CONTENT
I. Introduction
II. Materials and methods
III. Trophozoites of the enteric type
IV. The coelomic type
(a) The trophic phase: syzygy
(b) The gametocyst
(c) The zygocyst
(d) The sporocyst
V.
Infection
(a) Frequency of infection
(b) Dispersal of spores
(c) Digestion of spores by the host
VI. Discussion
VII. References cited
VIII. Description of plates
IX. Acknowledgments
page
I. INTRODUCTION
Gregarine parasitism has been extensively studied in
polychaetes (Ray, 1930; Miles, 1963; Dibb, 1938; Rees and Howell,
1966) cirratulids included (Cox, 1965). Most of these reports are
primarily concerned with gregarines found in the intestine. No
account is found of the acephaline form harbored in the coelom of
the cirratulid Cirriformia spirabrancha. Populations of C.
spirabrancha from Monterey, California are infected in high
frequency with a coelomic gregarine and at least one intestinal
sporozoan.
This communication describes some aspects of the
parasites of C. spirabrancha. Various stages of the life-cycle of
the coelomic type are demonstrated, and the trophic form of the
enteric type is introduced. Preliminary investigations into the
mechanism of infection are noted.
II. MATERIALS AND METHODS
Specimens of C. spirabrancha were dug from the intertidal
mud flats under the south edge of Fisherman's Wharf in the Monterey
Harbor. Worms were brought into the laboratory with a generous
portion of their substrate, placed in aquaria, and supplied with
aerated flowing sea water. They were examined within forty-eight
hours after collection.
Worms of average size (approximately 0.25 gram) were picked
at random from the aquaria, relaxed in isotonic MgCl,, and dissected
by dorsolateral longitudinal incision of the body wall. The sex of
the worms and stage of gametogenesis were noted, and the number and
location of parasitic forms in the coelom were estimated. Specimens
of gregarine forms were first examined in situ under the dissecting
microscope, and then transferred by pasteur pipet to microscope
slides and covered with a supported coverslip. Cytoplasmic structures
and cyst contents were freed for examination at high magnification by
crushing them gently under the coverslip.
The contents of at least four cysts from each worm were
examined to determine the extent to which each developmental stage of
the parasite was present. Each host was then assigned to one of three
groups according to maturation of gametes: 1) immature, having no
gametes; 2) maturing, males having spermatid clusters and females
having small eggs in sacs; and 3) mature, having fully ripe gametes.
ap6
Each worm was also classed according to the predominant developmental
phase of the parasite into one of six groups: 1) no parasites;
2) trophozoites; 3) associated sporadins; 4) gametocysts; 5) zygocysts;
and 6) sporocysts.
When coelomic forms had been examined, small portions of
the gut were excised, placed on microscope slides, teased apart with
needles, and pressed with the coverslip to spread the tissues and
reveal the attached trophozoites of the intestine.
Bouin-fixed and Masson's trichrome-stained serial paraffin
sections (12 microns thickness) of whole worms were examined for
attachment characteristics and evidence of entero-coelomic migration.
Methyl green and neutral red were used as vital stains. However,
unless otherwise noted all descriptions and illustrations in this
communication were made from untreated specimens,
An attempt was made to lyse the spores with egg white
lysozyme (Pentex), pronase (Calbiochem), dithioerythritol (DTE,
Sigma), DTE-pronase, sodium lauryl sulfate (Sigma), distilled water.
sea water, and extract of gut from the host. The gut extract was
prepared by homogenization of whole gastric tract at 0-5° C. in
buffered artificial sea water and subsequent centrifugation to
remove large particles. Twenty mature sporocysts were placed in each
culture chamber and about half were crushed with a glass rod to
release the spores. Each chamber was then filled with a lysing agent
and sealed with petroleum jelly under a coverslip. Cysts were
incubated with each lysing agent at 5P C., 14° C., and 21° C. up
to 24 hours, during which time they were examined frequently and compared.
ap7
III. TROPHOZOITES OF THE INTESTINE
The mature trophozoite (plate I, figure a) measures about
0.20mm in length and about 0.0l mm in diameter. In situ, they move in
a pendular manner from their anterior attachment point on the
intestinal epithelium; The mode of attachment is unknown, but no
penetration of host tissue is visible in fixed and stained paraffin
sections at high magnification. When detached, they assume a spiral
or sigmoid shape (plate I, figure b). Longitudinal striations in
the pellicle are pronounced, numbering between ten and twenty.
The nucleus usually lies between one-tenth and one-fifth
of the length from the anterior end; one or two prominent nucleoli are
visible. The size and shape of the nucleus are variable (plate I.
figure c), depending upon stresses applied to it by the anterior and
posterior cytoplasmic masses during peristaltic contractions along the
entire length of the trophozoite.
In a fixed and stained cross section of one attachment region
of a trophozoite (plate I, figure d), the anterior end seems to be
transversely septate; no other such examples were seen, although
other attachment regions were examined.
IV. THE COELOMIC TYPE
A complete life-cycle of the coelomic gregarine of C.
spirabrancha has not been followed in a single individual. But by
carefully observing all systematic changes of different individuals,
and using outlines (Kudo, 1946; Hyman, 1940; Calkins, 1933;
Mackinnan and Hawes, 1961) of the life-cycles of similar species,
one may hypothesize the continuum of events which brings about the
developmental cycle.
In other acephaline eugregarines, syzygy is followed by a
multiple fission of the haploid sporont nuclei. The resulting gametes
fuse, and a spore case develops around the resulting zygote. Meiosis
occurs, producing haploid sporozoites capable of reinfection and
development to mature trophozoites.
The parasite of the coelom of C. spirabrancha is presumedn
to have a similar life-cycle, and a similar logical progression has
been applied to what has been observed in this species.


(a) The Trophic Phase; Syzygy

The trophozoites are elongate and teardrop-shaped.
measuring 1.0 to 1.2 mm in length and about 0.1 mm in diameter at the
thickest region when mature. The pellicle is smooth and unstriated,
but sufficiently firm to buckle when the parasite is sharply bent
(plate II, figure a). Though the pellicle is thickest and has a conical
shape at the anterior end, no attachment organelles are evident.
The nucleus is nearly spherical, having a flexible membrane that
endures when the trophozoite is crushed; it lies very near the anterior.
No nucleoli are evident in unstained specimens.
Peristaltic contractions are in general more subdued in
the mature trophozoite than in the small ones (plate II, figure a:
about 0.12 mm long). These smaller individuals are capable of a sort
of autoencystment (plate II, figure b-g), a phenomenon induced by
adverse conditions on the microscope slide.
Trophozoites in situ associate in pairs in various ways
(plate III, figures a, e, f). After the initial contact they draw
their "tails" around one another and then gradually reduce them,
Eventually their original morphology is lost (plate III, figure a-d),
and the cyst appears approximately spherical, with a diameter of
0.35-0.40 mm; both nuclei are still clearly evident. If the trophozo-
ites are pressed apart just as they have begun to reduce their
"tails" (plate III, figure b) they retain much of their original form
except that their "tails" appear annulated (plate III, figure g).
But if they are pressed after their association has become firmly
established (plate IV, figure b), they rupture. Upon examination at
high magnification, the cytoplasm appears much like that of the
trophozoite itself (plate IV, figure c).
830
(b) The Gametocyst
In other cysts, the nuclear membranes have disappeared
(plate IV, figure d); but at high magnification the cytoplasm looks
much the same as before (plate IV, figure f). At the same time, a loose
membrane has formed about the cyst, to persist throughout the
development.
(c) The zygocyst
Still other cysts contain a large number of roughly
spherical bodies (plate IV, figure g-i) which measure about 10
microns in diameter. The contain large phase-dense nuclear bodies
near their center.
(d) The sporocyst
The slightly flattened (plate IV, figure j) mature cysts
contain navicular or biconical spores (plate IV, figure 1) similar
in sice to the zygotes. The number of phase-dense bodies per
spore varies up to eight; these are the nuclei of the sporozoites.
2
V. INFECTION
(a) Frequency of Infection
The stages of the coelomic form are found exclusively in
that region which, in worms carrying on gametogenesis, bears the
gametes. Some hosts contain predominantly one systematic stage;
others contain several stages in similar proportions. From as few as
none to more than 100 parasites have been seen in a single host.
Tabulation of the data from the population survey
(text figure) shows that there is no correlation between gamete
development in the host and life-cycle of the parasite. Approximately
the same infection pattern exists at all stages of gametogenesis.
That the stages between syzygy and spore formation are present in
such relatively low proportions indicates that this period of the
cycle is relatively short.
(b) Spore Dispersal

It is interesting to note that during spawning of both male
-
and female worms in the aquaria, a few cysts and trophozoites were
seen to emerge with gametes through the gonopores. These spawnede
animals were then dissected, and found still to contain a large
number of parasites. Since a low proportion of the sporocysts are dispersed
during a spawn, and since there is no synchrony between the processes
of gametogenesis and parasite development, it is likely that this
is a coincidental dispersion mechanism.
(c) Digestion of Spores by the Host
Unsuccessful attempts have been made to reinfect gregarine
hosts by feeding them mature cysts or spores. It was presumed that
reinfection is through ingestion of spores or sporocysts by the host:
the spore walls are digested by enzymes in the gut and the liberated
sporozoites migrate through the gut wall to the coelomic cavity.
where they mature.
Attempts to digest sporocysts and spores in vitro, using
proteolytic enzymes, polysaccharide degradingeagents, osmotic
shock, and extract of gut from the host all failed. It is possible
that the digestive process for spores is àn indûced one, and that
the capacity for enzyme production was lost during extraction of
gut enzymes. Further, no sporozoites have been observed in gut fluids,
and there was no evidence of entero-coelomic migration in the
serial paraffin sections.
23
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23/
VI. DISCUSSION
The form of the trophozoite and the presumed spore and
the apparent life-cycle plâce the coelomic type in the tribe acephalina
(Kölliker) of the suborder eugregarinida (Doflein). The determination
of famill rests on interpretation of the spore structure; the spores are
either biconical (monocystidae (Butschli))or rhynchocystidae (Bhatia))
or navicular (Urosporidae (Woodcock)). It is clear that some
criteria other than morphology must be established to clarify the
taxonomy of the order gregarinida.
A more efficient mode than spawning of the host for spore
dispersal must be sought. It must be firmly established that mature
spores can be digested by the host, or the mode of infection must be
seriously reconsidered.
Since there seems to be no relationship physiologically
between host gametes and the parasite, it is interesting that they
are found in the same region of the coelom. If infection is through the
gut, and entero-coelomic migration does occur, it is possible that
the region surrounded by gametes is the region of the gut at which
sporozoites are released. Perhaps nutrients required both for
gametogenesis and by the parasited are passed to the coelom in this
region exclusively.
3/
VII. REFERENCES CITED
CALKINS, G. N. (1933). The Biology of the Protozoa. Lea and Febiger,
Philadelphia.
COX, F. E. G. (1965).
Ditrypanocystis cirratuli (Sporozoa, Archi-
gregarinida) Parasitic in Cirratulus cirratus from Plymouth.
J. Mar. Biol. Ass. U. K. 45: 59-64.
DIBB, M. J. (1938). Selenocystis foliata (Ray) from Scolelepsis
fuliginosa and its Identity with Haplozoan Species.
Parisitology 30: 296-308.
HIMAN, L. H. (1940). The Invertebrates: Protozoa through Ctenophora.
McGraw-Hill, New York.
KUDO, R. R. (1946). Protozoology. Charles C. Thomas, Springfield, Ill.
MACKINNAN, D. L. & HAWES, R. S. J. (1961). An Introduction to the
Study of Protozoa. Clarendon Press, Oxford.
MILES, H. B. (1963). The Occurrence of Acephaline Gregarines in
British Earthworms. Arch. Protistenk. 106: 575-82.
RAY, H. N. (1930). Studies on Some Sporozoa in Polychaete Worms. I.
Gregarines of the Genus Selenidium. Parisitology 22, 370-98.
REES, B. (1962). Studies on Monocystic Gregarines. Two new Monocystic
Genera Cephalocystis and Dendrocystis. Parasitology 56: 305.
& HOWELL, M. J. (1966). Regection of the Generic Name Dendrocystis
Rees, 1962 and its Replacement by Arborocystis nov. nom.
V
Protozoa: Gregarinomorpha. Parasitolo
gx 21: 1-15.
236
VIII. DESCRIPTION OF PLATES
All figures are drawn with the aid of camera lucida.
The specimens were untreated unless otherwise npted.
Plate I.
Figure a. The enteric type. Mature trophozoites. Note nucleoli
and longitudinal striations. (x 400)
Figure b. The enteric type. In coiled state after detachment from
intestinal epithelium. (x 400)
Figure c: 1-8. Rhe enteric type. Region about the nucleus during
peristaltic contractions. Note deformation of nuclear shape.
(x 400)
Figure d. The enteric type. Trichrome-stained paraffin section
(12 microns) through the intestine of C. spirabrancha showing
attachment point of a trophozoite. Note ectoplasmic septa
near anterior of trophozoite. (x 200)
Plate II.
Figure a. The coelomic type. Trophozoites. Note smoothness and
rigidity of pellicle. (x 100)
Figure b.g. The coelomic type. Small trophozoite self-encysting
under stress of temperature, pressure, osmotic imbalance. (x 400)
8
Plate III.
Figure a-d. The coelomic type. Association and encystment of
sporadins over a period of about one-half hour. (x 100)
Figure e. The coelomic type. Anterior-to-posterior association of
sporadins. (x 100)
Figure f. The coelomic type. "Ball-and-socket" association of
sporadins, pressed apart slightly under a coverslip. (x 100)
Figure g. The coelomic type. One of a pair of encysted sporadins,
showing annulation in the "tail" region. (x 100)
Plate IV.
Figure a-c. The coelomic type. Encysted sporadins. (a, b: x 100;
c: X 1000)
Figure d-f. The coelomic type. Gametocyst after multiple fission of
sporadin nuclei. (d, e: x 100; f: x 1000)
Figure g-i. The coelomic type. EZygocyst. (g, h: x 100; i: x 1000)
Figure j-1. The coelomic type. Sporocyst. (j. k: x 100; 1: x 1000)
C.

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IX. ACKNOWLEDGMENTS
I wish to express my sincere gratitude to J. R. Pearse for
his generous guidance in the research and for his editorial assistance
with this communication; to D. Epel for his suggestions for the
spore lysis study; to R. Szal for his help in abtaining equipment and
supplies; to M. Jeffries for providing samples of active gut extract,
and to B. W. Belman for the excellent histological work.
This projeet was supported in part by the Undergraduate
Research Participation Program of the National Science Foundation
Grant G4-4369.