske, A. B., acknowledgment,
334.

Drew, A. D., acknowledgment, 2.

Index

Eetoplasm, in Balantidium, 263, 266,
267; in Councilmania lafleuri,
173; in C. muris and C. decumani,
499; in Bndamoeba ratti, 515; in
Leidyopsis, 102, 107, 111; in
Trichonympha, 48, 49, 52, 61, 62.

Ely, C. L., acknowledgment, 302.

Embadomonas, 151.

intestinalis, 147.

Encystment, 402, 527-528; in Council-
mania deeumani, 512; in C. la-
fleuri, 175, 186; in C. muris, 503;
in Endamoeba ratti, 516; in
Giardia, 217, 219, 221, 222; in
Trichomitus termitidis, 36.

Endamoeba dysenteriae in the
Lymph Glands of Man in Hodg-
kin’s Disease, 309-312.

Endamoeba blattae, 524.

coli, 170, 173, 177, passim, 324,
365-371, 411-430, 441, 490,

491, 507, passim.
dysenteriae, 118, 129, 130, 156, 170
173, passim, 301-307, 309-312,
366, 410-430, 504-505, passim;
conclusions from feeding ex-
periments, 413-421; chronic
type in rats, 414.
gingivalis, 306.
histolytica, 322,
legeri, 322, 323.
muris decumani, 491.
nana, 366.
ratti, 411, 491, 515-530.
williamsi, 184.

Endolimax buetschlii, 129, 130.
nana, 170, 411, 416, 418, 422.

Endoplasm, in Balantidium, 282; in
Councilmania lafleuri, 174; in C.
muris and C. decuwmani, 499; in
Endamoeba ratti, 515; in Leidy-
opsis, 105; in Menoidium, 450; in
Trichonympha, 48, 56, 60.

Endosome, in euglenoids, 463-464.

Entamoeba muris, 170, 434.

tetragena, 434.
undulans, 384.

Environment, not determining factor
in parasitic amoebae, 519, 523.

Epsom salt, purgative in rodent ex-
periments, 403, 405, 492, 517.

Euglena agilis, 451, 458.

viridis, 452, 462, 465, 466.

Euplotes, 64, 79.

patella, 270, 278, 281.
worcesteri, 283.

Eutrichomastix, 9, 33.

Experimental Infection of Rats and
Mice with the Common Intes-
tinal Amoeba of Man, 409-430.

Extra-nuclear motor apparatus, defi-
nition of, 21.

Extracts, alcoholic and alkaline, of
Leishmania, 479; table, 480.

497.

[547]

Faeces, routine examinations of, in
man and rodents, 401, 402, 408.

Fibers, in Balantidium: diverging,
268, cireumoesophageal, 285, 289,
290, radial, 286, 287, 289; in
Chilomastix, peristomal, 125; in
Giardia, 211, 212; in Leidyopsis,
oblique, 103, 111, as in Yricho-
nympha, 52, 53; in Paramaecium,
peripheral, 341; of cytosome and
eytopharynx in Paramaecium,
342-343, 352; fibrillar system
conduetile, 347-351.

Fission, binary, in Hndamoeba coli,
317; in Leidyopsis, 107; in Men-
oidium, 457, 460; in Pentatricho-
monas, 382; in Streblomastix, 10,
11; in Trichomitus, 35; in Tricho-
nympha, 44, 61; usual in motile
amoebae, 527.

multiple, in Pentatrichomonas, 382;
in Streblomastix, 12, 13; in
Trichomitus, 35, 37; usual in para-
sitic amoeba, 527; plane of, in
Giardia enterica, 215.

Flagella, in Chilomastixz, 128; in De-
vescovina, 373; in Giardia, 206,
207, 208, 418, 457; in Heteronema
acus, 457; in Hypermastigina,
373; in Leidyopsis, 103, 110, 111;
in Pentatrichomonas, 378, 383; in
Streblomastiz, 7, 8, 14, 16; in
Trichomitus, 27, 37; in Tricho-
monas, 158, 378; in Trichonympha,
47, 48, 50, 110; in Tritrichomonas,

378; bases of classification in
flagellates, 146, 158.
Flagellates, susceptibility to bio-

chemical changes, 29, 43; diplo-
zoie organization, 29; of human
intestine, 117-144, 145-168, 199-
234; method of feeding, 391-400.
Food. See Nutrition.
Force, J. N., acknowledgment, 477.
Fowler, Margaret B., acknowledg-
ment, 171, 375.
Giardia, comparison with Streblomas-
tix, 7; with Trichomitus, 22, 29;
with Trichonympha, 75; with
Chilomastiz, 118, 119, 136, 137;
description of genus, 160-164;
specific differences, 222, 225.
agilis, 161, 200.
alata, 161, 162.
eunieuli, 222.
duodenalis, 162, 200.
enterica, 162, 163, 164, 199-234,
366, 418.
intestinalis, 147, 211.
lamblia, 164.
microti, 162, 200, 206,
muris, 162, 163, 200, 204, passim,
406.
sanguinis, 200.

9

a4, a

999

one.

Index

Glycogen, vacuole, 178; in Cowncil-
mania lafleuri, 435; in C. muris,
439, 503, 505; in cytoplasm of
Endamoeba coli at encystment,
317; in E£. ratti, 517.

Gradients, axial, in Paramaecium,
347-348, 352.

Gyrodinium corallinum, 58.

Hall, R. P., 447-476.

Heterochromosome, in Trichonympha,
use of term, 72.

Heteronema acus, 457.

Hexamastix ardin delteili, 377.

Hexamita duodenalis, 160, 163.

Hexamitidae, 136, 137, 216, 217.

Hexamitus, 30.

muris, 406.

Hodgkin’s disease,
309-312.

Holomastigotes, 9, 15, 70.

Holomastigotoides, 78.

Hydrogen-ion concentration, in caeca
of rats, 520.

Hypermastigina, 80, 81, 112, 373.

Immunization to Leishmania, 478
483.

Intestine, human, parasites of, see
Amoeba; Flagellates.

Intradesmose, in Councilmania lafleuri,
177; in C. muris, 509; found in
Endamoeba dysenteriae, FE. coli,
and Councilmania lafleuri, 304,
319; characteristic in amoebae,
529.

Iodamoeba buetschlii, 174, 175, 366,
411, 416, 418, 422, 526.

Isogamy, in flagellates, 461.

Isotricha prostoma, 272, 273.

Joenia, 78.

Karyosome, in Councilmania decu-
mani, 441, 512; in C. laflewri, 174,
176, 432, 433, 435, 4387; im C.
muris, 439, 502, 506; in Enda-
moeba coli, 317; in E£, ratti, 517;
**nucleolo-centrosome,’’ 464.

Kessel, J. F., 401-408, 409-430, 489-
544,

Kofoid, C. A., 145-168; acknowledg-
ments, 245, 315, 334, 401, 411,
448, 477, 491.

303, 304, 305,

’

?

Kofoid, C. A., Boyers, L. M., and
Swezy, Olive, 301-312.
Kofoid, C. A., and Swezy, Olive, 1-

20, 2140, 41-98, 99-116, 117-144,
169-198, 199-234, 373-390.
Kofoid, C. A., Swezy, Olive,
Kessel, J. F., 431-445.
Kornhauser, 8. I, acknowledgment,
H9:
Lamblia, 161, 162, passim.
intestinalis, 200.
Legge, R. T., acknowledgment, 171.

and

[548]

Leidyopsis, morphology, 100-101; re-
lationships, 110-111; generie
diagnosis, 111; pseudopodial
method of feeding, 391-400.

sphaerica, 99-116; surface ridges,

103, 111.

Leiotropism, in polymastigotes, 9, 15.

Leishmania tropica, 477-488.

infantum, 477-488.

Leishmaniosin, preparation of, 478.

Literature cited, on: Balantidium,
295-297; Councilmania, 188, 371,
445; experimental infection with
rats, 408, 426-427; Leishmania,
485-486; Menoidium, 469-472;
mitosis, 138, 225-227, 312, 325-
326; nomenclature of human
intestinal flagellates, 165-168;
Paramaecium, 242, 353-355; para-
sites of the termites, 17, 37, 82-
83, 113, 400; pathological agent
in Hodgkin’s disease, 312; Pen-
tatrichomonas, 388-389; parasitie
amoebae of rats and mice, 531-

533k
Locomotion, in Couneilmania decu-
mam, 512; in C, laflewri, 171-

173, 435; in C. muris, 495-501,
512; in Endamoeba ratti, 515—-
516; in Pentatrichomonas, 382-
385; in Trichonympha, 43, 391-
400.

Lophomonas, 78.

Lung infection, among rats, 414, 425.

Lymph glands, presence of parasite
in, 309-312. ;

McDonald, J. D., 243-300.

Macronucleus in Balantidium coli and
B. suis, 259-260, 263, 282.

Macrostoma mesnili, 151.
MeVey, C. L., acknowledgment, 171.
Magnesium sulphate, purgative in ex-
periments with rodents, 408.
Mastigophora, direction of torsion in,
Medical zoology, value to, of cross-
infection, 423.

Megastoma entericum, 161, 163, 200,
211.

Menoidium inecurvum, 447-476.

Methods of Obtaining Amoeba-free
Rats for Experimental Infection
with Intestinal Amoeba, 401—

408.
Mice, infection experiments with, 406,
407, 414, 489-544; facultative

carriers of amoebiasis, 423.
Micro-dissection experiments, 235; on
Euplotes patella, 292; on Para-
maecium, 349.
Micro-Injection of Paramaecium, 235—
242, 336, 346.

Index

Micronucleus, in Balantidiwm,
283.

Microtus californicus, 222.

Mitosis in Endamoeba dysenteriae
in the Bone Marrow in Arthritis
Deformans, 301-307.

Mitosis, similar in all species of para-
sitic amoeba, 522; in Chilomastia,
133, 1387; in Councilmania decu-
mani, 441, 514; in C, laflewri, 177;
in C. muris, 4389, 509; in Enda-
moeba coli, 317-325, comparison

263,

with Councilmania laflewri, 322;
in FE. dysenteriae, 301-307; in
Euglena and Menoidium, 467,

468; in Giardia, 199-234; in hu-
man cell, 304-305; in Leidyopsis,
108, 111; in Noctiluca, 71; in Pen-
tatrichomonas, 382; in the Rhizo-
poda and Metazoa, 301; in
Streblomastiz, 10, 14; in Tricho-
mitus, 27, 31, 34; in Trichomonads,
27; in Trichonympha, 44, 47, 61,
64, 65, 69, 72, figures illustrating,
73.

Mitosis and Fission in the Active and
Encysted Phases of Giardia en-
terica (Grassi) of Man, with a
Discussion of the Method of
Origin of Bilateral Symmetry in
the Polymastigote Flagellates,
199-234.

Morphology and Binary Fission of
Menoidium incurvum (Fres.)
Klebs, 447-476.

Morphology, in Balantidium, 262, 263;
in Chilomastix, 119; differences
in, between Endamoeba coli and
Councilmania laflewri, 315; in
Giardia, 202, 223; in Leidyopsis,
100; in Menoidium, 450; in Para-
maecium, 338; in Pentatricho-
monas, 378; in Streblomastia, 4;
in Trichomitus, 23; in Trichonym-
pha, 44, 81; a criterion in iden-
tification, 306, 310; constancy of,
in transference of amoebae, 517,
519.

Motorium, in Balantidium, 285, 25%.

Multiple fission. See Fission, multiple.

Mus decumanus, 491.

musculus, 492.
norvegicus albinus, 492.
rattus, 410, 434, 491.

Myonemes, in Streblomastix, 7, 13, 14,
15, 16; in Trichonympha, 55, 56,
57; in Leidyopsis, 105, 111.

Naegleria gruberi, 323.

National Council of Research,
knowledgment, 119.

Neuromotor apparatus, definition and
extent of, in flagellates, 21, 22;
function of, 47-49, 350-351;
in Balantidium, 284-287; in

ac-

[549]

Chilomastix, 123, 133, 137; in
Diplodinium, 288; in Euplotes,
288, 289, 335; in Giardia, 203,

205; in Leidyopsis, 102, 111; in
Menoidium, 451, 468; in Para-
maecium, 333-364; in Streblo-
mastix, 7; in Trichomitus, 26, 27,
isolated, 30, during mitosis, 31,
32; in Trichonympha, 47-48, 62,
81.

Neuromotor Apparatus
maecium, 333-364.
Noectiluea, mitosis in, 71.
Nomenclature, human intestinal flag-

ellates, 145-168.
Novy, F. E., acknowledgment, 477.
Nuclear membrane, in Councilmania

of Para-

decumani, 441, 512; in C. la-
fleuri, 437, 443; in C. muris, 439,
502, 506; in Endamoeba ratti,

516; in Leidyopsis, 109, 111.
Nucleolo-centrosome, theory of, 464.
Nucleus, in Chilomastix, 121, 122, 131,

137; in Copromonas, 462; in

Councilmania decumani, 433, 441,

513; in C. lafleuri, 174, 175, 435;

in C. muris, 439, 502, 509; in
Endamoeba coli, 317, 320; in
Leidyopsis, 106, 107; in Meno-

idium, 4538, 462; in Pentatricho-
monas, 381; in Streblomastix, 10,
16; in Trichomitus, 24, 29, 37;
in Trichonympha, 57, 58, 81.

Nutrition, in Balantidium, 282; in
Councilmania muris, 496; in Lei-
dyopsis, 101, 391-400; in Men-
oidium, 452; in Paramaecium,
237, 238; in Streblomastia, 7, 13;
in Trichomitus, and Trichonym-
pha, 23, 56, 81, 391, passim; in
trichonymphid flagellates, 391-
400; comparison between milk
only and meat only diets in
parasitic amoebae, 521-522.

On Balantidium coli (Malmsten) and
Balantidium suis (Sp. Nov.),
with an Account of their Neuro-
motor Apparatus, 243-300.

On the Free, Encysted, and Budding
Stages of Councilmania lafleuri,
a Parasitic Amoeba of the Hu-
man Intestine, 169-198.

On the Genus Councilmania, Budding
Intestinal Amoeba Parasitic in
Man and Rodents, 431-445.

On the Morphology and Behavior of
Pentatrichomonas ardin delteili
Derrieu and Raynaud), 373-390.

On the Morphology and Mitosis of
Chilomastix mesnili (Wenyon),
a Common Flagellate of the Hu-
man Intestine, 117-144.

Operculum, 45.

Ophyroscolex, 267.

Index

Organelles, locomotor, in Balantidium,
290; in Diplodinium, 290; in Bu-
plotes, 290; in Leidyopsis, 103; in
Trichonympha, 50; doubling of,
in promitosis, 374.

Osmosis, in Streblomastia, 13.

Oxyrrhis marina, 464.

Parabasal body, in Chilomastix, 126;
in Giardia, 200, 209, 210; in
Trichomitus, 28, 29, 31, 32, 37.

Paradesmose, in Chilomastix, 118, 124,
133, 137; in Copromonas, 458; in

Euglena agilis, 458, 459; in
Giardia, 215, 224, 459; in Leidy-
opsis, 107, 108, 111; in Men-

oidium, 458, 459; in Trichomitus,
29, 32, 33, 37, 71; in Trichomonas,
71, 459; in Trichonympha, 47,
62, 71, 81; in contradistinction
to intradesmose, 177, 304, 319.

Paramaecium, 235-242, 249, 281;
neuromotor apparatus of, 333-
364.

Parasites of the termites, studies on,
1-116; of human intestine, see
Amoeba; Flagellates; specificity
of, or transference, 421-425, 519,
526; morphological constancy of,
in cross-infection, 424, 425, 519;
varied occurrence, 431; possible
source of infection of, in man,
526.

Parastyle, 127.

Pellicle, in Balantidium, 263, 265; in
Paramaecium, 349.

Pentatoma, 72.
Pentatrichomonas,
man, 432.
ardin delteili, 373-390.

bengalensis, 377.

Peristome, 275, 277.

Peromyscus maniculatus, 222.

Personympha flagellata, 216.

Pigs, hosts for Balantidium, 246, 293.

Plasmolysis, 265.

Plastids, in endoplasm of Menoidium,
450

Polymastigina, 15, 16, 81, 373.

Polymastigote flagellates, evolution

found only in

of, 373.

Protozoa, 1, 42. See also, Amoeba;
Flagellates.

Pseudopodia, in Councilmania de-

cumani, 439, 496, 497-501, 525;
in C. lafleuri, 172, 173, 432, 435,
497; in C. muris, 496, 497-501,
525; in Endamoeba coli, 497; in
E. dysenteriae, 410; in EF. histo-
lytica, 497; in HB. ratti, 515; in
Trichonympha, 391-400; as means
of nutrition, 391-400; as deter-
mining factor in establishing
species, 522-524, 525.

[550]

Pseudopodial Method of Feeding by
Trichonymphid Flagellates Para-
sitic in Wood-eating Termites,
391-400.

Pseudosynapsis (pseudotelosynapsis)
in Leidyopsis, 111; in Trichonym-
pha, interpretation of, 72-77, 81.

Pseudotrichonympha, 78.

Purgatives, in experiments with rats:
castor oil, 403; epsom salt, 403,
406, 408.

Pyrsonema, 78.

Pyrsonympha, 9, 78, 373.

vertens, 2, 14.

Rabbits, in experiments with Leish-
mania, 477-488.

Rats, infection experiments with,
401-430, 489-544; carriers of
amoebiasis, 423.

Rattus norvegicus, 411, 491, 492.

Rees, C. W., 235-242, 333-364.

Rhabdomonas incurva, 448.

Rhizoplast, in Chilomastix, 118, 131,
132, 137; in Giardia, 206; in
Menoidium, 451, 452; in Penta-
trichomonas, 379, 387; in Streblo-
mastix, 7, 16; in Trichomitus, 31,
32, 37; in Trichonympha, 52.

Rhodes, R. C., acknowledgment, 448.

Sarah Berliner Research Fellowship,
acknowledgment, 315.

Schizonts, arrangement of, in Tricho-
monas, 11.

Sexual behavior, absence of, in Streb-
lomastiz, 10, in Trichomitus, 35,
and in Trichonympha, 72.

Skin Reaction to Extracts of Leish-
mania tropica and Leishmania
infantum, 477-488.

Smith, Inez, acknowledgment, 411,
432.

Specificity of parasites, 421.

Sphaerita, as parasite in amoeba,

Spiral groove, in Chilomastix, 118,
121, 135, 137.

Spireme in Chilomastir, 134.

Spirostomum, 334, 349.

Spirotrichonympha, 9, 14.

Staining, intra vitam, in Paramaecium,
347. See also Congo red, re-
actions of cysts to; Technique.

Stentor coeruleus, 269, 334.

Streblomastigidae, 15, 16.

Streblomastix, morphology, 4-10; re-
lationships, 14-15; generic diag-
nosis, 15.

strix, 1-20; occurrence with Trich-
onympha, 43.

Striations, longitudinal, on Menoidium

ineurvum, 450.

Index

Studies on the Parasites of the Ter-
mites:

I. On Streblomastix strix, a Poly-
mastigote Flagellate with a
Linear Plasmodial Phase, 1-20.

II. On Trichomitus termitidis, a
Polymastigote Flagellate with
a Highly Developed Neuro-
motor System, 21-40.

III. On Trichonympha campanula,
Sp. Nov., 41-98.

IV. On Leidyopsis sphaerica, Gen.
Nov., Sp. Nov., 99-116.
Swezy, Olive, 313-332, 391-400; ae-

knowledgment, 411, 477, 491.

Taylor, C. V., acknowledgment, 334.

Technique, 3-4, 171, 245, 336-338,
401-408, 411-412, 492.

Termites, parasites of, studies on,
1-116.

Termopsis angusticollis, 2, 15, 16, 22,
36, 37, 48, 100, 111, 392; amount
of infection in single individual,
2009)

Tetratrichomonas, 9; synonym for
Trichomonas, 159.

bucealis, 156, 158, 159.

hominis, 156.

parisi, 159.

prowazeki, 159.

Torsion, direction and structural ex-
pression of, in polymastigotes, 9.

Tradescantia virginica, 465.

Tricercomonas intestinalis, 147.

‘«Trichomastix’’ condition, defined,
158.

Trichomitopsis, 36, 37.

Trichomitus, 9; short-lived in ecul-
tures, 23; nutrition in, 23; bio-
chemical changes of environ-
ment, 29; paradesmose in, 71.

parvus, 36, 37.

termitidis, 2140.

Trichomonas, 9, 21, 23, 151, 155,
passim; arrangement of schizonts
in, 11; multiple fission in, 13, 14;
paradesmose in, 71; centroble-
pharoplast in, 123, 124; distinet
from Pentatrichomonas, 377; in
rats, 405, 406.
augusta, 24, 160.
bucealis, 156, 159.
confusa, 156.
hominis, 155, 158, passim, 374, 375,
376, 377-386.
intestinalis, 147, 156, 158.
muris, 406.
pulmonalis, 156, 158.
sensu latu, 160; sensu strictu, 159.
vaginalis, 156, 158, passim.
Trichonympha, 2, 10, 22, 23; agility,
44, 393; key to, 80; relationship
with Leidyopsis, 110, 391-400.
agilis, first of genus discovered,
44; ridges on, 49; flagella of,
50; anterior part of, 60, 61;
minore and maggiore, 69, 78.
campanula, 41-98, 100; similarity
with Paramaecium, 339, with
Menoidium, 467.
hertwigi, 46, 47, 61, 70, 72, 78.
Trichonymphidae, 80, 81, 112.
Tritrichomonas, 160, 378.
augusta, 160.
Trypanosoma, 162.
Tyzzer, E. E., acknowledgment, 477.
Vacuoles, food, in Trichomitus, 24;
contractile, in Balantidium, 263,
280, 281.
Virgulina pirenula, 157.
Wagener, Edna H., 477-488.
Walker, E. L., acknowledgment, 411,
491,

ERRATA

Page 42, line 7 from bottom. Following efficiency, insert since the unicellular

state permits both,.

Page 264, labeling of figure 1. For mic. membr. read nuc. membr.
Page 378, line 9 from bottom. For while the three read while the four.
Page 418, line 15. For unknown ‘read a known.

[551] -

6.

11,

12.

13.
14

16.
16.

17.

21,

| 22,

23.

Vol. 21. 1.
2,
3.
4,

5.

UNIVERSITY OF CALIFORNIA PUBLICATIONS—(Continued)

. Studies on the Parasites of the Termites. III. Ox Trichon ympha campanula

sp. nov., by Charles Atwood Kofoid and Olive Swezy. - 41-98, pl
5-12, 4 figures in text. July, 1919 Pie Sevan

(Grassi) of Man, with a Discussion of the Method of Origin of Bilateral
Symmetry in the Polymastigote Flagellates, by Charles A. Kofoid and
Olive Swezy. Pp. 199-234, plates 23-26, 11 figures in text. March,
aE aN aR a TA OB Les BA ok eae Vea eee er at) oe AYO) pe ia kee gee Wa

. The Micro-Injection of Paramaecium, by Chas. Wm. Rees. Pp, 235-242.

Pat ta y.b ae Laat RSs i eat A i Ne RE NG CGR 0 OP

. On Balantidium coli (Malmsten) and Balantidium suis (sp. nov.), with

an account of their neuromotor apparatus, by J. Daley McDonald.
Pp. 243-300, plates 27, 28, 15 figures in text. May, 1922 0)

Mitosis in Endamoeba dysenteriae in the Bone Marrow in Arthritis de-
formans, by Charles Atwood Kofoid and Olive Swezy. Pp. 301-307,
7 figures in text.

Endamoeba dysenteriae in the Lymph Glands of Man in Hodgkin’s Disease,
by Charles A. Kofoid, Luther M. Boyers, M.D., and Olive Swezy. Pp. 309-
$12, 4 figures in text. ,

os, 11 /and 12 in.one-cover: April! 1922 isc05 ey Se re

Mitosis in the Encysted Stages of Endamoeba coli (Loesch), by Olive
Swezy.. Pp. 313-332, plates 29-31. May, 1922.00

The Neuromotor Apparatus of Paramaecium, by Charles William Rees.
Pp. 333-364; plates 32-36, 5 figures in text. November, 1922.00.00.

A Comparison of the Cysts of Endamoeba coli and Cowncilmania lajleurt in
Congo Red, by Nathaniel Bercovitz, M.D. Pp. 365-371. February, 1923.

On the Morphology and Behavior of Pentatrichomonas ardin deltgili (Der-
Tieu and Raynaud), by Charles Atwood Kofoid and Olive Swezy. Pp.
373-390, plate 37, 1 figure in text. August, 1923 20000. ee

The Pseudopodial Method of Feeding by Trichonymphid Flagellates Para-
sitic in Wood-eating Termites, by Olive Swezy. Pp. 391-400, 14 figs, in
GORY AUER SE, PLO ACs os he CS ee Ee at a Sh a

Methods of Obtaining Amoeba-free Rats for Experimental Infection with
Intestinal Amoebae, by John F, Kessel. Pp. 401-408.

Experimental Infection of Rats and Mice with the Common Intestinal
Amoebae of Man, by John F. Kessel, Pp. 409-430, plates 38-39.

Nos. 18 and 19 in one cover. October, 1923.22.20.

On the Genus Councilmania, Budding Intestinal Amoebae Parasitic in Man
and Rodents, by Charles A. Kofoid, Olive Swezy, and John F. Kessel
Pp. 431-445, 19 figures in text. October, 1923 2. .c.-..2. ee lees

Morphology and Binary Fission of Menoidium incurvum (Fres.) Klebs,
‘by Richard P. Hall. Pp. 447-476, plates 40-41, 2 figures in text. Novem-
[tyes shpat it 7-6 a a aw as BRAS oe ee NO Cer A) eee eRe ee D

A Skin Reaction to Extracts of Leishmania tropica and Leishmania in-

The Distinguishing Characteristics of the Parasitic Amoebae of Culture
Rats and Mice, by John F. Kessel. Pp. 489-544, plates 43-48, 3 figures
an texte PRC ary, 2024 co ee) Aa kh OE a ee ae

A Revision of the Microtus californicus Group of Meadow Mice, by Reming-
ton Kellogg. Pp. 1-42, 1 figure in text. December, 1918 —...............-...

Five New Five-toed Kangaroo Bats from California, by Joseph Grinnell.
Pp 48-4705 MATCH 1 a en ee race +

Notes on the Natural History of the Bushy-tailed Wood Rats of California,
by Joseph Dixon. Pp. 49-74, plates 1-3, 3 figures in text. December, 1919

Revision of the Avian Genus Passerella, with Special Reference to the Dis-
tribution and Migration of the Races in California, by H. 8. Swarth. Pp.
75-224, plates 4-7, 30 figures in text. September, 1920 —._..................

A Study of the California Jumping Mice of the Genus Zapus, by A. Brazier
Howell Pp. 225-238, 1 figure in text. May, 1920 —._....~...-.- yer

+25

UNIVERSITY OF CALIFORNIA PUEBLICATIONS—(Continued)

6. Two New Rodents (Genera Thomomys and Marmota) from the Eastern
Border of California, by Joseph Grinnell. Pp. 239-244, 6 figures in text.
INO Wember; 1927 piso ee oe a et eT a ae
7. A Study of the Californian Forms of the Microtus montanus Group of of
Meadow Mice, by Remington Kellogg. Pp. 245-274, 25 figures in text.
8, A Synopsis of the Microtus mordaz Group of Meadow Mice in California,
by Remington Kellogs. Pp. 275-302, plate 8, 29 figures in text.
Nos. 7 and 8 in one cover. April, 1922 .............. Soc eee
9. Behavior of the Leaf-nosed Snake, Phyllorhunchus decurtatus, by Sarah
Rogers Atsatt. Pp. 303-312. February, 1923 2220.
10. A Systematic List of the Mammals of California, by Joseph Grinnell.
Pp.31S-S24..Fanwary; 1928 5252.08 oN ah ee ee a
11. The Systematic Status of the Mountain Lion of California, by Joseph
Grinnell and Joseph Dixon. Pp. 325-332, plates 9-10. April, 1923....._
12, New Subspecies of Birds from Patagonia, by Alexander Wetmore. Pp.
SSS-837T eT PURE, WSLS Ved ws se gh ee ae ST ae
13. Revision of the Genus Lynx in California, by Joseph Grinnell and Joseph
Dixon. Pp. 339-354, plate 11, 1 figure in text. January, 1924 W000.
Vol. 22. 1. A Quantitative and Statistical Study of the Plankton of the San Joaquin
River and Its Tributaries in and near Stockton, California, in 1913, by
Winfred Emory Allen. Pp. 1-292, plates 1-12, 1 figure in text. June, 1920.
2. Variations in the Shell of Teredo navalis in San Francisco Bay, by Robert
Cunningham Miller. Pp, 293-328, plates 13-17, 6 figures in text. Novem-
ber, 1922 - it
8. Quantitative ‘Studies on Marine ‘Phytoplankton ‘at La Jolla in 191: r by
Winfred Emory Allen. Pp. 329-347, 2 figures in text. November, 4998 30
4, On the Effect of Low Salinity on Teredo navalis, by Harold Francis Blum.
Pp. 349-368, 4 figures in text. December, 1922... 35
5. Quantitative Studies on Inshore Marine Diatoms and Dinoflagellates of
Southern California in 1920, by Winfred Emory Allen. Pp. 369-878,
1 figure in text. ’
6. Stylarioides papillosa, sp. nov., a New Annelid from the San Diego Region,
by Christine E. Essenberg. Pp. 379-381, 8 figures in text.

Nos. 5 and 6 in one cover. December, 1922 -2 eee ee .25

7. The Digestion of Wood by Teredo navalis, by Walter H. Dore and Robert
©. Miller. Pp. 383-400, plate 18. February, 1923 ok 265

8. Variations in the Pallets of Teredo navalis in San Francisco Bay, by Robert
Cunningham Miller. Pp. 401-414, plates 19-20. February, 1923 _.._.......... -25

9. Some Tide-Water Collections of Marine Diatoms taken at Half-Hour Inter-

vals near San Diego, California, by Winfred E. Allen. Pp. 413-416,
iifigure:in text. June; 1923690 ee eS eS 25

10. Preliminary Statistical Report on the Occurrence of Marine Copepoda in

the Plankton at La Jolla, California, by Caivin O. Esterly. Pp. 417-433.
SUNG, WOAS se ee eh a ae eed 25

11. Studies on Marine Diatoms and Dinoflagellates Caught by Aid of the

Kofoid Bucket in 1922, by Winfred Emory Allen. Pp. 435-445, 5 figures
intext; - October, 1928 yao a eh PA ee ee ee 25

12. Statistical Studies of Marine Diatoms of the San Diego Region Collected

by U.S.S. Pioneer in Midwinter of 1923, by Winfred Emory Allen. Pp.

445-448"..; November, 1923 205-2) ee eee SR 265 ”
18. Life Cycle of Oithona nana Reared Experimentally, by Helen E. Murphy. ¥
Pp. 449-454, 5 figures in text. November, 1923 _ 0. oe 25 |
14, Morphology of the Digestive Tract of Teredo navalis, by Edgar Locke
Lazier. Pp. 455-476, plates 21-24. December, 1923... +35
Vol 28. The Marine Decapod Crustacea of California, by Waldo L. Schmitt. Pp.
1-470, plates 1-50, 165 figures in text. May, 1921 2.200002. 5.00
VoL24. LA Geographical Study of the Kangaroo Rats of California, by Joseph
Grinnell. Pp. 1-124, plates 1-7, 24 figures in text. June, 1922.0. 75

2. Birds and Mammals of the Stikine River Region of Northern British
Columbia and Southeastern Alaska, by H. 8. Swarth. Pp. 125-314, plate

8, 34: figures:in texts.d une; 1922 <2. Oe ae oe Nee 2.50
3. Birds and Mammals of the Skeena River Region of Northern British
Columbia, by Harry 8. Swarth. Pp. 315-394, plates 9-11, 1 figure in text.

January, 1924

VoL 25. A Bibliography of Eugenics, by Samuel J. Holmes. Pp.1-514. January, 1924 5.00
Vol. 26. 1. On the Family of Achiridae or Broad-Soles, with description of a new
species, Achirus barnharti, from California, by David Starr Jordan.

Py. 1-14, plate® F2December, : 1923 205 Son oo sa ea Ae 25
2. A Precipitin Test in Experimental Amoebic Dysentery in Cats, by Edna
Hannibal Wagener. Pp. 15-20, plate 2. January, 1924 22 25,

3. On the Distinctions between Endamdeba coli and Councilmania lafleuri, by
Charles A. Kofoid, Olive Swezy, and John F. Kessel. Pp. 21-39, 24
fignres iin text.” “DaAMMary, 19D oo Fie a SON a ot A5

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