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Molluscan Comparative Immunology (MCI) Group
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In vitro culture of the avian echinostome
Himasthla elongata
| Intramolluscan development of
the marine echinostome avian fluke, Himasthla elongata,
is similar to that of other members of family Echinostomatidae.
It involves the sequential
production of several distinct generations including Littorina
littorea-infective miracidia, primary or mother sporocysts,
mother and daughter rediae and cercariae, the infective larvae
for the intermediate host - blue mussel, Mytilus edulis.
Definitive host, seagulls, become infected by eating mussels
with encysted metacercariae (Werding 1969). Here we report the
primary in vitro culture of H. elongata rediae maintained
in axenic conditions. This is the first reported cultivation
of trematodes infecting marine prosobranch mollusc. |
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| Once transferred in the medium,
H. elongata rediae showed vigorous bending movements
as well as longitudinal and trans-verse contractions. Rediae
motility was maintained throughout the entire period of cultivation.
Dead or dying rediae were easily identified by their loss of
motility and tegument integrity. Long-term cultivation experiment
showed 50% survival level for up to 70 days and highly significant
between-"clone" differences in survival rate (ANOVA: F(2)=212.5,
P<< 0.001). To note, 50% of the rediae in the most robust "clone"
survived for up to 163 days, when the experiment was terminated.
During cultivation, moribund or dead cercariae, their lost tails
and dead rediae bodies (blue arrow) were the only source of
food for the rediae. In no case were rediae observed to attack
one another. No somatic growth was noted. |
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| Despite the fact that a large
part of H. elongata cercariae underwent elimination with
changed medium, some of the residuary larvae after about 24
hr of activity rounded off, encysted and transformed into metacercariae
(grey arrows). In very rare cases cercariae were observed to
encyst inside moribund rediae. As a rule, encysted larvae showed
sluggish and rare muscle contractions and were like that until
the cultures were terminated. However, some of them in one -
two weeks after the transformation became spontaneously active
and began to turn and twist under cyst envelopes in all directions.
Such larvae activity resulted in the excystment of juvenile
maritae (grey arrow) which were weakly motile in the beginning
and dead within 3-4 days. To note, if warmed up to 40-42°C,
maritae vigorous activated and showed specific body bending.
Attached to the substrate with both suck-ers, they lifted posterior
body part almost perpendicular to the plastic surface. |
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Generally, short-term cultures with
rediae derived from naturally infected periwinkles contained
only daughter rediae releasing cercariae. However, one redial
"clone" in the long-term cultivation experiment at day 90
post culture initiation was found to release several progeny
rediae producing germinal material (blue arrows). Though
these young rediae initially were very active, they did
not grow, their germinal balls did not increase in size
and they died within 3 weeks.
When pulsed with 14C-labeled aminoacids
mixture, rediae showed evident signs of high metabolic activity.
About 25 proteins were newly synthesized in the course of
24 hr in detectable quantity. Important to note the absence
of any differences in protein compositions and protein synthesis
between the 1st and the 6th days of cultivation. Means of
total proteolytic activity accumulated in culture medium
for 48 hr in 4 and 6-days cultures (A490= 0.015 and 0.013
accordingly) were near by detection level (for several enzymes
is about A490= 0.02 according to the PADK protocol) and
did not differ significantly (P>0.05). Both estimates were
significantly lower (P<< 0.0001) than that of positive control
(A490=1.01).
Our preliminary studies showed great potential
of L-15 medium for primary cultures of Littorina littorea
and Mytilus edulis hemocytes. They survive and demonstrate
some cell reactions adequately for up to one month. Having
in view uniform protein synthesis in the course of at least
the first week of rediae cultivation and minute proteolytic
activity of redia-conditioned medium, the culture system
suggested in this study is a valuable tool permitting us
not only to characterize the soluble factors the parasite
secretes, but also to investigate interference of these
factors with host cells.
by Alexander M. Gorbushin
& Tanya G. Shaposhnikova
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