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Comparison of Echinostoma caproni Mother Sporocyst Development In vivo and In vitro Using
Biomphalaria glabrata Snails and a B. glabrata Embryonic Cell Line
Author(s): Gennady L. Ataev, Annie Fournier and Christine Coustau
Source:
The Journal of Parasitology,
Vol. 84, No. 2 (Apr., 1998), pp. 227-235
Published by: The American Society of Parasitologists
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J. Parasitol., 84(2), 1998 p. 227-235
? American Society of Parasitologists 1998
COMPARISON
OF ECHINOSTOMA
CAPRONI MOTHER
SPOROCYST
DEVELOPMENT
IN
VIVO
AND IN VITRO USING BIOMPHALARIA
GLABRATA
SNAILS
AND A B. GLABRATA
EMBRYONIC
CELL
LINE
Gennady L. Ataev, Annie Fournier*,
and Christine Coustau*t
Department of Invertebrate Zoology, Saint-Petersburg University, 7/9, Univeritetskaja nab., Saint-Petersburg, 199034, Russia
ABSTRACT: Biomphalaria glabrata embryonic (Bge) cells have previously been shown to permit a successful cocultivation of
Schistosoma mansoni and Schistosoma japonicum from miracidia to mother sporocysts (MS) and then to the production of
daughter sporocysts (DS). To investigate further the properties of the Bge culturing system we used Echinostoma caproni under
identical in vitro conditions. In vitro-derived miracidia were used either for experimental infections of B. glabrata snails, or for
in vitro cultivation with Bge cells. Histological analysis showed that the development of MS in B. glabrata was similar to the
previously described development in Biomphalaria pfeifferi in terms of final site of infection, development dynamics, growth
dynamics, reproduction intensity, and life spans. Only short delays in migration dynamics were observed in B. glabrata. When
cultivated under in vitro conditions, E. caproni MS could live for up to 17 wk in the presence of Bge cells, as compared with
2 wk in cell-free Bge medium. The presence of Bge cells also permitted significant growth of MS and development through
complete embryogenesis of the next intramolluscan stage (embryos of 100-110 cells). However, degeneration of MS consistently
occurred before production of this second generation. During the entire cultivation period, no visible contact was observed
between MS and Bge cells, suggesting that development of MS was only triggered by soluble factors released by Bge cells.
The life cycle of digeneans is characterized by a complex
intramolluscan phase giving rise to the successive production
of sporocyst or redial generations or both. Although these
stages have been described for a tremendous number of species,
many important questions remain regarding specific events of
the development and the nature of host factors regulating par-
asite development.
Two methodological approaches are available to date for
studying intramolluscan development of digeneans. The first
approach consists of analyzing histological sections of infected
molluscs through time. These in vivo observations have pro-
vided essentially all the information constituting our present
knowledge on digenean development. The second approach in-
volves in vitro cultivation techniques (see review by Smyth,
1990). Although development of parasites under in vitro con-
ditions is usually suboptimal, this approach facilitates obser-
vations on certain events such as the transformation of mira-
cidia (Mi) to mother sporocysts (MS), or the emergence of
daughter sporocysts (DS) or rediae from MS. Furthermore, in
vitro cultivation permits the investigation of the nutritional,
physicochemical, and hormonal factors regulating developmen-
tal changes and determining host suitability. The main difficulty
with this approach is to elaborate in vitro systems suitable for
development, and current efforts are being made to attain this
goal.
A successful cultivation of a digenean species from Mi to
MS, and then to DS, was reported for the first time using the
human blood fluke Schistosoma mansoni (Yoshino and Laursen,
1995). In this work, MS were cultivated in the presence of a
snail cell line derived from embryos of Biomphalaria glabrata
(Hansen, 1976), a natural host for S. mansoni. Interestingly, this
B. glabrata embryonic (Bge) cell line also permitted compar-
able development of another human schistosome, Schistosoma
Received 12 August 1997; revised 5 November 1997; accepted 5 No-
vember 1997.
* Centre de Biologie et d'Ecologie tropicale et m6diterran6enne, UMR
5555 du CNRS, Universite, 52 Ave de Villeneuve, 66860 Perpignan
cedex, France.
t Corresponding author.
japonicum (Coustau et al., 1997). Despite the taxonomic dis-
tance separating B. glabrata from the natural snail host of S.
japonicum, Oncomelania hupensis (Rollinson and Simpson,
1987; Sobhon and Upatham, 1990), Bge cells were actually
providing soluble factors permitting, for the first time, a suc-
cessful cultivation of S. japonicum leading to the production of
daughter sporocysts.
To investigate further the potential use of Bge cells for co-
cultivating digeneans belonging to different taxa, we used, in
the present study, the digenean Echinostoma caproni, which is
known to develop in several species of the genus Biomphalaria,
including B. glabrata (Huffman and Fried, 1990; Fried and
Huffman, 1996). However, because the development of the first
intramolluscan stages of E. caproni has been previously de-
scribed only in Biomphalaria pfeifferi (Ataev et al., 1997), we
also examined its development in B. glabrata. The character-
ization of E. caproni MS development in B. glabrata and in
the Bge cocultivation system also represented a unique oppor-
tunity to compare the development of a single species in 2 snail
hosts as well as under in vitro conditions.
MATERIALS AND METHODS
Egg laying in vitro
The laboratory isolate of E. caproni (previously referred to as Echi-
nostoma liei) used in this study originated from Egypt (Jeyarasingam
et al., 1972) and has been maintained in the laboratory as described
previously (Trouve et al., 1996). The procedure to obtain eggs of E.
caproni in vitro was adapted from Reddy and Fried (1996). Briefly,
mice (Swiss OF1 stock) were dissected after 30-100 days of infection
and adult E. caproni were removed from the small intestine. Adults
were washed 3 times in Locke's solution containing an antibiotic/anti-
mycotic mixture (penicillin 100 units/ml, streptomycin 0.1 mg/ml, am-
photericin 0.025 jig/ml [PSA]; Sigma, St Louis, Missouri). They were
then transferred to wells of a 24-well sterile tissue culture plate con-
taining 1 ml of RPMI-1640 medium (GibcoBRL, Life Technologies,
France) plus the PSA mixture and maintained at 37 C. During the fol-
lowing 48 hr, RPMI-1640 medium was replaced every 12 hr. Eggs re-
leased during the first 48 hr were collected with siliconized pasteur
pipets, placed into 15-ml conical glass tubes, and washed 5 times with
sterile water containing the PSA mixture. After the last wash, the egg
suspension was aliquoted into 24-well sterile tissue culture plates. Plates
were wrapped with aluminum foil and kept in the dark at 26 C. After
3 wk at 26 C, plates were exposed to a light source until miracidia
227
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228 THE JOURNAL
OF PARASITOLOGY, VOL.
84, NO 2, APRIL 1998
50 umn
FIGURE
1. Light micrograph of 3-wk-old eggs of Echinostoma caproni (A, B). 1, Egg containing mature miracidium; 2, opening of the
operculum (arrow); 3, hatching of miracidium; and 4, empty egg after release of miracidium.
hatched from the eggs. Swimming miracidia were collected with a sil-
iconized Pasteur pipet and used either for experimental infection of B.
glabrata snails or for in vitro cocultivation with Bge cells.
Experimental infections
Biomphalaria glabrata snails used in this study came from a labo-
ratory strain previously selected for susceptibility to E. caproni (Lang-
and et al., 1997). Adult B. glabrata measuring 9-11 mm in shell di-
ameter were individually exposed to 10 in vitro-derived miracidia for
6 hr at 26 C. Snails were then transferred to 5-L tanks maintained in
an environmental temperature chamber at 26 C, on a 12L/12D cycle.
For histological analysis of E. caproni development or MS measure-
ments, 3-5 snails were dissected at 6 hr postinfection (PI), every day
from 1 to 21 days PI, and then at 27 days. The same experiment was
repeated 3 times.
In vitro cultivation of mother sporocysts
Newly hatched miracidia were transferred to 24-well culture plates
containing monolayers of Bge cells or cell-free Bge medium (Hansen,
1976) supplemented with 10% heat-inactivated fetal bovine serum
(complete or C-Bge medium). The Bge cell line used in coculture with
E. caproni was originally obtained from the American Type Culture
Collection (ATCC CRL 1494; Rockville, Maryland) and maintained in
50-ml culture flasks at 26 C under atmospheric conditions (Yoshino and
Laursen, 1995). Cells were plated in 24-well tissue culture plates 2 days
prior to addition of parasite larvae. A cell density of 350 + 100/mm2
was used in this study. The original density of the cells was approxi-
mately maintained by careful resuspension and selective elimination of
cells every other week. C-Bge medium in all cultures was replaced once
a week. Viability of sporocysts was assessed by counting exhaustively
living sporocysts once a week in each test well. Dead or dying sporo-
cysts were easily identified due to the loss of tegument integrity and
flame cell movements. Length and maximum width of approximately
30 MS were measured once a week with a calibrated ocular micrometer.
Volumes of E. caproni MS were estimated using the formula for a
parabolic-shaped object (Ataev et al., 1997): V = 2D2 X 7rh/15, where
D = maximal diameter and h = length. Three independent in vitro
cultivation experiments were performed. Photographs of living sporo-
cysts in cocultures were taken with the aid of an inverted microscope
(Leica DMIL) equipped with an automatic Leica (MPS28/32) camera
system.
Statistical analysis of sporocyst viability was performed after angular
(arcsine) transformation of percentage data. The significance of different
larval viability between groups was assessed using a Mann-Whitney
U-test for nonparametric data. Differences were considered significant
at P < 0.05.
Histology
Experimentally infected snails were fixed in Bouin's fixative, and
embedded in paraffin according to standard procedures. Sections (5-6
Ipm thick) were stained with Mayer's hematoxylin or Ehrlich's hema-
toxylin-eosin.
MS from cocultures were aseptically removed from cultures with
siliconized Pasteur pipets. They were washed in CBSS (Chernin, 1963)
until all Bge cells were removed and fixed in Bouin's fixative. The
technique of Langeron (1949), which is especially appropriate for his-
tological processing of small individuals, was used. Sporocysts were
embedded in 2% gelatin stained with 1% neutral red. The gelatin blocks
containing parasites were then dehydrated and embedded in paraffin.
Paraffin sections of 4-5 pm were stained with Ehrlich's hematoxylin-
eosin.
Scanning electron microscopy (SEM)
Prior to fixation for SEM, newly hatched miracidia were subjected
to sonication to remove adherent particles. Briefly, Mi were sonicated
for 5 min at 45 kHz using a Bransonic 321EH sonicator. When longer
sonication periods were used (10-15 min), a loss of cilia was observed
at the surface of Mi. Cultured miracidia were cleaned by careful suc-
cessive resuspensions in filter-sterilized CBSS. Mi were fixed in glutar-
aldehyde-osmic acid (4 C, pH 7.4, 425 mOsm) and subjected to further
preparation for SEM according to standard procedures (Fournier et al.,
1989).
RESULTS
Development in vivo
Eggs obtained after in vitro egg laying of adult E. caproni
were fully developed after incubating at 26 C for 3 wk. Most
(90-95%) hatched when exposed to a light source (Fig. 1). The
in vitro-derived Mi were infective for B. glabrata snails be-
cause 80% of the exposed snails became infected.
At 6-7 hr PI, transforming Mi could be found under the
epithelium of the head, foot, mantle collar, and mantle cavity
(Fig. 2A), indicating that penetration occurred at different sites
of the snail body, and that the migration of MS had not yet
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ATAEV ET AL.-IN VITRO DEVELOPMENT
OF E. CAPRONI 229
sc
15 gm ?. :
i *'%;M1
,?P .ISPA.^W
4
1
9
'._,a
!;
ql '-
:
' V
"
~*
-C
. - '
'
,- '
: r , . 9
, .-jg, 41
-
. '
*
.
'F
r
_*
- ,; W. t?'
25gm-
/
.. 69gm
FIGURE 2. Light micrographs of histological sections of developmental stages of E. caproni mother sporocysts (A-D). (A) Longitudinal section
of a mother sporocyst at 6 hr PI, under the head epithelium (arrow); gc, germinal cells; sc, secretory cells; sw, sporocyst wall. (B) Mother
sporocyst in the blood sinus at 1 day PI; es, eye spot. (C) Four-day-old mother sporocyst; dc, dividing cell; ec, embryonic cavity; em, embryonic
membrane; gb, germinal ball; pc, pycnotic cell. (D) Seven-day-old mother sporocyst showing well developed redial embryos; i, intestine; ph,
pharynx.
started. Although the transformation process from Mi to MS
started very shortly after penetration with the loss of epithelial
plates, the internal structures of Mi were unchanged at 6-7 hr
PI.
At 1 day PI, MS had not yet reached the final site of infection
(ventricle of the heart and proximal part of aorta). MS were
observed in the area of the rectal and genital ridges, near the
salivary glands, and the blood sinus system. They still pos-
sessed somatic features of Mi such as the neural mass, eyespots,
and apical gland (Fig. 2B).
The majority of MS had reached the final site of infection
and had transformed at 2 days PI. Mi internal structures had
completely degenerated. Division of undifferentiated and so-
matic cells resulted in a noticeable increase in the total number
of cells. In some MS, the cleavage of 1-2 of the primary ger-
minal cells (Ataev et al., 1997) could also be observed.
At 4 days PI, MS contained 5-6 embryos. One to 2 of them
had reached the germinal ball stage, consisting of 60-80 blas-
tomeres surrounded by an embryonic membrane (Fig. 2C).
Cavities surrounding individual embryos began to merge, form-
ing the general embryonic cavity (schizocoel). At this stage,
MS averaged 200 p.m in length and 70 pLm
in width.
MS, at 7 days PI, contained 5-7 large embryos, 2 or 3 of
them with primordia that are characteristic of mother rediae
(MR) organs such as pharynx and intestine (Fig. 2D). The
emergence of MR was observed for the first time after 8 days
of infection.
MS reached maximal sizes after 10 days PI, averaging 900
C
:^.sSI' ?.
.
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230 THE
JOURNAL OF PARASITOLOGY,
VOL.
84, NO.
2, APRIL
1998
' ?
'
~
. '
& ?
'o ? o
e. , * _.-
..4El
a**
' * ' -
- .
*;-. - ^
0
S ~~~'.'ts A1 ^ 'SS"'
~ D;
_-'s
, - ,,
. -1
*' ' '' . . , ' ' A
*rf
*'*.^ '?" ;^
^ -~~~ -{
'11
-.4
E?
wo^ I
43 gm
FIGURE
3. Light micrographs
of histological
sections of developmental stages
of E. caproni
mother
sporocysts
(A-C). (A) Ten-day-old
mother
sporocyst;
re, redial
embryo;
sch, schizocoel. (B) Fourteen-day-old
mother
sporocyst.
(C) Degenerated
21-day-old
mother
sporocyst.
pum
in length and 163 pum
in width (n = 13). The release of
MR resulted in the formation of empty spaces in the schizocoel
12- of MS (Fig. 3A), leading to the subsequent decrease in MS
??
- ,. _ volume (Fig. 4). Although maturation of new MR embryos was
- 10- in process, the degeneration of MS started and only 1-3 of these
x . - embryos developed before the end of the second week (Fig.
E 3B).
E -_ After 16-18 days PI, the degenerative process completely
E blocked further redial development. MR cells stopped dividing
: 6- T and became pycnotic. A strong decrease in MS size was also
> l
~_[ T observed (Fig. 4).
4 _ l|_ Tr Then, at 21 days PI, MS were degenerate: their cells were
lI more dense, very basophilic, and intercellular spaces had in-
0* )l 1 11 112 1 ed. The majority of MS started disappearing from the final site
0o lrV^I I ....... j IJ
X l
z -of infection by day 30 PI.
Days post-infection
FIGURE
4. Growth dynamics of E. caproni mother sporocysts
in
Biomphalaria glabrata. Each point represents mean estimated volume
+ SD (5 < n < 22).
In vitro cultivation
After transfer to C-Bge medium, Mi of E. caproni swam
actively for several hours. Mi gradually lost their motility, and
I
Ie1
o
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 27
.c
. ._
-1
-
b;;.
- tes
. ,>^X* -II ' ,t
I I j~~~~.
. A
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ATAEV ET
AL.-IN VITRO DEVELOPMENT OF E. CAPRONI 231
did not exert any obvious effect on cell spreading or adhesion
100 ------- Me to the substrate (Fig. 6).
90- Bge C Because Mi cultivated in Bge medium alone rapidly degen-
erated, we did not use them for further analysis, and the fol-
80- \lowing observations on development only refer to parasites cul-
> 70- \ tivated in the presence of Bge cells. The cultivation period
60- \needed for a complete shedding of epithelial plates was variable
.c - among Mi and could extend to the first 2-3 wk of cultivation.
> 50- - However, some changes in Mi morphology could be observed
40 - during the first day in culture. For example, after 1 day in cul-
-\ .ture, the ridge layers between epithelial plates increased as
30~ . _ \compared with newly hatched Mi (Figs. 7A, B, 8A). Few Mi
20- " also started shedding some epithelial plates at this time.
10 ' .
\ After 2 days in culture, many Mi had lost cilia on parts of
10-
\ %\ ^ --*-e their body. However, light microscopy and SEM observations
0 . , v
. v revealed that this did not always correspond to the shedding of
0 1 2 3 4 5 6 7 8 9 1 0 epithelial plates, but to a loss of cilia from the entire surface of
Weeks in Culture some epithelial plates. Most Mi had epithelial plates with and
FIGURE
5. Viability of E. caproni mother
sporocysts
cultivated
in without cilia (Fig. 7C). After the loss of cilia, the surface of
cell-free medium (Me) or in the presence of Bge cells (Bge C). Each epithelial plates showed numerous papillae (probably remains
point represents mean % + SD (3 < n < 10). of the basal bodies of cilia) (Fig. 7C). The appearance of epi-
thelial plates without cilia observed in our culture system was
similar to that of Mi subjected to extended periods of sonication
the majority of them were immobile at the bottom of the culture (Fig. 7D).
well by the end of the first day in culture with or without cells. After 3-4 days in culture, the degeneration of Mi soma could
When kept in C-Bge medium only, MS survived for a max- be observed (Fig. 8B); eye spots were breaking up into frag-
imum of 2 wk. As shown in Figure 5, the presence of Bge cells ments, peripheral ganglia detached from the neuropile, and pen-
in the cultures significantly increased MS viability. The longest etration and apical glands began to disappear. Cells involved in
life span recorded for MS in our coculture system was 17 wk. the development of sporocysts (germinal, somatic, and undif-
No particular interactions between Bge cells and E. caproni ferentiated) had not started dividing yet. Following 7 days in
sporocysts were observed during the entire period of coculti- culture, the degeneration of Mi structures was complete except
vation with cells. MS were not encapsulated by Bge cells and for shedding of epithelial plates, which was still in process.
_r . - ., ,.,-:.. -
FIGURE 6. Light micrograph
of E. caproni
mother
sporocyst
after 3 wk of cultivation
in the presence
of Bge cells.
FIGURE 6. Light micrograph of E. caproni mother sporocyst after 3 wk of cultivation in the presence of Bge cells.
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232 THE
JOURNAL OF PARASITOLOGY,
VOL.
84, NO.
2, APRIL
1998
FIGURE 7. Scanning electron micrograph of E. caproni (A-D). (A) Newly hatched miracidium. (B) Transforming miracidium after 1 day in
culture. rl, ridge layer between epithelial plates. (C) Transforming miracidium after 2 days in culture presenting epithelial plates with and without
cilia; c, cilia; p, papilla. (D) Newly hatched miracidium after 10 min of sonication. Notice the absence of cilia on epithelial plates.
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ATAEV
ET
AL.-IN VITRO
DEVELOPMENT
OF E. CAPRONI 233
A
gc,
B
es
D
15 m dc 16 gm
Is sw
.. .
^~cJ
em\..
em
20 gm
FIGURE 8. Histological sections of E. caproni miracidium and mother sporocysts cocultivated with Bge cells (A-E). (A) Miracidium after 6
hr in coculture; c, cilia; es, eye spot; gc, germinal cells; n, neural mass; sc, secretory cell. (B) Transforming miracidia after 4 days in culture. (C)
Mother sporocyst showing developing embryos after 14 days in coculture; dc, dividing cell; e, embryo; ep, remain of epithelial plates; es, eye
spot fragment. (D) Mother sporocyst after 21 days in culture; Is, laminar structures; sw, sporocyst wall. (E) Mother sporocyst after 50 days in
coculture; ec, embryonic cavity; em, embryonic membrane; gb, germinal ball; pc, picnotic cells.
One to 3 embryos of 5-10 blastomeres could be observed in
14-day-old MS (Fig. 8C). The division of the other primary
germinal cells had not yet started. A few epithelial plates could
still be seen at the surface of some MS (Fig. 8C).
Most MS contained 3-4 embryos of a maximum of 20 blas-
tomeres after 21 days in culture (Fig. 8D). Noticeable growth
began to occur after the third week of cultivation (Fig. 9). In
addition to growth, further development could be observed dur-
ing the following week in culture. Together with embryos, the
embryonic cavities increased in size. However, the numerous
laminar structures remained, preventing the cavities from merg-
ing to form the schizocoel.
After 8-9 wk in culture, most MS contained 4-6 embryos.
One or 2 embryos per sporocyst reached the germinal ball stage
(70-80 blastomeres) (Fig. 8E). Cells of these embryos remained
very similar in size and shape and did not differentiate. The
V
sc
13 ,gm
es
13 gim
C
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234 THE JOURNAL
OF PARASITOLOGY,
VOL.
84, NO. 2, APRIL
1998
0
x 1,5-
E
E
E 1,0-
0,5 - I
0o,o0 1 l
0 1 2 3 4 5 6
Weeks in Culture
FIGURE
9. Growth of E. caproni mother sporo
presence of Bge cells. Each point represents meat
SD (10 < n < 70).
other embryos were less advanced in theiJ
contained a maximum of 15-20 blastomere;
also contained 10-15 secondary germinal
1997). Further development of these secoi
observed.
MS reached maximal sizes of 300 p.m in
in width after 10 wk in culture. The most
served at this time showed embryos of 100-
these cells were condensed and pycnotic, st
eration. No further development was obser
culture. Sizes of MS decreased to an aver
length and 90 p.m
in width.
DISCUSSION
As previously shown by Reddy and Frie
by E. caproni adults in RPMI-1640 mediun
lent source of miracidial material. These M
B. glabrata snails and showed a typical d
and production of rediae. The comparison (
in B. glabrata with the previously described
pfeifferi (Ataev et al., 1997) showed very
tics. In particular, the timing of growth an
final site of infection, the reproduction intel
of MS were nearly identical. Only small di
tion dynamics were observed. For example
of Mi was longer in B. glabrata (6 hr) as
pfeifferi (5 hr). In addition, MS were obser
of infection at 2 days PI in B. glabrata, ins
B. pfeifferi. This longer period of migration
brata is probably linked to the larger size (
compared with B. pfeifferi.
When cultivated in the presence of Bge
roni transformed into MS and continued
through complete embryogenesis of the first
striking differences observed between in vi
velopment pertained to the transformation
caproni, as well as Mi of other species ol
are known to shed their epithelial plates when penetrating
through host tegument (see review by Semenov, 1991). It is
known that under in vitro conditions, several abiotic factors
(chemicals, ionic, or osmotic shocks) can also trigger shedding
of epithelial plates (Targett and Robinson, 1964; Wilson et al.,
1971; Basch and DiConza, 1974). In our coculture system, Mi
retained their epithelial plates for 2-3 wk. However, after 2 days
in culture, the majority of Mi lost cilia on parts of their surface.
| Sonication-induced breakage of cilia also occurs at the level of
the basal plate, suggesting that it is a weak point. Although
ciliary loss can obviously be induced by a variety of chemical
and physical factors, it is likely that in our system the loss of
cilia is related to the degeneration of epithelial plates. This is
supported by the fact that the presence or absence of cilia only
varies from 1 plate to another.
Although shedding of epithelial plates was delayed, another
7 8 9 1 0 characteristic of transformation, the degeneration of Mi soma,
could be observed after 3-4 days in culture. When Mi pene-
cysts cultivated
in the trates a snail host, degeneration of Mi soma occurs after shed-
n estimated volume + ding of epithelial plates and the formation of MS primary body
wall. Under in vitro conditions, the complete degeneration of
Mi soma and the beginning of embryogenesis actually occurred
before the complete shedding of epithelial plates. These results
s. At this time, MS suggest that the degeneration of Mi soma and embryogenesis
cells (Ata
ths te, can be triggered independently of the shedding of epithelial
cells (Ataev et al., pt
aidary GC was not The first steps in the development of germinal material were
similar under in vivo and in vitro conditions. In both cases, 1-
length and 100 obm 2 germinal cells (GC) distinguished by their larger sizes in the
develolped MS ob- Mi started dividing and formed the first embryos. Only then
110 cells.t Howeve, did the other 4-5 primary GC develop into embryos. The de-
gved after 10 wk in velopment of embryos was considerably slower under in vitro
aved ofte 00 m in conditions. The absence of development of the secondary ger-
rage of 200 Fm in minal balls even in the most developed 10-wk-old MS supports
the assumption that secondary MS start developing only after
the first redial embryos have reached maturity (Ataev et al.,
1997).
d (1996), eggs laid It is clear from this cultivation experiment that the presence
n provide an excel- of Bge cells in the cultures was essential for Mi survival and
[i were infective for MS development because Mi maintained in cell-free medium
levelopment of MS could not survive more than 2 wk in culture. However, in con-
of MS development trast to what was shown for S. mansoni (Yoshino and Laursen,
I development in B. 1995) and S. japonicum (Coustau et al., 1997), Bge cells did
similar characteris- not permit development of E. caproni MS leading to the pro-
d development, the duction of the next intramolluscan generation. Some redial em-
nsity, and life spans bryos reached the stage of 100-110 cells, but their development
ifferences in migra- stopped at this stage and was followed by a slow degeneration
, the resting period of MS. The differential success in cocultivating E. caproni, S.
compared with B. mansoni, and S. japonicum with Bge cells is probably because
ved in the final site E. caproni has different physicochemical requirements as com-
stead of 1 day PI in pared with the 2 schistosome species. One important difference
observed in B. gla- in the development of these species lies in their final sites of
of this snail host as infection. The MS of both schistosomes develop in various snail
tissues, whereas E. caproni MS strictly develop in the central
cells, Mi of E. cap- blood system. It has been shown that E. caproni MS acciden-
their development tally settling in tissues such as the buccal mass or muscle un-
embryos. The most dergo abnormal development potentially leading to death
ivo and in vitro de- (Ataev et al., 1997). This suggests that factors specifically pres-
process. Mi of E. ent in the hemolymph of the central blood system are strictly
f Echinostomatidae, required for E. caproni development. It is known, for example,
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ATAEV
ET
AL.-IN VITRO
DEVELOPMENT OF E CAPRONI 235
that the oxygen pressure is very low in tissues as compared
with the hemolymph (Ginetsinskaya, 1968). In addition to dif-
ferences in physicochemical requirements, it is also possible
that the very slow shedding of epithelial plates observed for E.
caproni has a negative effect on development. Although the
epithelial plates retained at the surface of MS during the first
week in culture were no longer functional, they may prevent a
proper uptake of nutrients through the sporocyst tegument, thus
significantly decreasing the rate of growth and development.
Previous studies have shown differential interactions between
Bge cells and schistosome MS. Whereas Bge cells were shown
to adhere rapidly to and encapsulate S. mansoni MS (Yoshino
and Laursen, 1995), they did not exhibit such a surface-aggre-
gating response with S. japonicum MS (Coustau et al., 1997).
However, because of the high motility of S. japonicum MS in
culture, it was not possible to determine whether the lack of
encapsulation was the result of this mechanical factor or of
nonrecognition of S. japonicum MS. In the present study, the
limited motility of E. caproni MS (similar to S. mansoni mo-
tility) was not likely to prevent a Bge encapsulation reaction.
However, echinostomes have been reported to interfere with B.
glabrata hemocyte functions (Adema and Loker, 1997), and it
is possible that E. caproni excretory-secretory products inhibit
the Bge cell encapsulation reaction. Future studies will inves-
tigate whether this lack of encapsulation of E. caproni MS is
due to a nonrecognition or to a parasite-mediated interference
with Bge cell function. This lack of encapsulation reaction sug-
gests that development of E. caproni MS was triggered by sol-
uble factors. The nature of the Bge cell factor(s) that trigger
development of S. mansoni, S. japonicum, and E. caproni MS
remains to be characterized. In particular, it is unknown whether
all of these parasite species benefit from identical Bge factors
for their development. This Bge cocultivation system can now
be used for analyzing nutritional, physiological, and hormonal
factors involved in the development of 3 digenean species, as
well as for investigating mechanisms underlying the differential
adhesion of these B. glabrata cells to schistosomes/echinosto-
mes MS.
ACKNOWLEDGMENTS
We thank T. Yoshino (School of Veterinary Medicine, Mad-
ison, Wisconsin) Sandrine Trouvd, and Juliette Langand
(CBETM, Univ. Perpignan, France) for supplying Bge cells,
infected mice, and susceptible B. glabrata snails, respectively.
We are grateful to E. S. Loker for critical reading of the manu-
script. This work was supported by the French CNRS.
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