Scanning electron microscopy of Echinostoma revolutum (Trematoda) during development in the chick embryo and the domestic chick

SPANNING ELECTRON MICROSCOPY OF EC~~~O~TO~~ REVOLUTUM (TREMATODA) DURING DEVELOPMENT IN THE CHICK EMBRYO AND THE DOMESTIC CHICK BERNARD FRIED* and TAKAHIRO FUJINO* j-Department

*Department of Biology, Lafayette College, Easton, PA 18042, U.S.A. of Parasitology, Faculty of Medicine, Kyushu University 60, Fukuoka

812, Japan

(Received 5 January 1983) electron microscopy Of Echinostoma revoiutum and the domestic chick. ~~ter~utio~~~Journal for Parasitology 14: 75-81. Scanning electron microscopy @EM) was used to study the development of chemically excysted metacercariae of Echj~ostomu revolutum on the chick chorioallantois. SEM studies were also made on preovigerous adults of E. revoiutum grown in the domestic chick. During worm development on the chorioallantois the tegument changed from smooth to granular and sensory papillae on the suckers became well-defined. As worms developed on the chorioallantois the cephalic collar spines became thicker and more curved and the tegumentary spines showed marked changes in shape, size and distribution on both ventral and dorsal aspects of the body. Changes in the surface uhrastructure of worms grown on the chorioallantois were essentially similar to those observed in preovigerous worms from chicks.

Abstract-FRIso B. and FUJINO T. 1984. Scanning (Trematoda) during development in the chick embryo

INDEX KEY WORDS: SEM; Trematoda; chorioallantois; development; ultrastructure;

Echinosfoma revolutum; chick embryo; growth;

INTRODUCTION Pentz (1983) recently reported light microscopical observation on excysted metacercariae of Echinostoma revolutum cultivated in chick embryos. Studies using scanning electron microscopy @EM) on trematodes cultivated in chick embryos are not available. Furthermore, SEM studies on excysted metacercariae or adults of E. revolut~m are not available. This study uses SEM to examine excysted metacercariae, worms grown in chick embryos and preovigerous worms of E. revolt&urn grown in the domestic chick.

tegument;

domestic

chick;

spines; papillae.

Fe-1 100 ion-sputtering apparatus and then examined with a JEOL JSM-U3 scanning electron microscope at an accelerating voltage of I5 kV.

FRIED &

RESULTS

Excysted metacercariae. Low magnification SEW’s of excysted metacercariae show the prominent oral collar with its cephalic spines (Figs. 1, 2) and the tegumentary spines which appear to end at the midacetabular level. However, higher magnification SEM’s show the presence of tegumentary spines posterior to the acetabulum on the dorsal and ventral surface, which are small and peg-like (Figs. 3, 4). The cephalic spines on the oral collar are coneshaped and bluntly pointed (Figs. 5, 6). The oral sucker is thick-lipped and the lip contains sensory papillae that are not well-defined (Fig. 7). The papillae around the oral sucker are platelike with a ringed-base (Fig. 6 inset). The acetabulum is thicklipped with sensory papillae on the lip that are not well-defined (Fig. 8). The tegument is rather smooth with transverse and longitudinal grooves. The ventral and dorsal tegumentary spines anterior to the acetabulum are flat with rounded tips and broad bases (Fig. 9). Day-old chorioaflantoic-worms. The tip of the oral collar spines is round. The lip of the oral sucker

MATERIALS AND METHOUS Excysted metacercariae of E. revoi~tum were obtained as described in Fried & Butler (1978) and transferred to the chick chorioallantds using the procedure of Zwilling (1959) as described by Fried & Pentz (1983). Worms were removed from the chorioallantois on days 1, 4 and 7 days postinoculation. Some preovigerous worms were grown in domestic chicks for 7 days as described in Fried & Weaver (1969). Excysted metacercariae, chorioallantoic-worms and chick-worms were washed rapidly in three changes of cold sterile Locke’s solution and then fixed in cold 10% neutral buffered formalin. For SEM studies, worms were postfixed for 2 h in 1% osmium tetroxide buffered to pH 7.4 with 0.1 M sodium cacodylate. They were dehydrated through a graded series of ethanol and critical-point dried. The specimens were coated with gold, using a JEOL 75

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SEM of

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Echinostoma revolutum in chick embryos

is thinner than in the metacercaria, although papillae on the lip are still not well-defined. The bases of the papillae around the oral sucker are swollen. The acetabulum and papillae on the acetabular lip are essentially as described for the metacercaria. The tegument is somewhat granular. The tegumentary spines on the dorsal and ventral surface anterior to the level of the acetabulum are triangular with blunt-pointed tips (Fig. 10). Tegumentary spines in the posterior aspect of the body are as described for the metacercaria. Four-day-old chorioallantoic-worms. The cephalic spines are thicker and more curved than seen previously (Fig. 11). The papillae on the lip of the oral sucker are swollen, well-defined and are cup-orbutton-shaped (Fig. 12). The lip of the acetabulum is thinner than seen previously and sensory papillae on the lip are now well-defined, round and have rough surfaces (Fig. 13). Tegumentary spines anterior to the acetabulum are flat and broad, with blunt tips and curved lateral edges with sculptured surfaces (Fig. 14). The tegumentary spines in the posterior aspect of the body are essentially as described previously. The tegument contains a granular or knobbed surface. Seven-day-old chorioallantoic-worms. A low magnification SEM of a 7-day-old chorioallantoicworm is shown in Fig. 15. The acetabulum is in the anterior-third of the body compared with its location in the posterior-half of the body of the excysted metacercaria (see Fig. 2). The cephalic collar spines are as described for 4-day-old worms. Papillae on the lip of the oral sucker are now well-defined, single, or in groups of two or three (Fig. 16). The acetabulum is essentially as described for 4-day-old worms. The surface of the tegument is granular or knobbed as seen in 4-day-old worms. The tegumentary spines behind the oral collar are broad and more elongate than in the 4-day-old worms (Fig. 17). There is a

FIGS. 1-8. Excysted

71

reduction both in the number and size of the tegumentary spines in the posterior aspect of the body (Fig. 18) (see Figs. 3,4). Preovigerous (7-day-old) chick-worms. Low magnification SEM’s of 7-day-old chick-worms show the acetabulum in the anterior-third of the body as seen in 7-day-old chorioallantoic-worms (see Fig. 15). The cephalic collar spines are thick with a wide gap between spines and wrinkles are apparent on the spines. The lip of the oral sucker is not well-defined and papillae on the lip are similar to those seen on other parts of the body. The papillae around the oral sucker are swollen and contain step-like elevations. The lip of the acetabulum is swollen with grooves and papillae associated with the acetabulum appear granular (Fig. 19). The tegument appears granular or knobbed. The dorsal tegumentary spines are thick, scale-like and contain broadly rounded tips (Fig. 20). The ventral tegumentary spines are also thick, scale-like and have indented tips (Fig. 21). The tegumentary spines have transverse ridges on their surfaces. DISCUSSION

Bennett (1975b) and K$ie, Nansen & Christensen (1977) used SEM to study growth of Fasciola hepatica in the mouse. They observed marked changes in tegumentary spines during postmetacercarial development of this fluke in the mouse. SEM studies on postmetacercarial development of flukes in chick embryos are not available. We observed ultrastructural changes mainly on the tegument, tegumentary spines and sensory papillae of E. revolutum excysted metacercariae grown on the chick chorioallantois. Whereas the tegument of excysted metacercariae was smooth with shallow wrinkles, that of chorioallantoic-worms contained knob-like protuberances, which gave the surface a

metacercariae.

FIG. 1. Whole body in dorsal view. Scale bar = 50 pm. FIG. 2. Whole body in ventral view. Scale bar = 50pm. FIG. 3. Dorsal tegumentary

spines in the posterior

aspect of the body. Scale bar = 5pm.

FIG. 4. Ventral tegumentary

spines in the posterior

aspect of the body. Scale bar = 5pm.

FIG. 5. Anterior

part of the body in ventral view, showing Scale bar = 20 pm.

the oral sucker and cephalic

collar.

FIG. 6. Enlarged view of cephalic collar spines which are bluntly pointed. Scale bar = 5 pm. Inset: enlarged view of one sensory papilla around the oral sucker. Scale bar = 1 pm. FIG. 7. Enlarged

view of rectangular

FIG. 8. Acetabulum

area of Fig. 5, showing sensory Scale bar = 2 pm.

with swollen lip, showed no defined sensory

papillae

which are not well defined.

papillae.

Scale bar = 5 pm.

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SEM of Echinostoma revoiuium in chick embryos

granular or cobblestone-like appearance. Tegumentary spines changed during worm development on the chorioallantois. Those anterior to the acetabulum showed marked changes in shape, whereas spines posterior to the sucker became irregular and reduced in number. Sensory papillae on the lip of the oral sucker and acetabulum were not well-defined in the excysted metacercaria, but became obvious and welldifferentiated during worm development on the chorioallantois. The ultrastructural changes seen in worms on the chorioallantois were essentially similar to those observed in preovigerous worms grown in the domestic chick. These findings provide further evidence that the chick embryo is a favorable model to study postmetacercarial growth and development of E. revolutum (see Fried & Pentz, 1983). Beaver (1937) used light microscopy and reported that posterior tegumentary spines tend to disappear in E. revoIutum adults grown in w~m-blooded hosts. He noted that the reduction in spines was more apparent in birds than mammals. Our SEM study showed that in flukes grown on the chorioallantois both the number and size of posterior tegumentary spines were reduced. Sensory papillae in juvenile and adult trematodes have been reported in ~~h~tosorna rna~~o~i by Morris & Threadgold (1967); in Cyathocotyle bushiensis by Erasmus (1967); in Megalodiscus tempera&s by Nollen & Nadakavukaren (1974); in F. hepatica by Bennett (1975a) and K#ie et al. (1977); in Philophthalmus megalurus by Edwards, Nollen & Nadakavukaren (1977); in ~lo~orch~ sinensis by Fujino, Ishii & Choi (1979); in Leu~ochioridi~morpha constantiae by Font & Wittrock (1980); and in Gorgoderina vitelliloba by Hoole & Mitchell (1981). We observed three types of papillae in E. revolutum. One is the ciliated knob-like papilla, which is common in juvenile flukes. A second type is the nonciliated papilla with a step-like elevation of the tegument, and is present mainly on the lip of the

79

oral sucker. The third is rounded, swollen, or sometimes spherical and was seen mainly on the lip of the acetabulum of worms cultivated on the chorioallantois. This last type of papilla is similar to the domed one in F. hepatica described by Bennett (1975a) and the “C” type reported by Fujino et al, ( 1979) in C. sinensis.

REFERENCES P. C. 1937. Experimental studies on Echinostoma revalu~um(Froelich), a fluke from birds and mammals. I&nois Biologicalmonographs 15: l-96. BENNETTC. E. 1975a. Surface features, sensory structures, and movement of the newly excysted juvenile Fasciola hepaticaL. Journal of Parasitology61: 886-891. BENNETTC. E. 1975b. Scanning electron microscopy of Fasciola hepatica L. during growth and maturation in the mouse. Journal of Parasitology61: 892-898. EDWARDSH. H., NOLLENP. M. & NADAKAV~KAREN M. J. 1977. Scanning and transmission electron microscopy of oral sucker papillae of Philophthalmus megalurus. InternationalJournalfor ParasitologyI: 429-437. ERASMUSD. A. 1967. The host-parasite interface of Cyathocotyle bushiensis Khan, 1962 (Trematoda : Strigeoidea) II. Electron microscope studies of the tegument. Journal of Parasitolow 53: 703-714. FONTW. F. & WITTROCKD. D. 1980. Scanning electron microscopy of Leucochioridiomorpha constantiaeduring development from metacercaria to adult. Journal of Parasitology66: 955-964. FRIED B. & BUTLERM. S. 1978. Infectivity, excystation, and development on the chick chorioallantois of the metacercaria of Echinostoma revolutum (Trematoda). Journal of parasitology64: 175-177. FRIED B. & PENTZ L. 1983. Cultivation of excysted metacercariae of Echinostoma revolutum (Trematoda) in chick embryos. International Journal for Parasitology 13: 219-223. FRIEDB. & WEAVERL. J. 1969. Exposure of chicks to the metacercaria of Echinostoma revolutum (Trematoda). Proceedings of the Helm~nt~oIogical Society of Washington36: 153-155. BEAVER

Fro. 9. Ventral tegumentary spines behind cephalic collar. Spines are flat with rounded tips. Scale bar = 2 pm. Fro. 10. One-day-old chorioallantoic-worm. Ventral tegumentary spines behind cephaiic collar. Spines are flat and triangular with bluntly pointed tips. Scale bar = 2 pm. FIGS. 11-14. Four-day-old chorioallantoic-worms. FIG. 11. Enlarged view of cephalic collar spines, which are thick and curved posteriorly. Scale bar = 5 w. FIG. 12. Oral sucker. The papillae on the lip are swollen, rounded and well-defined. Scale bar = 5 pm. FIG. 13. Acetabulum, of which the lip is thin with rounded and well-defined papillae. Scale bar = 10 pm. FIG. 14. Ventral tegumentary spines anterior to the acetabulum, which are flat, broad with curved lateral edges. Scale bar = 2 pm.

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SEM of Echinostoma re*volutum in chick embryos

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FUJINO T., ISHII Y. & CHOI D. W. 1979. Surface ultrastructure of the tegument of Clonorchis sinensis newly excysted juveniles and adult worms. Journal of Parasitology 65: 579-590. HOOLE D. & MITCHELL J. B. 1981. Ultrastructural observations on the sensory papillae of juvenile and adult Gorgoderina viteliiloba (Trematoda : Gorgoderidae). International Journalfor Parasitology 11:41l-417. K#IE M., NANSEN P. & CHRISTENSEN N. 8. 1977. Stereoscan studies of rediae, cercariae, cysts, excysted metacercariae and migratory stages of Fasciola hepatica. Zeitschrift fiir Parasitenkunde 54: 289-297.

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MORRIS G. P. & THREADG~LD L. T. 1967. A presumed sensory structure associated with the tegument of Schistosoma mansoni. Journal of Parasitology 53: 537-539. NOLLEN P. M. & NADAKAVUKAREN M. J. 1974. Megalodiscus temperatus: scanning electron microscopy of the tegumental surfaces. Experimental Parasitology 36: 123-130. ZWILLINO E. 1959. A modified chorioallantoic grafting procedure. Transplantation Bulletin 6: 115-l 16.

FIGS. 15-18. Seven-day-old

chorioallantoic-worms.

FIG. 15. Whole body in ventral view. Scale bar = 1OO~m. FIG. 16. Enlarged

view of triple papillae

FIG. 17. Tegumentary

FIG. 18. Posterior

on the lip of the oral sucker.

spines behind the oral collar, which are elongated Scale bar = 2 pm. aspect of the body showing

small dispersed

FIGS. 19-21. Preovigerous FIG. 19. Acetabulum FIG. 20. Dorsal

FIG. 21. Ventral

tegumentary

tegumentary

with swollen rounded

Scale bar = 2 pm. with rounded

tips.

spines. Scale bar = 20 Mm.

chick-worms. papillae.

Scale bar = 5 pm.

spines behind the collar which show rounded tegument under the spines. Scale bar = 5 pm.

tips and a depression

spines behind the oral collar, which are flat, scale-like indented tips. Scale bar = 5 pm.

and wrinkled

in the

with