comparative studies on two similar species of haplorchis...

Proc. Helminthol. Soc. Wash.56(1), 1989, pp. 35-41

Comparative Studies on Two Similar Species of Haplorchisand Metagonimus (Trematoda: Heterophyidae)—Surface Ultrastructur e of Adults and Eggs

T. FUJINO,1 H. HlGO,1 Y. ISHII,1 S. SAITO,2 AND E. R. CHEN3

1 Department of Parasitology, Faculty of Medicine, Kyushu University, Fukuoka 812, Japan,2 Department of Parasitology, Faculty of Medicine, Yamagata University, Yamagata 990-23, Japan, and3 Department of Parasitology, Kaohsiung Medical College, Kaohsiung, Republic of China

ABSTRACT: The surface ultrastructure of adults and eggs of Haplorchis pumilio and H. taichui, and of Meta-gonimus yokogawai and M. takahashii, are each compared by scanning electron microscopy. The adults of thesespecies bear scalelike spines with a posterior serration. It is difficul t to distinguish the similar species by theirsurface ultrastructure, such as the shape of the tegumental spines, the number and localization of sensory papillae,and the shape of the opening of the ventrogenital complex. The eggs of H. pumilio which bear surface granulartubercles are different from those of H. taichui which are characterized by slender coiled projections. The eggsof both Metagonimus species are marked by granular tubercles on the surface, and they appear difficul t to separatefrom each other.

KEY WORDS: Haplorchis pumilio, H. taichui, Metagonimus yokogawai, M. takahashii, Heterophyidae, ultra-structure, scanning electron microscopy, adult worms, eggs, heterophyidiasis.

Adult trematodes in the family Heterophyidaeare small intestinal parasites of birds and mam-mals including man. Besides species in the genusHeterophyes producing heterophyiasis, mem-bers of this family include other medically im-portant species in the genera Haplorchis and Me-tagonimus. Two species of Haplorchis, If. pumilio(Looss, 1896) and H. taichui (Nishigori, 1924),whose lif e cycles and morphologies were de-scribed by Faust and Nishigori (1926) and Pear-son (1964), have similar morphological features.Two species of Metagonimus, M. yokogawai(Katsurada, 1912) and M. takahashii Suzuki,1930, are also similar to each other and remainedundefined specifically, although Takahashi (1929)and Ochi (1957) showed some morphological andbiological differences between them. Recentstudies showed that they are separate species(Takahashi, 1967; Saito, 1972, 1973; Saito andTsuji, 1973).

Recent investigations using scanning electronmicroscopy (SEM) have revealed the surface ul-trastructure of several trematodes (Fujino et al.,1979) and eggs (Ishii and Miyazaki, 1971). Withrespect to the species of Haplorchis and Meta-gonimus there have so far been no reports of SEMwork except for the comparative study of cer-cariae of M. yokogawai and M. takahashii (Fu-jino et al., 1976) and observations on eggs of M.yokogawai (Ishii and Miyazaki, 1971; Ishii, 1972).

The present investigation was undertaken tocompare using SEM the surface ultrastructure of

the adult worms and eggs of 2 similar species ofeach of the genera Haplorchis and Metagonimus;H. pumilio and H. taichui, and M. yokogawaiand M. takahashii.

Material s and Methods

The metacercariae of Haplorchis pumilio were re-moved from the natural freshwater fish host, Hemi-culter leucisculus, at Chenching Lake, KaohsiungCounty, southern Taiwan, and the adults were re-covered from a golden hamster 7 days after oral in-oculation. The metacercariae of//, taichui, taken fromunidentified freshwater fish in Suimen Village, Santi,Pingtung County, southern Taiwan, were inoculatedinto a golden hamster and the adults were recovered14 days later. The metacercariae of Metagonimus yo-kogawai, obtained from the muscle of the freshwaterfish, Plecoglossus altivelis, in the Ota River, HiroshimaCity, central Japan, were inoculated into a golden ham-ster which yielded adults 14 days later. Metagonimustakahashii adults were taken from a golden hamster10 days after inoculation of the metacercariae whichwere removed from the freshwater fish, Carassius ca-rassius, taken at the Toyano Lake, Niigata City, north-ern Japan.

For SEM preparation adult worms and eggs whichwere naturally shed were rinsed well with Ringer's so-lution. The specimens were then fixed with phosphatebuffered (pH 7.3) 4% glutaraldehyde for 2-4 hr, fol-lowed by 2 hr postfixation in 1% osmium tetroxide.They were dehydrated with an ethanol series, and thencritical-point dried. After coating with gold, the spec-imens were examined under a JEOL-U3 scanning elec-tron microscope at 15 kV. Ten specimens each wereexamined, measured, and their averages were taken.

For the examination of eggs by transmission electronmicroscopy (TEM), part of the uterus was dissected

35

Copyright © 2011, The Helminthological Society of Washington

36 PROCEEDINGS OF THE HELMINTHOLOGICAL SOCIETY


OF WASHINGTON, VOLUME 56, NUMBER 1, JANUARY 1989 37

and fixed for 3 hr in Karnovsky's (1965) fixative. Aftera wash in cacodylate buffer the material was osmicated,dehydrated, and embedded in Spurr Resin or Quetol812 (Nisshin EM, Tokyo). Cut sections were viewedin a Hitachi HS-9 electron microscope at 75 kV.

Results

Adul t worms

Haplorchispumilio resembled H. taichui in thesurface ultrastructure of the body. The body wasoblong or slightly elongate, and encircled by rowsof flat, oval, or scalelike spines with a serratedposterior edge (Figs. 1, 3-6). The spines mea-sured approximately 2.0 ^m long by 2.3 ^m inH. pumilio (Figs. 3, 5, 6), and 2.0 ^m long by2.5 um in H. taichui (Fig. 4). The surface of thespines was almost flat with 6-8 shallow furrowsextending from near the basal part of the spineto the distal edge. The spines near the middle ofthe body were larger than the anterior and pos-terior ones. The posterior spines were short andnarrow and simple or double- or triple-pointed(Figs. 5, 6). There were no spines around theexcretory pore. No marked difference was foundin shape and size between the dorsal and ventralspines. There were many sensory papillae, eachwith a short cilium, on the lip of and around theoral sucker. Two, 3, or 4 papillae often occurredclose together. Several pairs of papillae were ar-ranged bilaterally dorsally and ventrally. Theopening of the ventrogenital complex, which wasdevoid of spines, appeared as a round or slitlikedepression of the tegument in about the anteriorthird of the body (Fig. 2).

Metagonimus yokogawai and M. takahashiiwere similar to each other in the surface finestructure of the body. The body was flat and ovalor pyriform, and was covered with flat, oblongor oval spines with a serrated posterior end (Figs.7-12). The spines measured approximately 1.5/urn long by 1.7 yum in M. yokogawai and 1.5 /umlong by 1.5 /um in M. takahashii (Figs. 11, 12).The posterior serration of the spines was morebluntly pointed in M. takahashii than in M. yo-

kogawai. The spines were smaller and narrowerand simple or double-pointed posteriorly, andno spines were present around the excretory pore.The ventrogenital complex occurred in the an-terior third of the body (Fig. 7), and the openingappeared as a round swelling directed anteriorly(Fig. 8). Small knobbed protuberances and sen-sory papillae were scattered on or around the lipof the oral sucker (Fig. 9). Sensory papillae werearranged bilaterally in the anterior half of thebody dorsally and ventrally. Each papilla had ashort cilium.

Eggs

The eggs of Haplorchis pumilio, measuring 22-23 /vim long by approximately 10 /um, taperedtoward the abopercular end which had a tuber-cular nodule. The operculum was prominent. Thesurface of the eggshell including the operculumwas coarse with minute tubercles measuring 0.2-0.3 yum by 0.1 /am (Fig. 13). There were longi-tudinal irregular ridges of various lengths whosesurfaces were also tuberculate. TEM showed ir-regular minute tubercles on the egg surface withoccasionally raised extensions or projectionswhose surface was tuberculate (Fig. 14). The bas-al part of the operculum and the shell edge bor-dering the operculum were raised to formlow "shoulders."

The eggs of H. taichui, measuring approxi-mately 18 ^m by 8 /urn, were smaller than thoseof H. pumilio and the abopercular end wasbroader than in H. pumilio. The egg surface wasmarked by irregularly coiled ropelike ridges, eachof which was approximately 0.1 /um wide (Fig.15). The edge of the eggshell bordering the oper-culum and the basal part of the operculum wereslightly raised, but not forming "shoulders." Thenarrow area along the eggshell edge was devoidof ridges. TEM showed the presence of irregu-larly curved slender projections covered with afilamentous coat (Fig. 16).

The eggs of Metagonimus yokogawai, mea-

Figures 1-6. 1. Haplorchis pumilio. Dorsal aspect of the body. The body is flattened, elongate, and spinousalmost all over the surface. Spermatozoa from Laurer' s canal opening are seen (arrowhead). Bar = 20 fim. 2. H.pumilio. The opening of the ventrogenital complex. No spines are present around the opening. Bar = 2.0 jim . 3.H. pumilio. Spines of middorsal area. The spines are scalelike with longitudinal furrows and a serrated posterioredge. Bar = 1.0 um. 4. H. taichui. Spines of middorsal area. The spines are similar to those of H. pumilio. Bar= 1.0 /tm. 5. H. pumilio. Spines of posteroventral area. The spines are narrower than those of middorsal area.Bar = 1.0 pm. 6. H. taichui. Spines of posteroventral area. The spines are simple or double-pointed. The tegumentalsurface is granular. Bar = 1.0 pm.



suring about 20 /urn by 12-13 jon, were ellipsoi-dal with a broad abopercular end which had asmall knoblike projection occasionally. The eggsurface was coarse with small granular tuberclesmeasuring 0.2 /tm by 0.1 /j-m (Fig. 17). The oper-culum was prominent. The basal part of the oper-culum and the shell edge were raised to formnarrow ridges. The eggs of M. takahashii resem-bled those of M. yokogawai, being slightly longerand wider than M. yokogawai. The surface fea-tures of M. takahashii were very close to thoseof M. yokogawai except that the granular tuber-cles appeared more conspicuous than those ofM. yokogawai (Fig. 18).

Discussion

The adults of 2 species of Haplorchis and ofMetagonimus are revealed to be very similar toeach other under SEM observations. All of thespecies examined bear scalelike tegumental spineswith a serrated posterior edge. Similar spines arereported in the heterophyid Cryptocotyle linguaby K0ie (1977). It seems possible that species ofthis family commonly bear this type of spine.Heterophyid worms are known to inhabit theintestines of birds and mammals (Faust and Ni-shigori, 1926). These serrated spines on the bodysurface probably help the abrasion of host tissuefor feeding and also function in attachment tothe host or for easy movement among the intes-tinal villi (K0ie, 1977).

Although it is hard to distinguish M. pumiliofrom H. taichui by the shape of the tegumentalspines, the shape and localization of the sensorypapillae, and the opening of the ventrogenitalcomplex, eggs of the 2 species differ markedly.

The Metagonimus species, M. yokogawai andM. takahashii, are also very similar in the surfaceultrastructures of the adults except for a slightdifference in the serration of the tegumentalspines. Unlike the eggs of the Haplorchis species,the eggs of the Metagonimus species are so sim-ilar that it seems difficul t to distinguish them

from each other. Saito (1972) compared both ofthe species in their developmental stages, cer-caria, metacercaria, and adult, including eggs,under the light microscope. He found differencesin the number of oral spines and the shape ofthe penetration gland cells of the cercaria, thebody color of the metacercaria, and the body sizeof all the stages. Fujino et al. (1976) confirmedwith SEM observations of the cercariae that thenumber and size of the oral spines are the mostdistinguishing characteristics and that other fea-tures are too similar to use in separating thespecies from each other.

Africa et al. (1935, 1936a, b, 1937) reportedthat some heterophyids including H. taichui andH. pumilio were recovered from human intes-tines and that their eggs caused cardiac, cerebral,or spinal heterophyiasis. In these cases, the eggshave been found associated with lesions in theorgans. Africa et al. (1935, 1936a) mentionedthat heterophyid eggs plugged capillaries andother vessels and caused rupture of these vessels.It seems possible that the eggs of these species,which are comparatively small, are easily carriedby the blood stream from the intestine to theabove-mentioned regions. The egg surfaces of//.taichui and H. pumilio, as observed in SEM, arecoarse with surface granular tubercles and slen-der coiled projections, respectively. These struc-tures of the eggs may be responsible for the em-bolism of eggs in the capillaries of various organs.

Acknowledgment

We thank Dr. M. K0ie of the Marine BiologicalLaboratory, Helsingor, for her interest and crit-ical reading of the manuscript.

Literatur e Cited

Africa, C. M., W. De Leon, and E. Y. Garcia. 1935.Heterophyidiasis: II. Ova in sclerosed mitral valveswith other chronic lesions in the myocardium.Journal of the Philippine Islands Medical Asso-ciation 15:583-592.

Figures 7-12. 7. Metagonimus yokogawai. Ventral aspect of the body. The body is flattened, elongate, ovalor pyriform , and spinous. The ventrogenital complex is marked (arrowhead). Bar = 50 jim. 8. M. yokogawai.Enlarged view of the opening of the ventrogenital complex. The swollen wall of the complex is covered withspines and the opening is directed anteriorly. Bar = 10 /am. 9. M. yokogawai. Grouped ciliated papillae near theoral sucker. Bar = 1.0 tan. 10. M. yokogawai. Spines of middorsal area. The scalelike spines have a finelyserrated posterior edge. Bar = 1.0 jim. 11. M. takahashii. Spines of middorsal area. The posterior edge is serratedbluntly . Bar = 1.0 /urn. 12. M. takahashii. Spines of posteroventral area. The spines are simple or double-pointed.Bar = 1.0 fim.



Figures 13-18. 13. Haplorchis pumilio. The operculum and the distal part of the eggshell. Note "shoulders"of the basal part of the operculum and eggshell edge (small arrowheads). The eggshell is marked by minutetubercles all over the surface. Longitudinally running irregular ridges are characteristic. Bar = 1.0 tan. Inset:Enlarged view of the shell surface. An arrowhead indicates a tubercular ridge. Bar = 0.5 jtni. 14. H. pumilio.TEM of part of the eggshell showing irregular minute tubercular projections and longer extensions (arrowhead)on the surface. Bar = 0.5 jim. 15. H. taichui. The operculum and part of the eggshell. The surface is characterizedby irregularly coiled ridges. "Shoulders" are inconspicuous. Bar = 1.0 jtin. Inset: Enlarged view of the eggshellsurface. Bar = 0.5 fan. 16. H. taichui. TEM of part of the eggshell showing irregularly curved projections witha filamentous cover. Bar = 0.5 pm. 17. Metagonimus yokogawaL The operculum and part of the eggshell. The



and 1936a. Heterophyidi-asis: III . Ova associated with a fatal hemorrhagein the right basal ganglia of the brain. Journal ofthe Philippine Islands Medical Association 16:22-26.

-, and . 1936b. Heterophyidi-asis, V. Ova in the spinal cord of man. PhilippineJournal of Science 62:393-399.

-, and . 1937. Heterophyidiasis:VI . Two more cases of heart failure associated withthe presence of eggs in sclerosed valves. Journalof the Philippine Islands Medical Association 17:605-609.

Faust, E. C., and M. Nishigori. 1926. The lif e cyclesof two new species of Heterophyidae, parasitic inmammals and birds. Journal of Parasitology 13:91-128.

Fujino, T., Y. Ishii , and D. W. Choi. 1979. Surfaceultrastructure of the tegument of Clonorchis si-nensis newly excysted juveniles and adult worms.Journal of Parasitology 65:579-590.

, , and S. Saito. 1976. Studies on thecercariae of the genus Metagonimus with the scan-ning electron microscope (Trematoda: Hetero-phyidae). Japanese Journal of Parasitology 25:175-185.

Ishii , Y. 1972. Morphology of helminth ova throughthe scanning electron microscope. Fukuoka ActaMedica 63:419^131.

, and I. Miyazaki . 1971. Morphology of hel-minth ova revealed by the scanning electron mi-croscope. Chinese Journal of Microbiology 4:123-131.

Karnovsky, M. J. 1965. A formaldehyde-glutaral-

dehyde fixative of high osmolality for use in elec-tron microscopy. Journal of Cell Biology 27:137A-138A.

ie, M. 1977. Stereoscan studies of cercariae, meta-cercariae, and adults of Cryptocotyle lingua (Crep-li n 1825) Fischoeder 1903 (Trematoda: Hetero-phyidae). Journal of Parasitology 63:835-839.

Ochi, G. 1957. Studies on Metagonimus species inJapan. Tokyo Iji-Shin-Shi 74:591-599.

Pearson, J. C. 1964. A revision of the subfamilyHaplorchinae Looss, 1899 (Trematoda: Hetero-phyidae). I. The Haplorchis group. Parasitology54:601-676.

Saito, S. 1972. On the differences between Metagon-imus yokogawai and Metagonimus takahashii. I.The morphological comparisons. Japanese Jour-nal of Parasitology 21:449^158.

. 1973. On the differences between Metagon-imus yokogawai and Metagonimus takahashii. II .The experimental infections to the second inter-mediate hosts. Japanese Journal of Parasitology22:39-44.

-, and M. Tsuji. 1973. Immunoelectrophoreticcomparisons of the rediae-cercariae of Metagon-imus yokogawai and Metagonimus takahashii.Medical Journal of Hiroshima University 21:19-22.

Takahashi, S. 1929. On the life-history of Metagon-imus yokogawai, a new species of Metagonimusand Exorchis major. Okayama-Igakkai-Zasshi 41:2687-2755.

. 1967. Studies on genus Metagonimus. Oka-yama-Igakkai-Zasshi 79:43—49.

surface is coarse with round or oval granules. Bar = 1.0 /im. Inset: Enlarged view of the eggshell. Bar = 0.5 /im.18. M. takahashii. The operculum and part of the eggshell. The surface is coarse with round or oval granules.The opercular base and the edge of the eggshell are slightly raised. Bar = 1.0 /um. Inset: Enlarged view of theeggshell. Bar = 0.5 pm.


comparative studies on two similar species of haplorchis...

Documents