International Journal of Marine Science, 2016, Vol.6, No.18, 1-14
3
Scanning electron microscopy (SEM)
Parts from the intestine of
Styela plicata
and
Ciona intestinalis
were fixed in PAF (picric acid-formaldehyde) 1200
mOsm pH 7.5. These parts were dehydrated in a graded ethanol series. The dehydrated larvae were critical point
dried, mounted on specimen holders, and subsequently sputter-coated with gold. Specimens were examined and
photographed using a FEI Quanta 200 SEM at 15 kV.
Transmission electron microscopy (TEM)
Parts from the intestine of
Styela plicata
and
Ciona intestinalis
were fixed in 2.5% Glutaraldehyde in 0.05 M PBS
containing 0.33 M NaCl (1 h, 4°C). The fixative was removed by washing specimens several times with PBS.
Post-fixation was carried out using 2% Osmium tetroxide in PBS for 30-60 min at 4°C. Specimens were
subsequently washed with PBS, dehydrated in a graded ethanol series, and propylene oxide and embedded in
araldite resin. Ultrathin (60-70 nm) sections were obtained using Leica UC6 microtome equipped with diamond
knives. Ultrathin sections were picked with formvar-coated singleslot copper grids, stained automatically with
uranyl acetate and lead citrate in a Nanofilm TEM STAINER, and examined on a Phillips CM 120 transmission
electron microscope at 60 kV.
Results
Styela plicata
(Fig.1) can be differentiated from other neighbouring ascidians as follows: the size ranges10-90 mm
in length and 40-50 mm in width, siphons (oral or atrial) are quadrilobed, the later siphon is subterminal, stigmata
straight, oral tentacles are simple, four pairs of branchial folds, dorsal lamina is continuous, stomach without
hepatic diverticula and gonads are 4 in the right side and 1 or maximally 2 in the left side.
Ciona intestinalis
(Fig.2) was brownish-white coloured and transparent to the extent that one can see the internal viscera while alive.
It is large-sized measuring about 40-100 mm in length and 20-35 mm in width. The body is cylindrical,
longitudinal muscles of branchial basket are conspicuous, oral siphon is terminal with 8 lobes and red or orange
pigment spots between lobes, atrial siphon is subterminal with 6 lobes and pigment spots, stigmata straight,
stomach and intestinal loop behind branchial sac, longitudinal branchial bars are papillated, ovary is compact and
situated in the intestinal loop whereas testis diffusely spread over stomach and intestine.
The gastrointestinal tract of solitary ascidians begins with the oral opening which lies at the base of oral siphon
and leads to an immense pharynx. This chamber serves both respiration and filter feeding. The pharynx is
perforated by dorsoventral rows of numerous gill slits called stigmata. Blood vessels traverse the pharyngeal
wall between the slits. Water passes through the gill slits to the atrium and then is expelled through the atrial
opening. Along the ventral margin of the branchial basket is a specialized organ called endostyle. The endostyle
secretes large quantities of mucus which is distributed as a thin sheet over the inner surface of the pharynx by the
flagella and pharyngeal cilia. Food particles become entangled in the mucus, are collected along the dorsal wall of
the pharynx and are propelled by ciliary action to the oesophagus behind the pharynx. The digestive tract leads to
a stomach at the bottom of the U-shaped digestive loop and an intestine terminates at the anus which opens in the
atrial cavity.
Artificial infection of the ascidian with parasite did not carried out because of difficulties into rearing ascidians for
a long time. This work lasted about four years.
Gametic cycle of the coccidian parasite
Gamonts were observed while spreading along the intestinal epithelial cells inside white blood cells of an infected
of
Ciona intestinalis
.
Merozoites were released from the gamonts after complete destruction of the host intestinal epithelial cell. They
wandered freely in the circulatory system and penetrated new host blood cells (Figs.3-8). Merozoites later
transformed from the cylindre banana motile form to spherical form and lost characters from its apical complex
represented by micronemes, rhoptries and polar ring. Early formed gamonts were surrounded by parasitophorous
vacuole which was spherical in shape and began small sized (5.5 X 3.8 µm). Gamonts later differentiate to macro-
