Proc. Helminthol. Soc. Wash.
47(1), 1980, p. 118-121
Fertilization in the Coccidia: Fusion of
Sarcocystis bovicanis Gametes
HARLEY G. SHEFFIELD AND RONALD PAYER
Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases,
National Institutes of Health, Bethesda, Maryland 20205 and Animal Parasitology Institute,
USDA-SEA, Beltsville, Maryland 20705
Coccidian parasites of a.nimals and man include the genera Eimeria, Isospora,
Toxoplasma, Sarcocystis, and others. The life cycle of each of these parasites
includes both asexual and sexual stages of multiplication. The sexual stages of
nearly all coccidian species develop intracellularly in the intestine of the final
host. Differentiation of the parasite into microgametes (male) and macrogametes
(female) is followed by fertilization and development of the zygote into an oocyst
stage. Thousands of species of coccidia have been described from numerous
hosts, but the act of fertili2:ation has never before been recorded, perhaps because
of the concealed location of the gametes within host cells. However, eimerian
microgametes have been observed on the surface of macrogametes by light (Marquardt, 1966) and scanning electron microscopy (Madden and Vetterling, 1977),
and within a macrogamete by transmission electron microscopy (Hammond and
Scholtyseck, 1970). The present study reports for the first time the process of
coccidian fertilization by fusion of the plasma membranes of a micro- and a
macrogamete and the transfer of nuclear material. This observation indicates that
fertilization in the coccidia resembles that reported for other invertebrates (Epel
and Vacquier, 1978) and for mammals (Bedford and Cooper, 1978), where membrane fusion occurs, rather than penetration of the female gamete by the male
gamete as previously assumed for the coccidia.
Intramuscular cysts of Sarcocystis bovicanis develop in bovine hosts after
ingestion of sporocysts from canine feces. Following ingestion of S. bovicanis
cysts containing mature asexual zoites, infection is established in the small intestine of the dog (Payer, 1974). Zoites enter goblet cells and differentiate, without further multiplication, into sexual stages which mature within 6-12 hr after
ingestion (unpublished observations). During a study of specimens collected at
these time periods, fertilization was observed.
Materials and Methods
Myocardium containing mature cysts of Sarcocystis bovicanis was taken from
a bovine host experimentally infected with sporocysts from dogs and ground in
a commercial meat grinder. The ground myocardium was fed, within an hour
after death of the bovine host, to coccidia-free beagle puppies that had never
before eaten meat. The dogs were killed at 6 and 12 hr after ingesting the ground
myocardium. Segments of small intestine were fixed and processed for study by
both light and electron microscopy.
Results
Fertilization of macrogametes was observed in specimens of canine small intestine fixed 12 hr after dogs were fed infected bovine heart (Fig. 1). The gametes
Copyright © 2011, The Helminthological Society of Washington
OF WASHINGTON, VOLUME 47, NUMBER 1, JANUARY 1980
119
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Figure 1. Electron micrograph of Sarcocystis bovicanis gametes in dog small intestine. Portions of
intestine were fixed in 5% glutaraldehyde buffered at pH 7.3 with 0.05 M phosphate and then were
postfixed in 2% OsO.,. Tissues were dehydrated, embedded in Epon, sectioned, and stained with lead citrate
and uranyl acetate. Section is through a microgamete (upper left) and a portion of a macrogamete. The
microgamete lies inside the host layers and is fused with the macrogamete through a discontinuity in the
raised parasite membrane layer. At the point of fusion, the gamete membranes are thickened (arrows)
and an aggregate of dense material lines the cytoplasmic side of the fusion area. A dense mass of
microgamete nucleoplasm (NM) has passed through the area of fusion into the macrogamete cytoplasm.
Microtubules (T) and a mitochondrion (M) remain within the microgamete. The macrogamete nucleus
(N) is visible at the lower right. Scale bar, 1 /AITI.
were extracellular within the lamina propria. The macrogamete was enveloped
by several layers. Outermost was a nonmembranous thick layer, beneath which
was a closely adherent membranous layer; both are thought to be of host cell
origin. Another membranous layer, presumably derived from the parasite, was
present beneath the two outer layers and separated them from the pellicle of the
macrogamete. The fertilizing microgamete was found between the two outermost
layers and the membranous layer of parasite origin. The latter appeared to be
discontinuous at the point of fusion of the two gametes.
The body of the microgamete, which contained a typical mitochondrion and
several microtubules, was surrounded by a single-membrane plasmalemma and
was oriented tangential to the surface of the macrogamete. The two gametes were
connected to one another by a small necklike region through which there was
cytoplasmic continuity. In the necklike region, at the point of their fusion, the
Copyright © 2011, The Helminthological Society of Washington
120
• PROCEEDINGS OF THE HELMINTHOLOGICAL SOCIETY
gamete membranes formed an electron-dense, thickened ring. Through this ring,
nucleoplasm from the microgamete passed into the macrogamete cytoplasm.
Microgamete nucleoplasm was also present in the cytoplasm of macrogametes in
which oocyst wall formation had begun, and no evidence of residual microgamete
structure was observed. Fusion of the microgamete nucleoplasm with the macrogamete nucleus was not seen.
In several specimens, microgametes were seen in association with macrogametes but fusion was not apparent, although it could have occurred outside the
plane of the sections. In each case, the microgamete was located beneath the two
outermost layers as described above. Flagella were not present on any microgametes observed in association with macrogametes.
Discussion
Observations of fertilization of Sarcocystis bovicanis differ from those reported
for other coccidia by earlier workers. The first ultrastructural observation of
fertilization of coccidia was made by Hammond and Scholtyseck (1970), who
found an intact microgamete within a macrogamete of Eimeria bovis. The microgamete appeared to lie within a vacuole delimited by a membrane. Two flagella
were observed, one of which was enclosed by a membrane that also covered the
microgamete nucleus. Penetration of a macrogamete by a microgamete was reported in a scanning electron microscopy study of Eimeria tenella (Madden and
Vetterling, 1977). Microgametes have also been seen by light microscopy on the
surface of macrogametes of Eimeria nieschulzi, but neither penetration nor fertilization was reported (Marquardt, 1966). These studies imply that the intact
coccidian microgamete attaches to the macrogamete surface, penetrates its cell
membrane, and enters its cytoplasm during fertilization.
In contrast, fertilization in S. bovicanis, as reported herein, proceeds by fusion
of the microgamete plasmalemma with that of the macrogamete and subsequent
entry of microgamete nucleoplasm into the macrogamete cytoplasm. A similar
process has been observed for three other genera of related protozoans. For
Parahaemoproteus velans, the presence of the nucleus and axoneme of the microgamete free within the cytoplasm of the macrogamete suggested that fusion
of the gamete membranes occurred during fertilization, resulting in entry of the
"contents" of the microgamete into the macrogamete (Desser, 1972). For Haemoproteus coluinbae, fusion of the gamete membranes resulted in one continuous
membrane surrounding their combined contents (Gallucci, 1974). It was also noted that microgamete axonemes did not persist within the macrogamete cytoplasm
for more than two hours after fertilization. For Plasmodium yoelii nigeriensis,
fusion of the gamete plasmalemmas allowed the naked axoneme and nucleus of
the microgamete to pass into the cytoplasm of the macrogamete (Sinden et al.,
1976).
Interpretation of the differences observed in fertilization among the coccidia
is difficult because so few observations have been made. The lack of microgamete
flagella at the time of fusion in our study may be explained as follows. Two layers
of host origin surround the macrogamete and must be penetrated by the microgamete before fusion with the macrogamete. Madden and Vetterling (1977) may
have observed a flagellated microgamete penetrating these layers rather than
penetrating the macrogamete membrane. We suggest that the microgamete fla-
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OF WASHINGTON, VOLUME 47, NUMBER 1, JANUARY 1980 •
121
gella are lost at the time of fusion or slightly thereafter. The observation of a
flagellated microgamete in a membrane delimited vacuole within a macrogamete
(Hammond and Scholtyseck, 1970) differs from the other cited reports on fertilization, but resembles the entry of mammalian sperm into somatic cells where
phagocytosis occurs and the sperm is membrane enclosed (Bedford and Cooper,
1978).
Only two electron microscope studies of sexual stages of Sarcocystis utilizing
in vitro techniques have been reported (Vetterling et al., 1973; Mehlhorn and
Heydorn, 1979). Although neither reported evidence of fertilization, Mehlhorn
and Heydorn (1979) published a micrograph (fig. 22) of an oocyst with its cytoplasm connected to a cytoplasmic mass through a ringlike opening at its periphery, similar to Figure 1 (present study). However, they interpreted their micrograph as the beginning of sporogony.
Because of the difficulty of making observations in vivo, clarification of the
fertilization process will probably necessitate further utilization of in vitro cultivation of the parasite.
Acknowledgments
The technical assistance of Mr. Taylor Chestnut and Mr. Donald C. Davis is
gratefully acknowledged.
Literature Cited
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eggs. Pages 65-125 in G. Poste and G. L. Nicholson, eds. Membrane Fusion. Elsevier/NorthHolland Biomedical Press. Amsterdam.
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(=Haemoprotens) velans (Coatney & Roudabush) (Haemosporidia: Haemoproteidae): an ultrastructural study. J. Protozool. 19:287-296.
Epel, D., and V. D. Vacquier. 1978. Membrane fusion events during invertebrate fertilization.
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Copyright © 2011, The Helminthological Society of Washington