IN VITRO CULTURE OF REDIAE OF ECHINOSTOMA CAPRONI
LOKER E.S.*, COUSTAU C.**, ATAEV G.L.*** & JOURDANE J.**
S u m m a r y :
Rediae of Echinostomo caproni (Egyptian strain) were dissected from Biomphalaria glabrata snails at intervals from 13-34 days post-exposure and co-cultured for up to 5 1 days with cells of the B. glabrata embryonic (Bge) cell line. Rediae readily ingested Bge cells and survived longer when co-cultured with cells than in cell- free cultures. Rediae released mostly motile cercariae throughout the observation period when in Bge medium and cells. Rediae cultured in 199 medium with Bge cells also produced progeny throughout most of the observation period. In the latter medium, progeny were much more likely to include rediae as well as cercariae. Some cercariae produced in vitro encysted as metacercariae. Rediae consumed cercariae released into culture but were not observed to attack one another or rediae of a different echinostome species.
KEYWORDS: Digenea, Echinostomatidae, Echinostomo caproni, Biomphalaria glabrata, rediae, in vitro culture.
Résumé : CULTURE IN VITRO DE RÉDIES D'ECHINOSTOMA CAPRONI Les rédies du trématode Echinostoma caproni (souche égyptienne) ont été isolées des tissus de Biomphalaria glabrata entre 13 et 34 jours après infestation, mises en culture avec la lignée cellulaire Bge (Bge celisi dérivée d'embryons de B. glabrata, et maintenues jusqu'à 51 jours dans ces conditions expérimentales.
Les rédies ingéraient activement des cellules Bge, et présentaient une meilleure survie en présence de cellules dans le milieu de culture. Les rédies cultivées avec des cellules Bge dans du milieu Bge produisaient des cercaires mobiles tout au long de la période d'observation. Lorsqu'elles étaient cultivées avec des cellules Bge mais en présence de milieu 199, elles produisaient à la fois des rédies et des cercaires. Certaines cercaires produites in vitro se sont enkystées en métacercaires. Le suivi des cultures a mis en évidence l'ingestion par les rédies de cercaires produites in vitro, mais jamais de phases de cannibalisme entre rédies appartenant ou non à la même espèce.
MOTS CLÉS : Digenea, Echinostomotidae, Echinostoma caproni, Biomphalaria glabrata, rédies, culture in vitro.
Several fundamental aspects o f the intramolluscan b i o l o g y o f d i g e n e t i c t r e m a t o d e larvae remain poorly understood. T h e basic developmental pat- terns o f larval d i g e n e a n s require further clarification.
Intra- and interspecific interactions b e t w e e n digenean larvae also remain understudied. Such studies have b e e n i m p e d e d in part by our inability to provide cul- ture conditions that allow c o m p l e t e and normal in vitro d e v e l o p m e n t o f d i g e n e a n larval stages.
Most previous w o r k o n culturing o f larval digeneans has focused o n Schistosoma mansoni. It is possible to transform axenically miracidia into m o t h e r sporocysts ( V o g e & Seidel, 1972; B a s c h & DiConza, 1 9 7 4 ) . Mira- c i d i u m - d e r i v e d m o t h e r s p o r o c y s t s will p r o d u c e daughter sporocysts ( Y o s h i n o & Laursen, 1 9 9 5 ) w h e n co-cultured in the p r e s e n c e o f cells o f the Biompha-
* Department of Biology, University of New Mexico, Albuquerque, New Mexico 87131, USA.
** Laboratoire de Biologie Animale, Université de Perpignan, Perpi- gnan, France.
***Department of Invertebrate Pathology, Academic Centre of Com- parative and Ecological Pathology, 5/10 Volzhsky pereulok, St.
Correspondence: Eric S. Loker.
Tel.: 505 277-5508 - Fax: 505-277-0304 - E-mail: firstname.lastname@example.org
laria glabrata embryonic cell line ( B g e cells) originally d e v e l o p e d by Hansen ( 1 9 7 6 a ) . Daughter sporocysts derived from infected snails will release additional daughter sporocysts w h e n cultivated with a mosquito cell line (Hansen, 1 9 7 3 ) , B g e cells (Hansen, 1976b), m e d i u m c o n d i t i o n e d b y B g e c e l l s ( H a n s e n et al, 1 9 7 4 a ) o r e v e n in a x e n i c medium ( H a n s e n et al, 1974b). Early cercarial e m b r y o s freed from daughter sporocysts will d e v e l o p into swimming but noninfec- tive cercariae w h e n cultured with B g e cells (Hansen,
1975; B a s c h & DiConza, 1 9 7 7 ) . Coustau et al. ( 1 9 9 7 ) have also reported the d e v e l o p m e n t o f S. japonicum mother sporocysts and their production o f daughter sporocysts in the p r e s e n c e o f B g e cells.
Little work has b e e n undertaken with the in vitro cul- ture o f other digeneans, particularly those s p e c i e s that feature redial stages in their larval development. Rediae o f Fascioloides magna w e r e maintained for up to ten days in simple media containing a m i n o acids and sugars (Friedl, 1961 a, and B a s c h & DiConza ( 1 9 7 5 ) reported that Echinostoma paraensei rediae can b e maintained for m o r e than a month in a x e n i c culture.
T h e y noted that E. paraensei rediae dissected from snails at 18 days p o s t - e x p o s u r e subsequently released small rediae within the first few w e e k s o f culture but
Parasite, 1999, 6, 169-174
Note de recherche 169
Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/1999062169
LOKER E.S., COUSTAU С , ATAEV G.L. & JOURDANE J .
did not p r o d u c e cercariae. Rediae w e r e also o b s e r v e d to eat S. mansoni m o t h e r and daughter sporocysts in vitro ( B a s c h & DiConza, 1 9 7 5 ) . Augot et al. ( 1 9 9 7 ) cul
tured rediae o f Fasciola hepatica axenically for inter
vals up to 16 days to quantify the n u m b e r o f daughter rediae and cercariae p r o d u c e d in vitro.
As so little previous work has b e e n d o n e to study rediae in culture, the present study was undertaken to observe rediae o f Echinostoma caproni. Recently, Ataev et al. ( 1 9 9 8 ) reported that sporocysts o f this s p e c i e s lived for up to 17 w e e k s in the p r e s e n c e o f B g e cells as c o m p a r e d to only two w e e k s w h e n in cell-free medium. For the present study, E. caproni rediae w e r e p l a c e d into culture wells in which B g e cells w e r e b e i n g propagated in two types o f media. W e sought to determine if 1) survival o f rediae was prolonged by the p r e s e n c e o f B g e cells; 2 ) rediae would c o n s u m e B g e cells; 3 ) progeny rediae or cercariae would b e released and over what interval o f time; and 4 ) if E. caproni rediae would attack or prey u p o n other rediae o r cercariae o f the s a m e or different e c h i n o - s t o m e s p e c i e s .
MATERIALS AND METHODS
E c h i n o s t o m a caproni (Egypt) was maintained in hamsters and a l b i n o Biompbalaria glabrata (Brazil). An isolate o f Echinostoma sp. from Niger was maintained in hamsters and Bulinus glo- bosus. Biomphalaria glabrata o f 5-10 m m shell dia
meter w e r e individually e x p o s e d to 2 0 - 3 0 miracidia o f E. caproni. At intervals ranging from 13-34 days post
e x p o s u r e ( d p e ) , rediae w e r e harvested from these snails. Snails were s o a k e d for o n e hour in water contai
ning 1 % penicillin, s t r e p t o m y c i n a n d f u n g i z o n e , s w a b b e d in 7 0 % ethanol, and then crushed in a p o o l o f serum-free m e d i u m 199 diluted to half normal strength for use with snails (Loker et al., 1 9 9 2 ) . Rediae were transferred to fresh medium and rinsed to remove snail debris. Concurrent dissection o f infected snails and e x a m i n a t i o n o f the redial populations present indicated that rediae harvested before or o n 20 dpe w e r e m o t h e r rediae, w h e r e a s those c o l l e c t e d at later d a t e s r e p r e s e n t e d m i x e d g e n e r a t i o n s . F r o m 1 to 22 rediae w e r e placed in individual flat- or round-bot
t o m e d wells in 96-well culture plates ( B e c t o n Dic
kinson Co., Franklin Lakes, NJ).
Most wells w e r e s e e d e d with B g e cells in B g e medium (Hansen, 1 9 7 6 a ) s u p p l e m e n t e d with 10 % fetal calf serum ( F C S ) and gentamycin, the day before rediae w e r e to b e added. T h e source and maintenance o f B g e cells w e r e as described by Y o s h i n o & Laursen ( 1 9 9 5 ) . At the time rediae w e r e added, the medium in e a c h well w a s c h a n g e d , either to B g e or 199 medium.
D e p e n d i n g o n the well, the m e d i u m c o n t a i n e d 0, 5 or 10 % heat-inactivated FCS. All m e d i a c o n t a i n e d gentamycin ( 4 3 p g / m l ) . Cultures w e r e incubated at 24- 2 7 ° C u n d e r n o r m a l a t m o s p h e r i c c o n d i t i o n s a n d medium in 0.75 ml volumes was c h a n g e d o n a w e e k l y basis. No additional B g e cells w e r e a d d e d o n c e t h e cultures w e r e initiated. Six cultures lacking B g e cells w e r e also set up. e a c h receiving a different c o m b i nation o f m e d i u m ( B g e or 1 9 9 ) and FCS ( 0 , 5, o r 10 % ) . All cultures w e r e e x a m i n e d immediately after initial set up and at w e e k l y intervals thereafter. An additional 11 cultures w e r e e s t a b l i s h e d , e a c h c o n taining B g e cells and a single redia e a c h o f E. caproni a n d o f Echinostoma sp. T h e n u m b e r and c o n d i t i o n o f larvae in e a c h culture w a s m o n i t o r e d as d e s c r i b e d a b o v e .
Rediae in cultures lacking B g e cells, regardless o f the medium or amount o f FCS present, w e r e ini
tially motile and released cercariae and rediae within the first four days o f culture. B y o n e w e e k , they p o s s e s s e d a gut that w a s abnormally inflated, their b o d y wall was b r o w n and rough instead o f clear and smooth, and they w e r e only weakly motile. B y 2 0 days they w e r e dead.
All remaining efforts focused o n cultures containing B g e cells. B g e c e l l s grew well in b o t h B g e a n d 199 media. Rediae in B g e medium initially d e v e l o p e d a bloated gut and o p a q u e b o d y wall (Fig. 1), but s e e m e d to recover and w h e n the cultures w e r e ter
minated, many contained developing progeny. Rediae in cultures containing medium 1 9 9 did not b e c o m e bloated and released m o r e progeny, particularly pro
g e n y rediae, than did rediae in cultures with B g e medium ( T a b l e I ) . No o b v i o u s effect o n B g e cell pro
duction or progeny production by rediae w a s noted in cultures w e a n e d o f FCS.
Rediae cultured with B g e cells readily ingested the cells (Fig. 2 ) and in general, did m u c h better than rediae from cultures lacking cells. S o m e p r o d u c e d germinal balls (Fig. 3 ) that increased in size and s o m e released p r o g e n y e v e n at 51 days, the longest interval that cul
tures w e r e followed. As long as B g e cells w e r e pre
sent, rediae routinely survived for longer than 3 0 days in culture.
Mother rediae that w e r e relatively y o u n g w h e n t a k e n from snails ( 1 3 d p e ) did not r e l e a s e p r o g e n y in cul
ture, but after 27 days o f o b s e r v a t i o n , o n e c o n t a i n e d an e l o n g a t e d e v e l o p i n g redia that c o n t i n u e d to g r o w for t h e r e m a i n i n g 2 0 days o f o b s e r v a t i o n . Individual m o t h e r r e d i a e c o l l e c t e d f r o m s n a i l s at 2 0 d p e r e l e a s e d as m a n y as 37 daughters o v e r a 4 0 - d a y
IN VITRO CULTORE O F REDIAE O F ECHINOSTOMA CAPRONI
Figs 1-8. - In vitro cultivation of Echinostoma caproni rediae.
Fig. 1. Rediae taken from snail with 24 day-old infection, in Bge medium with Bge cells for three days. Note enlarged, empty gut (*) and opaque body wall. Scale bar = 145 µm.
Fig. 2. Mother redia taken from snail with 13 day-old infection, in culture with 199 medium and Bge cells for six days, with gut contai
ning ingested Bge cells (arrow). Scale bar = 143 µm.
Fig. 3. Redia taken from snail with 19 day-old infection, in culture with 199 medium and Bge cells for 29 days, containing germinal balls (arrow). Scale bar = 145 µm.
Fig. 4. Progeny redia (arrow) released from a redia taken from snail with 30 day-old infection and held in culture with 199 medium and Bge cells for 10 days. Scale bar = 109 µm.
Fig. 5. Numerous (approximately 40) progeny rediae released over a 39 day period of culture, derived from 12 original rediae (see dark, opaque rediae) taken from snail with 30 day-old infection, in 199 medium and Bge cells. Scale bar = 439 µm.
Fig. 6. Recently released cercariae, some tail-less, in a culture established for 41 days. The original rediae were taken from a snail with a 19 day-old infection, and were cultured in 199 medium and Bge cells. Scale bar - 139 µm.
Fig. 7. Ingestion of tail (arrow) of recently-produced progeny cercaria by a redia (R), 33 days after initiation of culture. The original rediae were taken from a snail with a 30 day-old infection and cultured in Bge medium with Bge cells. Scale bar = 107 µm.
Fig. 8. Encysted metacercariae (arrows) derived from cercariae released in vitro, from a four day-old culture containing 199 medium and Bge cells. The original rediae were taken from a snail with a 30 day-old infection. Scale bar = 417 µm.
Parasite, 1999, 6, 169-174
Note de recherche 171
Age of infection in snails (days) when rediae to be cultured were collected 13-34 13-34
Number of of cultures set up 25 14
Average * of rediae/culture (± SE) 4.68 ± 1.04 4.0 ± 0.95
* of cultures in which no progeny were released 10 7
* of cultures in which only rediae were released 4 1
* of cultures in which only cercariae were released 5 6
* of cultures in which both rediae and cercariae were released 6 0
Average * of progeny released per cultured redia (± SE) 2.89 ± 0.78 0.94 ± 0.41
Total * of rediae released 127 1
Average # of rediae released per cultured redia (± SE) 1.48 ± 0.69 0.01 ± 0.01
Total * of cercariae released 160 54
Average * of cercariae released per cultured redia (± SE) 1.40 ± 0.48 0.93 ± 0.41 Longest observed culture interval (days) after which new progeny were noted
progeny rediae 45 51
progeny cercariae 41 45
Longest observed interval (days) during which progeny were produced within
a single culture- > 44 > 44
Table 1. - Summary of results for Echinostoma caproni rediae cultures containing Bge cells and either 199 or Bge medium.
interval. No c e r c a r i a e w e r e r e l e a s e d b y t h e s e m o t h e r rediae.
Cultures with rediae derived from snails > 2 0 dpe likely c o n t a i n e d mixtures o f mother and daughter rediae which released progeny rediae (Figs. 4 and 5 ) and cer
cariae (Fig. 6 ) , or both ( T a b l e I ) . In n o c a s e was a single redia o b s e r v e d to release b o t h cercariae and rediae. In most cases, progeny w e r e p r o d u c e d 10 days or longer after culture establishment and in several, progeny w e r e released 4 0 days or longer.
Progeny rediae ingested B g e cells and in s o m e c a s e s c o n t a i n e d developing germinal balls and several w e r e living w h e n the cultures w e r e terminated. Motile cer
cariae w e r e released as late as 4 5 days after initiation o f cultures. Cercariae w e r e o b s e r v e d to b e ingested by rediae (Fig. 7 ) . Cercariae also e n c y s t e d in culture (Fig. 8 ) , and the resultant cysts w e r e c o v e r e d by B g e cells. Moribund cercarial b o d i e s separated from their tails w e r e also noted.
In n o c a s e w e r e rediae o b s e r v e d to attack o n e a n o ther. B g e cells w e r e not o b s e r v e d sticking to living rediae or cercariae but occasionally w e r e o b s e r v e d to encapsulate rediae believed to b e moribund or dead.
After several w e e k s o f culture, s o m e rediae w e r e sur
rounded by what a p p e a r e d to b e remnants o f B g e cells. It is not k n o w n if these w e r e cells that died as a normal part o f B g e cell culture, if they w e r e killed by the p r e s e n c e o f rediae, or if they had b e e n regur
gitated from redial guts.
W h e n rediae o f E. caproni and Echinostoma sp. w e r e cultured together, they s h o w e d n o t e n d e n c y to attack e a c h other. Consumption o f released cercariae, inclu
ding cercariae o f the other species, was observed.
As n o t e d by Y o s h i n o & L a u r s e n ( 1 9 9 5 ) for S. mansoni sporocysts, the p r e s e n c e o f B g e cells clearly had a salutary effect o n cultured rediae o f E. caproni. Many o f the rediae cultured with B g e cells w e r e alive and a p p e a r e d to contain d e v e l o ping progeny w h e n observations w e r e terminated at 51 days. In s o m e cultures, p r o g e n y w e r e released o n as many as s e v e n separate o c c a s i o n s over intervals as long as 4 4 days. Consumption o f B g e cells by cultured rediae was prominent but the beneficial effects o f B g e cells w e r e not derived from eating cells a l o n e . S o m e rediae consistently had guts p a c k e d with B g e cells but did not undergo germinal cell d e v e l o p m e n t and others w e r e o b s e r v e d to eat few cells yet p r o d u c e d progeny.
Laursen & Y o s h i n o ( 1 9 9 9 ) noted that cultured rediae o f F. magna c o n t a i n e d particulate matter but did not o b s e r v e rediae to feed on B g e cells.
Y o s h i n o & Laursen ( 1 9 9 5 ) o b s e r v e d that B g e cells attached to and then e n c a p s u l a t e d S. mansoni mother sporocysts and suggested that the intimate contact with cells was a r e q u i r e m e n t for d e v e l o p m e n t o f daughter sporocysts. Ataev et al. ( 1 9 9 8 ) o b s e r v e d n o contact b e t w e e n B g e cells and E. caproni sporocysts in their culturing experiments. B g e cells also did not attach to or encapsulate F. magna rediae (Laursen &
Y o s h i n o , 1 9 9 9 ) . Similarly, in the present study, B g e cells did not attach to or encapsulate viable E. caproni rediae although moribund rediae and metacercariae w e r e encapsulated. This pattern is similar to that noted with e c h i n o s t o m e sporocysts, rediae and metacercariae L O K E R E . S . , C O U S T A U С , A T A E V G.L. & J O U R D A N E J .
IN VITRO CULTURE OF RF.DIAE OF ECHINOSTOMA CAPRONI
and host h e m o c y t e s in vivo (Loker et al., 1 9 8 7 ) , sup
porting the c o n c e p t o f Y o s h i n o & Laursen ( 1 9 9 5 ) that B g e cells have a least s o m e properties reminiscent o f h e m o c y t e s . T h e t e n d e n c y o f B g e cells to adhere to schistosome mother sporocysts but not to e c h i n o s t o m e sporocysts and rediae is similar to the pattern noted by Loker ik Adema ( 1 9 9 5 ) with respect to in vitro inter
actions b e t w e e n s c h i s t o s o m e and e c h i n o s t o m e larvae and h e m o c y t e s .
As the rediae placed in culture w e r e all obtained from infected snails, it is difficult to k n o w with certainty which, if any. o f the progeny they released had deve
l o p e d exclusively under in vitro conditions. However, it is likely that many o f the p r o g e n y released in cul
tures containing B g e cells had d e v e l o p e d largely in vitro. As compared to rediae cultured without Bge cells, rediae co-cultured with cells released m o r e progeny over m u c h longer intervals. Also, the a p p e a r a n c e in vitro o f germinal balls that d e v e l o p e d into elongate redial e m b r y o s i n d i c a t e s that larval d e v e l o p m e n t o c c u r r e d in vitro.
Medium 199 supported production o f B g e cells for over 50 days in culture. This medium was m o r e favorable to rediae during the early stages o f culture and sup
ported greater overall production o f progeny, especially o f progeny rediae. In contrast, after initially looking quite abnormal in the presence o f B g e medium, rediae s e e m e d to recover and survived and remained pro
ductive for longer time intervals in this medium than in medium 1 9 9 .
B a s c h & DiConza ( 1 9 7 5 ) noted that E. paraensei rediae dissected from snails at 18 dpe released progeny rediae over t w o to three w e e k s in cultures lacking B g e cells.
T h e y did not o b s e r v e cercariae or a d v a n c e d cercarial e m b r y o s to b e produced. In the present study, motile cercariae w e r e released as late as 4 5 days after initia
tion o f culture, and s o m e encysted. Stein & B a s c h ( 1 9 7 7 ) o b s e r v e d that cercariae o f E. paraensei released from infected B. glabrata w e r e c a p a b l e o f normal e n c y s t m e n t in cultures c o n t a i n i n g B g e cells or in medium conditioned by B g e cells, but not in uncon
ditioned medium. T h e y also observed metacercariae to b e encapsulated by B g e cells in vitro.
In agreement with B a s c h ik DiConza ( 1 9 7 5 ) , w e noted n o t e n d e n c y for cultured rediae to attack other rediae.
W e n o t e d that rediae c o n s u m e d cercariae, both o f the s a m e or o f a different s p e c i e s , but their t e n d e n c y to d o s o under the conditions o f culture e m p l o y e d can only b e considered as moderate.
O u r results provide e v i d e n c e that the p r e s e n c e o f B g e cells has a distinctly beneficial effect o n both survi
vorship and progeny production for cultured redial stages. Additional studies with cultured rediae should b e undertaken b e c a u s e the prospects for considerably improving culture conditions are high, and b e c a u s e it
will o p e n up interesting possibilities for study with res
pect to interactions b e t w e e n larval digeneans o f the s a m e or different species.
This study was supported by NIH grant AI24340 and Centre National de la R e c h e r c h e Scienti
fique. T h e authors thank Ms. Pascale Léonard for assistance with translations into French and Dr. C o e n Adema and Ms. Lynn Hertel for critically reading the manuscript.
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