The Journal of Protozoology

Volume 10 February, 1963 Number 1

J. PROTO,ZOOL.
lO(l), 1-6 (1963).

Differentiation in Protozoa*
WILLIAM TRAGER
The Rockefeller Institute, New York 21, N . Y

SYNOPSIS. Two main kinds of morphogenetic events occur
among protozoa: (1) reproduction ; ( 2 ) processes of reorgani-
zation, including encystment, excystment, sexual processes,
structural and physiological adaptations exhibited in the
course of complex life cycles, and restorative events elicited
by environmental stimuli. Examples of such events are dis-
cussed with reference both to their own intrinsic interest and
to their value in the study of cellular differentiation in gen-
eral. Stomatogenesis in ciliates provides a remarkable in-
stance of extensive and rapid differentiation. The phenomenon
may be studied to advantage not only in reproduction hut also
in the macrostome-microstome transformation. Other simple
transformations in protozoa providing unparalleled material
for experiments on cytodifferentiation under controlled condi-
tions are excystment and encystment, the amebo-flagellate
transformation, and the leishmania-leptomonad change. The
more complex transformations occurring in the life cycles of
trypanosomes and in the appearance of sex under the influ-
ence of an insect hormone provide additional material of basic
significance to an understanding of differentiation.

ARS have been fought over words-words such Cytodifferentiation( 3 1) refers to the process by
W as “nation” or “liberty.” These are words mean-
ing different things to different people, words from
which a cell is transformed into a specialized structure
as rated by the tangible results of this transformation.
which we tend to expect more than we have put into Differentiated cells may produce visible secretions (as
them. In science, such a fighting word is “differentia- in neurosecretory cells) or they may produce secretions
tion.” Some would discard the word completely. And which cannot be seen but the effects of which are de-
it is true-as soon as you attempt to define it pre- monstrable (as hormones), or they may form products
cisely, you are lost. Yet it is a very useful word- which remain part of the cell, as myofibrils or cilia, or
as long as we remember that its meaning must be the tail of a spermatozoon.
rather vague and loose, in keeping with the even more It is important to realize that our recognition of
vague and incomplete state of present knowledge as cytodifferentation depends entirely on the techniques
to how living things come to be the way they are. As we have for its detection. We think of a ciliate like
Paul Weiss has said, it is “idle to indulge in definitions Euplotes as being more highly differentiated than an
of such terms as differentiation and dedifferentiation. ameba because it has so many more structures visible
The task is rather one of identification and precision with the light microscope. A muscle cell has more
of the facts underlying such concepts”(31). visible structure than a fibroblast, and so on. Perhaps
The entire complex process of morphogenesis and if we rated differentiation by some enzymatic criterion,
growth of a multicellular organism involves interac- or by the complexity of mitochondria1 structure as
tions among its constituent cells as well as changes in seen by electron microscopy, we would consider the
the cells themselves, changes whereby originally pluri- ameba more highly differentiated than the ciliate.
potent cells are channeled into one of several courses The essential aspect of differentiation is change-
of transformation. If we can agree that the cells sur- change in the chemistry and morphology of a cell
rounding an individual cell in a multicellular organism making it distinctly different from its previous state,
are in fact an important part of the environment of despite the fact that its nucleus still contains the same
that cell, then the development of that cell in response ehromosomes with the same genetic information. Such
to its environment is the basic phenomenon to be changes occur in protozoa as well as in multicellular
studied. For this the term cytodifferentiation has organisms. It will be the aim of this paper to exam-
been used. ine the kinds of differentiation exhibited by protozoa
* Address of Past President, Society of Protozoologists, with reference both to their own intrinsic interest and
delivered at Corvallis, Oregon, August 31, 1962. especially to their value in the study of cellular differ-

1
2 DIFFERENTIATION
IN PROTOZOA
entiation in general. That the protozoa are cellular
can no longer be argued. Even in details of fine struc-
ture exhibited by the electron microscope they are
obviously comparable to metazoan cells and the term
“acellular” should be abandoned (see 1 2 ) .
Two main kinds of morphogenetic events may be
observed among protozoa under natural conditions:
( I ) reproduction; ( 2 ) processes of reorganization. I n
the latter category may be placed encystment, excyst-
ment, sexual processes, all the specialized structural
and physiological adaptations exhibited in the course
of complex life cycles, as well as restorative events
elicited by various environmental stimuli. All of these
may or may not be accompanied by reproduction.
I n the reproduction of a ciliate protozoon a more
or less transverse division of the cell gives rise to 2
daughter cells. But since the original cell has a dis-
tinct antero-posterior axis with quite different struc-
tures at each end, simple transverse fission such as
occurs in bacteria would produce two very different
and incomplete daughters. both incapable of sustained
life. Before division occurs, there must take place a
complex process of formaton of new organelles of the
prospective daughter cells. Most striking and of course
most important of those organelles is the mouth. The
neoformation preparatory to division of the specialized
cilia and membranelles comprising the mouth of the
posterior daughter in the reproduction of ciliates is
one of the most dramatic of all niorphogenetic events.
This has been studied especially in certain large, free-
living ciliates which lend themselves to extensive surgi-
cal operations( 23.26,32). In one of these. Stentor,
the anlage of the new mouth arises in a particular re-
gion of the cell along a line of sharp contrast between
the broad stripes of the dorsal part of the body and
the narrow stripes of the ventral part. Tartar ( 2 3 ) has
truly called it “an astonishing achievement of differen-
tiation.” In less than 4 hours 15.000 basal bodies are
formed, become aligned in rows by twos or threes, and
produce cilia, ciliary rootlets and a system of connect-
ing fibrils. Cutting and graftng experiments have
shown that. as in multicellular organisms, formed parts
inhibit neoformation of their like. l-et in cell division
the inhibitory effect of the esisting mouth must some-
how be temporarily suspended to permit development
of the new anlage of the posterior daughter. Xeither
the nature of the inhibition, nor of its removal prepara-
tory to cell division, are understood. T h e controlling
mechanisms, however. have definitely been shown to
reside in the ectoplasm and certainly to be associated
with the development of kinetosomes.
The kinetosomes or basal bodies of ciliates have
been assumed to be autonomous, self-duplicating nior-
phogenetic agents(lS), but there is no proof of this
( 1 2 ) . It is interesting that the behavior of kineto-
somes in the formation of dfferentiated structures in
the cell of a ciliate exhibits some striking analogies to
the behavior of cells in regeneration in multicellular
Organisms. Thus, the kinetosomes become more nu-
merous and form fields of kinetosomes. These then
become arranged and give rise to the new structures.
Likewise, the differentiation of eggs of the mosaic type,
as found in insects, presents some striking analogies
to the differentiation of Stentor(23). In both, certain
regions of the cytoplasm appear to be predetermined
for the formation of particular structures. I n the
egg these structures are ultimately expressed through
the formation and activities of numerous cells: in the
ciliate they find expression through the activities of
kinetosomes. These analogies may be superficial. But
there can be no doubt that if we could find out what
makes kinetosomes behave the way they do, we would
have made an important advance toward understand-
ing the mechanisms of cellular differentiation.
I n their fine structure the kinetosomes of ciliates
resemble closely the basal body or centriole of other
protozoa and of animal cells in general. All have a
fundamental unit consisting of a short cylinder con-
taining in its periphery 9 fibrils which may be either
double or triple( 2,3). Further elaborations may be
present. There is good evidence that one centriole
can give rise to another but just how is not known
( 1 0 ) . The morphogenetic role of the centriole in all
cells is well shown by its function in spindle formation
during mitosis. But in some flagellate protozoa one
or two centrioles synthesize all the organelles of the
cell. The studies of Cleveland( 7,8) on a giant Tricho-
m o m s and on the large hypermastigote flagellates of
C‘ryptoccrcus and of termites have shown this espe-
cially well. The hypermastigotes are among the few
cells whose centrioles are so large that we can follow
their development with the light microscope in living
organisms. I n these flagellates each resting cell has
two centrioles, an old one and a new one. From pro-
phase onward, two new ones and two old ones are
present. These new centrioles, in their first genera-
tion. produce only extranuclear organelles (flagella,
parabasals. asostyles, etc.) ; in their second and later
generations they produce a new centriole and the
achromatic figure.
In view of the apparent morphogenetic potency of
kinetosomes and centrioles in protozoa these structures
certainly deserve more study than they have so far
received. Cytodifferentiation must involve some con-
cept of molecular morphogens. As an example of a
molecular niorphogen. one may cite sickle cell hemo-
globin. for this molecule, in an environment lacking
osygen. causes red cells to assume a characteristic
shape. I t seems possible that in the formation of
various fibrillar structures from centrioles we have a
system which will ultimately be susceptible to descrip-
tion in terms of such a concept. The importance of
 IN PROTOZOA
DIFFERENTIATION
fibrous proteins in morphogenesis has been thoroughly
treated by Berrill( 1).
The differentiative processes discussed so far are
concerned with the formation of new individuals. Per-
haps of even greater interest are differentiations in-
volving change in form or function, often not accom-
panied by reproduction. I n this category belong the
changes occurring in the life cycles of protozoa. Some
of these life cycles, as I need hardly tell this audience,
are extremely complex. A very simple cycle is illus-
trated by certain free-living ciliates which can exist
either as small forms with a relatively small mouth
(microstomes) , or as larger forms with a relatively very
much larger mouth (macrostomes). The microstomes
generally feed on bacteria, the macrostomes on other
ciliates. The adaptive significance of the large mouth
for carnivorous feeding is obvious. This polymorph-
ism is well known in Tetrahymena vorax and has re-
cently been studied in T . patula. Interestingly enough,
the latter when grown axenically exhibited both forms
in a medium with lettuce extract; in a medium with-
out lettuce extract, in which growth was slow, only
macrostomes were present(33). Clones started from
either type produced both in an appropriate environ-
ment. When given washed T . pyriformis as food, the
T . patula became carnivorous and only macrostomes
were present. Under axenic conditions both tempera-
ture and p H affected the proportion of macrostomes,
which was always greatest toward the end of the
growth cycle. I n the phase of logarithmic multiplica-
tion only microstomes were present. I n the stationary
phase a t p H 7.0 and a temperature of 20“C, 100% of
the organisms had changed to macrostomes, whereas
at 25” only 50% were macrostomes(22). The con-
version from microstome to macrostome involves re-
organization: the old oral apparatus is not directly
transformed into the new; on the contrary, the old oral
apparatus is resorbed and replaced by the differentia-
tion of a new mouth from a field of kinetosomes which
arises just posterior to the old mouth. Actually this
is formed in about the same place in which a new
mouth would be farmed preparatory to division. N o
division occurs, however, and the new mouth moves
forward to replace the resorbed old one. But the
change from macrostome to microstome always in-
volves division, one macrostome giving rise to two
microstomes. Here we have available for study under
controlled environmental conditions a reversible dif-
ferentiation which occurs in nature and is of functional
significance to the organism.
Another such reversible differentiation is the process
of encystment. Protozoan cysts may be highly differ-
entiated structures: yet the organism within often loses
all its extranuclear organelles, only to regain them be-
fore excystment. Numerous factors tend to produce
encystment: deficiency of food, excess of food, excre-
3
tion products, drying, lack of oxygen, hormones of the
host. Excystment may follow merely placing a dry
cyst in water, but more often special stimulatory sub-
stances are needed, such as sugars and organic acids.
Visible changes during the encystment process include:
(1) secretion of cyst wall; (2) storage of reserves; (3)
resorption of organelles and dedifferentiation of struc-
tures such as the contractile vacuole system; (4) loss
of water with increase in density of the protoplasm.
Vickerman( 27,28) has recently reported interesting
differences in fine structure between mitochondria of
trophic and encysted Acanthamoeba. Whereas the
mitochondria of the trophic amebas contain a single
intracristal spherical inclusion of 500-1000 A . diameter,
in the encysted amebas these inclusions are 3000-5000
A diameter and occupy about half the mitochondrion.
Physiological changes occur too. Pigon & Edstrom
( 17) have measured the respiration of individual cili-
ates (Colpoda cucullus) during encystment and ex-
cystment. They found that the animal settled down
and secreted a delicate cyst membrane before there
was any marked decrease in respiratory rate or rate
of pulsaton of the contractile vacuole. The respira-
tory rate then dropped, at first rapidly and then more
slowly, from an average of 11.3 X l o p 5 pl 02/hr to
only 1.3 after 24 hours. It remained constant a t this
low level for a t least 4 weeks. When a so-called “un-
stable” resting cyst of this type was put in hay infu-
sion medium, it showed within 60 to 100 minutes a n
increase in respiration, and the contractile vacuole re-
appeared. Excystment occurred in 2 to 3 hours and
respiration reached the pre-encystment level after an-
other hour or two.
Still a third reversible differentiating system in pro-
tozoa is provided by the amebo-flagellate transforma-
tion. This phenomenon, first seen by Schardinger( 19)
in 1899, was described in detail by Bunting(5) and
by Hollande( 13) for Tetramitus rostratus. The cyst
of this species gives rise to a uninucleate ameba which
can multiply as such indefinitely under appropriate
environmental conditions. If the medium is diluted
enough, or the O2 tension reduced, the amebas change
to flagellates. At NaCl concentrations of ~ / 1 6to
N/20 Tetramitus then multiplies as a flagellate. At
concentrations of N/8 to ~ / 1 4it again becomes ame-
boid. Recent experiments( 16) indicate that the con-
trol of transformation in Tetramitus is more complex
than had been supposed. Some culture strains have
lost the ability to become ameboid. Cell density itself
seems to be an important factor. When a n ameba is
about to change to a flagellate, two blepharoplasts ap-
pear and migrate rapidly to the posterior pole of the
ameba; each produces two flagella. T h e pseudopods
of the ameba retract, its body becomes conical, a cyto-
stome forms at what is now the new anterior end, and
the flagellate swims away. T h e contractile vacuole
4 DIFFERENTIATION
I N PROTOZOA
stays in place: hence it is posterior in the ameba, an-
terior in the flagellate. This reversal of the antero-
posterior pole may not involve a fundamental change
in polarity, for Hollande notes that the ameba ingests
food at its posterior end, the end which becomes an-
terior in the flagellate and which now has the cyto-
stome, where food is again ingested. The whole change
from ameba to flagellate takes about 30 min. The
reverse change is slower. T h e flagella stop moving,
the flagellate loses its definite contours, suddenly be-
comes ameboid and changes direction with the appear-
ance of a large anterior pseudopod. The flagella. cysto-
stome, rhizoplast and the blepharoplast disappear.
Very similar processes have recently been described
by Bovee(4). I n a related species, n’aegleria gruberi,
although the flagella appear a t the posterior end. they
migrate to the anterior end. For this species \Yillmer
(34) has shown that osmotic pressure per se is not the
determining factor in the change from ameba to flagel-
late. Certain ions, including S a but especially JIg.
a t appropriate concentrations are specifically effective
in suppressing the change from ameba to flagellate.
The change will nevertheless occur in non-electrolyte
solutions as concentrated as M ’ 5 . With methods now
available for growing these ameba-flagellates axenically
in defined medium. with concomitant studies by elec-
tron microscopy and of changes in enzyme activities
and in nucleic acids, we might get some surprising in-
formation. Fortunately. such studies have already
been begun by Balamuth and his associates( 1 6 ) .
A phenomenon superficially resembling the one just
discussed is the transformation from an aflagellate to
a flagellate state of certain parasitic flagellates, such
as Leishmania donovani. This organism. which causes
kala-azar in man, grows inside reticuloendothelial cells
as an intracellular ovoid body. It is transmitted by
sand flies, and within the alimentary tract of an in-
fected fly it grows extracellularly as an actively motile
flagellate, the so-called leptomonad. In this stage it
can also grow in culture.
The change from leishmania to leptomonad can be
easily produced in vitro. It suffices to grind up the
spleen of an infected hamster and prepare a suspension
of leishmanias in nutrient broth. If this is left a t
25-28”C, within a few hours flagellate forms begin to
appear. The reverse change. however. does not occur
in vitro. Flagellates placed at body temperature ( 3 7 )
die. unless supplied with living cells; if they get inside
a living host cell they change to leishmanias and multi-
ply as such. The change here is more profound than
in the amebo-flagellate transformation: at room tem-
perature not only does the form change and a flagellum
appear, but the organism acquires the ability to grow
extracellularly in an appropriate nutrient medium. At
3 7 ” the flagellum is resorbed and key synthetic proc-
esses must be inactivated, for the organism can now
grow only inside another living cell( 24). Recent elec-
tron microscope studies(l8) show that in the change
from leishmania to leptomonad the kinetoplast enlarges
and seems to be forming much new mitochondria1 ma-
terial. This would fit with the greater synthetic ac-
tivities of the leptomonad form.
Trypanosomes are yet another group of hemoflagel-
lates exhibiting profound changes in metabolism as
well as form during their life cycle. The blood stream
forms of the African trypanosomes metabolize sugar
without the intervention of cytochromes, whereas the
forms in the midgut of the tsetse fly respire with a
cytochrome system. T h e end products of sugar me-
tabolism are quite different in the two stages (29). The
midgut forms are unable to infect a mammalian host
(or even to survive at 3 7 “C) but when they reach the
salivary glands of the fly some morphogenetic effect
is here exerted which transforms them so that they
resemble blood stream forms and are able to infect the
mammalian host. This effect has been duplicated in
vitro only sporadically ( 11,30). For Trypanosoma
vizlax cultivation in the presence of living tsetse By
tissue and exposure to a temperature of 38°C brought
about the appearance of infective trypanosomes( 2 5 ) -
Here we have a cellular differentiation which results in
the dramatic change from a harmless symbiote of in-
sects to a pathogenic parasite of mammals.
.4n analogous change has already been studied in
more detail in a trypanosome of frogs, Trypanosoma
mega. This parasite grows in culture as a crithidia,
presumably corresponding to stages in an invertebrate
vector. I n the blood of the frog it lives as a larger
trypanosome form with a conspicuous undulating
membrane. Steinert (20) found that a few trypano-
somes would appear in the crithidial cultures if a high
concentration of serum was added to the medium.
Contrary to expectation, i t turned out that the active
substance in serum was not a protein but urea. Urea
added a t a physiological concentration of only 0.016
11 could bring about the appearance in zritro of typical
blood stream trypanosomes. T h e percentage of trans-
formed organisms depended on the stage of growth
of the culture. During the phase of logarithmic in-
crease the addition of urea had no effect. I n the sta-
tionary phase up to half of the crithidia changed to
trypanosomes in the presence of urea. Here we have
beautifully illustrated the phenomenon called “compe-
tence” by the embryologists. Only in stationary
phase do the flagellates in the culture become compe-
tent to transform into trypanosomes under the influ-
ence of urea. The detailed analysis which this system
deserves has only begun. The Steinerts( 2 1) have found
that urea added to a competent culture stops the up-
take of tritiated thymidine by those cells which trans-
form but not by those which remain as crithidia.
 DIFFERENTIATION
IN PROTOZOA
Whether this is a cause or an effect of the transforma-
tion, we cannot yet say.
Finally, I should like to consider the morphogenetic
effect of an insect hormone, ecdysone, on protozoa.
The flagellates of the hindgut of the wood-feeding
roach Cryptocercus ordinarily reproduce by mitotic
division, but, as Cleveland(6) has shown, they all ex-
hibit sexual phenomena associated with the molting
of their host. There will be time to review these phe-
nomena only for the single genus Trichonympha.
About 6 days before the roach is going to molt, the
asexual, haploid flagellated cells of Trichonympha
round up, form a thick cyst wall and lose all their
extranuclear organelles except the centrioles, nuclear
sleeve, rostral tube and rostral lamella. Within the
cyst haploid gametogenesis occurs with the formation
of one male and one female gamete. These can be
distinguished immediately by the paler staining of the
chromatin of the female. New complete sets of or-
ganelles are formed for each gamete and the gametes
excyst just after the roach has molted. The male
gamete penetrates the female and all of its extranuclear
organelles are digested. This is a remarkable phe-
nomenon, this ability of the cytoplasm of the female
gamete to digest only the organelles of the male
gamete and not its own, which seem to be of identical
origin, appearance and function. T h e male and fe-
male pronuclei fuse to form a zygote and this under-
goes typical 2-division meiosis to form four haploid
asexual cells. T h e process is complete 2 days after
molting .
T h e details of gametogenesis, fertilization and meio-
sis, as well as the timing of these processes, differ from
one genus of flagellate to another. The exciting fact
is that all of these changes can be brought about by
the injection of crystalline ecdysone(9). This is true
even if the hormone is injected into an adult roach or
an intermolt nymph in such dosage that the host itself
does not undergo ecdysis. Some genera of the flagel-
lates begin gametogenesis within only 3 hours after
the injection of 100 units (0.75 mg) of ecdysone.
There is now some evidence that ecdysone affects di-
rectly the chromosomes of insect cells (14). Further
analysis of its action on protozoa should tell us more
about this powerful differentiating agent, this rela-
tively simple molecule which can have such profound
effects.
One of the paradoxes of biology is that relatively
simple molecules, or even small changes in physical
conditions such as temperature, can have profound
morphogenetic effects. All indications are that these
effects must come about via complex processes involv-
ing complex molecules, notably proteins and nucleic
acids. Studies on bacteria are beginning to provide
an insight into what may be the nature of these proc-
esses which go on within the cell when i t is induced
5
to form a material it has not previously been produc-
ing. Ultimately we will have to know much more
about the intracellular milieu-the medium within
which the nucleus and the cytoplasmic organelles in-
teract to form specialized substances and structures.
I n the meantime, I hope that this brief review has
indicated the special opportunities provided by pro-
tozoa in this central problem of biology.
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6 DISTRIBUTION
OF Plasmodium berghei SCHIZONTS
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l O ( l ) , 6-10 (1961).
Distribution of Schizonts of PZasmodium berghei in Tissues of Rats,
Mice and Hamsters"
NELDA E. ALGER
Depart vietit of Prevenlive Medicitit-, S e w York Cnivrrsity School o j Medicine,
See, York 16, Sew York
SYXOPSIS. .4dult mice, adult hamsters. ?L-day-old rats, and
240-250 gram rats were each infected with one million para-
sitized red cells. Significant differences in the distribution of
schizonts in the tissues of the four groups of animals ranked
from the lowest to the highest were found to be as follows:
LTHOVGH the unequal distribution of malaria
14 in its avian, human and rodent hosts has been
well known for many years, little attention has been
given the possible variatons in the distribution of the
dividing forms. The fact that the segmenting stages
of Plasmodium fakiparum are confined to the capil-
laries of the visceral organs is well established. Talia-
ferro 'and Taliaferro( 14) and Taliaferro and Cannon
( 13 ) , while attempting the transmission of P. falci-
parum to howler monkeys, noted a scarcity of seg-
menters in the peripheral blood in spite of the fact
that the parasite numbers continued to increase. They
concluded that the segmenters must exist in the capil-
laries of the internal organs. During the crisis, seg-
menters were observed in the perpheral blood in
greater numbers than at any other time. This sug-
gested to them that adverse conditions elsewhere
tended to drive them into the blood. They concluded
that the bone marrow and the spleen were the actual
sites of segmentation.
Hegner and Eskridge(4) found even distribution of
schizonts throughout the bodies of canaries infected
with P . cathemerium, whereas Hewitt(5) using a
Mexican strain of P . relictum, concluded that parasites
were unequally distributed throughout the body of
the canary a t different times during the asexual cycle.
* Presented in partial fulfillment of the requirements for
the degree of Dvctor of Philosophy at S e w York rniversity.
1961.
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ation. The Developmental Biology Conference Series, Na-
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R e v . Biol. 29, 207-29.
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Tetrahymt-na patula. J . Protozool. 7, 10-17.
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Biol. 33, 583-603.
young rats-blood, spleen, liver, bone marrow ; older rats-
blood. liver, spleen, bone marrow ; adult mice-no significant
difference between blood and liver, next in rank bone mar-
row, spleen; hamster-blood, but no significant differences
among the organs.
He found that immediately before maximum segmenta-
tion, more segmenters occurred in the liver than in
the peripheral blood, bone marrow, or spleen. At all
times in the cycle he found more parasites in the liver
than in any other part of the body. Hewitt(6) in a
later investigation, noted that in P . relictum and P .
cathemerium the concentration of parasites was not
constant, but that the spleen and bone marrow usually
harbored fewer parasites than did the liver or periph-
eral blood.
Schizonts have been observed after blood inoculation
of P . berghei in the internal organs of rats and mice
by Galliard and Lapierre(3), Vargues and Fabiani
( 1 5 ) and Baldi( 1 ) . N y e ( 9 ) working with a strain
having a n asynchronous cycle, found the dividing
forms irregularly in the blood, but she found no con-
centration in the organs of the hamster.
The following investigation was a comparative study
of the numbers of schizonts found in the blood, liver,
spleen and bone marrow of rats, mice and hamsters
infected with P. berghei. T h e roles played by immu-
nity and age resistance were also considered.
MATERIALS AND METHODS
The S e w York University strain (NYUz) of P . bevghei ob-
tained from Dr. Meir Yoeli of the New York University Dr
partment of Preventive Medicine was maintained in Carworth
Farms X o . 1 strain (CF-I) female adult mice by weekly intra-
peritoneal blood passage. LVeekly passage was made by the
collection of several drops of tail blood in normal sterile saline