Mitochondrial Behaviour during the Life-cycle

of a Sporozoon (Monocystis).
By
E. S. Horning, D.Sc.
Department of Zoology, University of Melbourne.

With Plate 9.

INTRODUCTION.
AT the present time the behaviour and function of mito-
chondria within the animal and plant cell is a matter of con-
troversy among cytologists, and, in order to help to elucidate
the nature of these bodies, observations were made upon these
cytoplasmic inclusions within a common sporozoon (Mono-
c y s t i s ) . The changes which mitochondria undergo during the
life-cycle of this organism, together with the role they appear
to play in cellular metabolism, is also discussed.

LIFE-CYCLE OF MONOCYSTIS.
If the sperm-sacs of the common Australian (European)
earthworm are examined, it will be noticed that they are occa-
sionally infected with M o n o c y s t i s . The life-cycle of the
parasite is well known. When reproduction is about to take
place the two trophozoites or adult individuals come together,
become rounded off, and finally secrete a cyst, in which two
gametocytes become enclosed. The two nuclei of the adult
gametocytes divide repeatedly until a large number of nuclei
are formed. Later, each individual nucleus becomes sur-
rounded by a small quantity of protoplasm. From this stage
small minute cells (gametes) are finally formed. These minute
gametes subsequently combine in pairs, and from these the
zygotes or spores arise, so that the cyst at this phase of the life-
cycle contains many such bodies. The nucleus of each zygote
136 B. S. HORNING

undergoes fission giving rise to a number of fusiform sporo-

zoites, generally eight in number. The spore coat finally rup-
tures and these young fusiform sporozoites are freed, and then
make their way to the developing clumps of sperm, eventually
becoming surrounded by sperm-cells. Finally, they grow into
the adult organism.

METHODS OF INVESTIGATION.

In order to observe the behaviour of mitochondria during the

life-cycle of this Sporozoon, the sperm-sacs of the common
European earthworm, which occasionally swarm with Mono-
c y s t is, were fixed in osmo-chroim'c fluid or else in a Champy
solution. The fixed material was then washed in running water
for a period of twenty-four hours, brought up through the
alcohols and eventually embedded in paraffin, and sectioned.
The sections were then cut 2-5 p in thickness and stained
with Heidenhain's iron haematoxylin, and occasionally counter-
stained with eosin.
An examination of these sections with high magnifications
shows that the cytoplasm of the trophozoite or adult individual
contains numerous small, bent, rod-shaped bodies, together with
a smaller number of spherical bodies scattered irregularly
throughout the medullary region of the organism (see fig. 1,
PI. 9). These cytoplasmic inclusions certainly present a
bacterial appearance, but their subsequent reaction to certain
stains and fixing reagents shows quite clearly that they cannot
be foreign organisms. Also, at various stages of the life-cycle,
these bacteria-like bodies stained a bluish green when
treated i n t r a v i t a m with a Janus green B solution of about
1:10,000. It has been frequently shown by various authors
that this i n t r a v i t a m method does not appear to have any
corresponding selective action upon bacteria, while Cowdry (1)
and many other investigators have found that mitochondria
in living cells stain specifically with Janus green B. Various
other tests for mitochondria were also carried out, similar to
those I have already described in some of my previous
 MITOCHONDRIA OF MONOCYSTIS 137

papers (4, 5, 6, 7, 8, 9, 10), and the results show quite clearly

that these protoplasmic bodies are true mitochondria reacting
in the same manner as mitochondria present in other animal
and plant cells.
MlTOCHONDRIAL BEHAVIOUR DURING THE LlFE-CYCLE.
When the adult organism or trophozoite, cut in longitudinal
section and stained with the Heidenhain process, is examined
under high magnifications, the cytoplasm is found to contain a
large number of bent rod-shaped and spherical mitochondria,
scattered irregularly throughout the protoplasm of the medul-
lary region of the parasite (see fig. 1, PI. 9). A closer study of
these mitochondria shows that they increase in number by
means of transverse binary fission. A more detailed observation
of the general arrangement of these bodies will reveal a dense
aggregation of mitochondria in the vicinity of the nucleus (see
fig. 1, PI. 9) lying in close contact with the outer surface of the
nuclear membrane. When dealing with this phenomenon in
previous papers (9,10), I have pointed out that it may possibly
be a surface-tension effect which is apparently dependent upon
the phosphatidal nature of the mitochondria. When stained
sections of the next stage of the life-cycle—the early sexual
phase—are examined, the cytoplasm of the encysted gameto-
cytes is observed to contain numerous rounded mitochondria,
often varying in size; while on the other hand the number of
rod-shaped bodies, such as were seen in the previous stage, have
undergone a considerable decrease in number (see fig. 2, PI. 9).
These larger rounded bodies probably arise through a fusion of
several mitochondria. This apparent fusion of mitochondria
has been observed by several authors, among whom is W. J. M.
Scott (13), who describes a similar phenomenon occurring in
the pancreas ; and Cowdry believes that the larger spherical
bodies are the result of a coalescence of several or more mito-
chondria. Later, I was able to demonstrate the actual fusion of
mitochondria in living amoebae (7). This constant fusion of mito-
chondria during certain phases in many cells may possibly be
explained in terms of surface-tension, and may be similar to the
138 E. S. HORNING

fusion of oil-drops in suspensions of oil in water, and it does

not seem likely that any physiological function can be attached
to it. If sections showing the small gametes within the cyst
are now examined, it will be noticed that the protoplasm of
these minute cells contains small spherical mitochondrial bodies
varying in size (see fig. 3).
During the true sexual phase of the life-cycle of the organism,
when union of the gametes occurs, a noticeable fusion of
mitochondria takes place (see fig. 3, PI. 9), since the rounded
mitochondria during this process tend to lose their spherical
form and become irregular clumps. I have observed this effect
on several occasions, and it is of interest to note that the same
phenomenon occurs during the sexual phase in the binucleate
O p a l i n a (5) when conjugation of the gametes occurs.
The protoplasm of the two encysted gametocytes is not
entirely used up to form the young gametes, and a surplus of
residual cytoplasm is always left over and is termed by many
authors the ' cystal residuum '.
An examination of this surplus cytoplasm within the cyst
reveals the presence of several types of granules within it (see
fig. 3, PI. 9), mitochondria, lipoidal droplets, together with a
third type of granule, which appears light blue after staining
with Heidenhain's iron haematoxylin. These latter types
of grains have the appearance of vegetative bodies. As the
cyst becomes more mature these granules disappear as the
residual cytoplasm becomes absorbed (see fig. 4, PI. 9).
In order to ascertain the nature of these granules within the
' cystal residuum ' the sperm-sacs of many earthworms in-
fected with Monocystis were placed in a saline solution, teased,
and examined for cysts. A portion of the sperm-sacs, containing
several fairly large cysts, was removed on to a clean slide, and
with the aid of needles the cyst walls were then ruptured. In
order to test for mitochondria a Janus green solution of about
1:10,000 was introduced under the cover-slip containing the
cyst. Several granules (the mitochondria) stained a bluish
green, while other granules within the residual protoplasm, pre-
sumably the lipoidal and vegetative grains, did not show
 MITOCHONDRIA OP MONOCYSTIS 139

a selective action to this stain. In order to ascertain the nature

of the former type of granule a solution of Soudan III was run
under the cover-slip containing another such ruptured cyst, and
in this case the fatty droplets were observed to stain a light
yellowish red, which demonstrated their lipoidal nature.
If the young zygote is examined in a fairly mature cyst the
cytoplasm is seen to contain a variable number of rounded mito-
chondria. But later examination of the young spores, which
are derived from the zygotes before the chitinous case is fully
secreted around them, reveals that the mitochondria during this
stage have undergone a notable decrease in number, and when
finally the spore has reached maturity it is found to be entirely
devoid of mitochondria. Sections cut through many such
mature zysts show the complete disappearance of mitochondria
both within the spores themselves and also within the cyst, and
the ' cystal residuum ' at this stage of the life-history of the
parasite has become completely absorbed.
It cannot be suggested that this effect is clue to a faulty
fixation or technique, nor that the fixing fluids were unable to
penetrate the chitinous case of the mature spores ; because in
every case the individual nuclei in each spore stained sharply
and clearly, while the protoplasm of the sporozoite stained
a palish blue when treated with the Heidenhain process.
In several cases material was fixed in a solution containing
4 per cent, osmic instead of the usual 2 per cent., and each time
the same results were obtained. Material was also left in both
Champy and osmo-chromic fixatives for a period of forty-eight
hours, and even in over-stained cells no traces of mitochondria
could be seen.
The next step in the life-history occurs when in another earth-
worm the mature spore-coat finally ruptures and the young
sporozoites within are liberated. They immediately become
active and make their way to the developing sperm, where they
gradually increase in size, and develop into the adult individual
or trophozoite.
Stained sections of these young organisms on examination
once more reveal the presence of small rounded and rod-shaped
140 E. S. HORNING

mitochondria scattered irregularly throughout the medullary

region of the cell. As the sporozoite gradually develops into the
trophozoite the mitochondria increase in number. Continued
observation of progressive stages of development has shown
that the awakening of the cell is accompanied by a reformation
and rapid reproduction of the mitochondria.
The behaviour of the mitochondria during the life-cycle of
this organism, and especially their apparent origin de n o v o
during chemical resynthesis, do not lend any support to
Wallin's (14) contention that mitochondria are symbiotic
organisms.
ON THE BOLE PLAYED BY MITOCHONDRIA DURING CELLULAR
METABOLISM.
That mitochondria play an important part in the metabolism
of the cell was first put forward by Guillermond (3), who sug-
gested that starch and other plastids produced in plant cells
owe their origin indirectly to mitochondria. Cowdry (2) also
formulated the theory that these substances are originally
formed within the mitochondria which later enlarge to form
the body of the plastid, while Eegaud (12) suggested that mito-
chondria are able to select certain materials from the surround-
ing protoplasm of the cell and build them up into various
products. Later, while studying the behaviour of mitochondria
within a common endoparasite protozoon—Opalina (5)—I
was able to detect vegetative granules arising in close connexion
with the surface of each single mitochondrium. These observa-
tions tend to support the speculation that mitochondria are the
loci of protein and of general protoplasmic synthesis within the
living cell. More recently the work of Marston (11) has suggested
that the mitochondria contain concentrated within them the
enzymes which bring about cell synthesis, their action being to
build up protein at their surface. Moreover, the behaviour of
mitochondria appears to illustrate the capacity of enzymes to
reverse their activity in accordance with conditions, i.e. syn-
thesis or hydrolysis, according to the concentration of the
substrate. For example, in O p a l i n a we see that mitochondria
 MITOCHONDRIA OF MONOCYSTIS 141

synthesize vegetative products at their surface; while in

P a r a m o e c i u r n (6), N y c t o t h e r u s (8), and A m o e b a (7)
the function of mitochondria is also one of hydrolysis, where it
is believed that the mitochondria which come to be included
within the food vacuoles bring about the digestion of the food.
We see that O p a l i n a and M o n o c y s t i s feed in the same
way by means of endosmosis through the cuticle of the proto-
plasm. One would therefore expect to observe a similar pheno-
menon in M o n o c y s t i s . But observations on this organism
show that the actual origin of these food-storage products from
the surface of the mitochondria cannot be detected. The mito-
chondrial grains present in the protoplasm of M o n o c y s t i s
are very much smaller than those of O p a l i n a , and it is quite
probable that the latter organism, owing to its large size,
is the more favourable object for studying this phenomenon.
But it is highly suggestive that the vegetative granules, which
we previously observed in the residual protoplasm of the early
cyst, arise in the same manner as similar plastid and vegetative
products in other animal and plant cells.
Bearing in mind the evidence that mitochondria appear to
be the centres of general protoplasmic synthesis within the
living cell, it is of interest to observe that in M o n o c y s t i s
there occurs a stage in the life-cycle of this organism—namely, the
spore phase—wherein synthetic activity and digestive activity
do not occur, and moreover, at this period previous observations
have shown that mitochondria can no longer be detected within
the protoplasm of the cell. The study of the behaviour of mito-
chondria throughout the life-history has shown that these bodies
make their reappearance in the free active sporozoite at a time
when chemical synthesis takes place. Later, the growth of the
sporozoite into the adult individual is accompanied by an in-
crease in the number of cell mitochondria. This evidence seems
to suggest that the reawakening of cellular activity within the
spore is dependent upon the reformation of mitochondria.
142 B. S. HOENING

SUMMARY.
1. Observations show that mitochondria are capable of aris-
ing d e n o v o in the freshly liberated sporozoite stage of the
life-cycle of M o n o c y s t i s .
2. Mitochondria are present in large numbers throughout the
course of the asexual phase of the life-cycle. During the con-
jugation of the gametocytes the rod-like mitochondria give rise
to numerous spherical bodies. At fertilization the mitochondria
within the gametes appear to fuse, resulting in the formation of
larger clumps.
At the commencement of the spore phase the mitochondria
gradually decrease in numbers, and are totally absent within the
mature spore. Later, the growth of the liberated sporozoite
or young trophozoite is accompanied by a reformation and rapid
reproduction of mitochondria.
3. The disappearance and reformation of mitochondria during
certain stages of the life-cycle may be correlated with their
apparent synthetic activity.
I am greatly indebted to Professor W. E. Agar, F.E.S., for
his valuable suggestions.
This investigation was carried out during the tenure of a
Government Research Scholarship in Zoology at the University
of Melbourne.
REFERENCES.
1. Cowdry, E. V.—' The American Nat.', vol. lx (1926), p. 135.
2. ' General Cytology.' Chicago, 1924.
3. Guillermond.—' Ann. Soi. Nat., Bot.', vol. x (1919) a.
4. Horning, E. S.—' Australian Journ. of Exper. Biol. and Med. Science ',
vol. ii (1925), p. 135.
5. Ihid., p. 167.
6. Ibid., vol. iii (1926), p. 89.
7. Ibid., pp. 89, 149.
8. Ibid., vol. iv (1927), p. 69.
9. Ibid., p. 75.
10. Ibid., p. 187.
11. Marston, H. R.—' Bioehem. Jourh. of Exper. Biol. and Med. Science ',
vol. 17 (1923), p. 851.
 MITOCHONDEIA OF MONOCYSTIS 143

12. Regaud, C, and Mawas.—' Comptes rend. Soc. Biol.', vol. 66 (1926).
13. Scott, W. J. M.—' American Journ. of Anat.', vol. 20 (1916), p. 37.
14. Wallin, I. E.—Ibid., vol. 32 (1924), p. 467.

DESCRIPTION OF PLATE 9.
Sections of M o n o c y s t i s at various stages of the life-cycle.
All figures are drawn from material fixed in either Flemrning's
solution without acetic or else in a Champy fixative and stained
with Heidenhain's iron haematoxylin, and occasionally counter-
stained in eosin.
Fig. 1.—Longitudinal section of adult individual or Trophozoite, showing
mitochondria scattered throughout the medullary region of the organism.
Note mitochondria lying close to and in intimate contact with outer surface
of nucleus.
Fig. 2.—Section showing association of two adult organisms within the
cyst. Note the increase in the number of spherical mitochondria, which
have arisen by fusion of the rod-shaped mitochondria of the previous stage.
Fig. 3.—Section through cyst showing mitochondria within the proto-
plasm of the gametes, showing conjugating gametes and larger granular
clumps (x and z) formed by fusion of the mitochondria. Within the residual
protoplasm are seen the three types of granules, M, mitochondria ; L,
lipoidal droplets ; v, vegetative grains.
Fig. 4.—Section showing mature cyst. The residual protoplasm has been
completely absorbed. Note the absence of mitochondria within the spores.
The nuclei of spores are stained clearly.
Fig. 5.—Longitudinal section through two spores as seen under higher
magnification. Note how the nuclei of each spore stains clearly, and also
the total absence of mitochondria.
Fig. 6.—Section cut through a sporozoite showing the reappearance of
mitochondria.
Fig. 6 A.—Section of same as seen with higher magnification. Note
the rod- and spherical-shaped mitochondria in cytoplasm, together with
sharply stained nucleus.