Key-words: Opisthorchis viverrini. Laos. Developmental stages. Surface morphology. Electron microscopy. Mots-clés : Opisthorchis viverrini. Laos. Stades larvaires. Mor­ phologie des téguments. Microscopie électronique.

Ann. Parasitol. Hum. Comp., 1992, 67: n° 3, 82-90. Mémoire.

STUDY ON THE SURFACE MORPHOLOGY OF THE DEVELOPMENTAL STAGES OF THE LIVER FLUKE, OPISTHORCHIS VIVERRINI (TREMATODA: OPISTHORCHIIDAE) T. SCHOLZ, O. DITRICH, M. GIBODA Summary ------------------------------------------------------------------------------

The external morphology of some developmental stages of the fluke Opisthorchis viverrini (Trematoda: Opisthorchiidae), parasi­ tizing humans in Southeast Asia was studied for the first time using electron microscopy. The surface structure of the egg, as

well as the rediae, cercaria, metacercaria, and adult found in natu­ rally infected hosts from Laos are described herein and their mor­ phological characteristics discussed.

R ésumé : Étude morphologique de la surface des stades larvaires de la douve hépatique Opisthorchis viverrini (Trematoda: Opisthorchiidae).

La morphologie externe des stades larvaires d'Opisthorchis viverrini (Trematoda: Opisthorchiidae) est étudiée pour la première fois

INTRODUCTION

en microscopie électronique. La morphologie superficielle des œufs, rédies, cercaires, métacercaires et adultes est décrite.

and have been observed in the scanning electron micro­ scope (SEM) by Suzuki (1977), Ditrich et al. (1990a), and Ditrich et al. (1992).

Current morphological studies widely use electron micros­ copy for observation of the fine surface structures of many digenetic trematodes, including those of medical importance (Miller et al., 1972; Bennet, 1975a, b ; Kuntz et al., 1976, 1977; Sakamoto and Ishii, 1977). These studies also deal with some members of medically important opisthorchiid flukes, the species Clonorchis sinensis (Cobbold) and Opis­ thorchis felineus (Rivolta) (Fujino et al., 1979; Li et al., 1988; Beer et al., 1990a, b). Another species of opisthorchiid fluke, Opisthorchis viver­ rini (Poirier, 1886) is endemic in some areas of Southeast Asia, i. e. Thailand (Sadun, 1955; Wykoff et al., 1965; Upatham, 1988), Laos (Segal et al., 1968; Giboda et al., 1991), and Malaysia (Bisseru and Chong, 1969). The life cycle of O. viverrini has already been detailed by Wykoff et al. (1965), and the morphology of the cer­ caria and metacercaria of this parasite has also been stu­ died in detail by Vajrasthira et al. (1961) and Wykoff et al. (1965). However, these studies were limited to obser­ vations by the light microscope. With the exception of the egg, there is no stereoscan study on the external morpho­ logy of any of the developmental stages of O. viverrini. The surface structures of the eggs play an important role in the determination of small fluke eggs in the human stool

The rediae and young cercariae were obtained by dissecting the snail species, Bithynia siamensis goniomphalus (Morelet) (Ditrich et al., 1992), which is the first intermediate host, and mature cerca­ riae shed by these snails were also used. Metacercariae, encysted in the flesh o f the cyprinid fish, Hampala dispar Smith (Scholz et al., 1990), were isolated from cysts by needles or by slight pressure from a coverglass. Adult trematodes containing eggs were obtained from the liver of autopsied domestic and stray cats (Felis catus L.) (Giboda et al., 1991). Mature eggs for SEM study were obtained from the proximal part of the uterus of the same flukes. Eggs were fixed directly in 4 % solution of paraformaldehyde in a phosphate buffer.

Institute of Parasitology, Czechoslovak Academy of Sciences, Branišovská 31, 370 05 České Budĕjovice, Czechoslovakia. Accepté le : 24 mars 1992.

(1) This study was a part of the project of bilateral scientifictechnical cooperation between Czechoslovakia and Laos « Studies on the epidemiology and morbidity o f some parasitic diseases in Laos ».

During the study on opisthorchosis (1) carried out in Laos, developmental stages of O. viverrini were obtained from naturally infected hosts (Ditrich et al., 1990b, 1992; Scholz et al., 1990; Giboda et al., 1991). The aim of the present study is to describe the external morphology of these stages using the scanning electron microscope (SEM) and to supplement this description by details of the inner structures of the tegument documented in the transmission electron microscope (TEM).

MATERIALS AND METHODS

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Article available at http://www.parasite-journal.org or http://dx.doi.org/10.1051/parasite/199267382

SURFACE M O RPH OLO GY OF THE D EVELOPM ENTAL STAGES OF OPISTHORCHIS

The rediae, cercariae and metacercariae were washed in water, transferred to a 0.1 M cacodylate buffer (pH 7.2) for 5 min, then fixed with 4 % glutaraldehyde in a cacodylate buffer, dehydrated with an alcohol series, transferred into aceton, mounted on stubs, dried using critical point method (CO2), and coated with gold. A Tesla BS-300 scanning electron microscope was used for the examination. For the transmission electron microscopical study (TEM), small pieces of the hepatopancreas from heavily infected snails were fixed with 2.5 % glutaraldehyde in 0.1 M cacodylate buffer and postfixed with 2 % OsO4. After dehydration in acetone, the material was embedded in Durcupan. Ultrathin sections were stained with uranyl acetate and lead citrate and examined with Philips EM 420 microscope.

RESULTS E gg Egg measuring 25.8-31.0 x 12.4-17.6 µm, operculated; operculum well developed (Figs. 1, 2), about 5-6 µm in dia­ meter. Outer surface of egg provided with net of ribs up to 5 µm long, forming typical musk-melon structure (Fig. 1). Wall of egg-shell surrounding operculum thickened, thus forming narrow, pronounced « shoulder » (Figs. 1, 2). Internal margins of aperture smooth, without any identation. In most eggs, apparent knob developed in abopercular end.

Cercaria Cercaria pleurolophocercous, pipe-form (concave on the ventral side and convex on the dorsal side), measuring 100-250 X 33-68 µm. Body surface covered with tegumental folds, either arranged as transverse depressions (Fig. 18) or panel-shaped (paved) (Fig. 16). Tegument armed with minute, single-pointed spines (Fig. 11), alternating with these folds. Size and shape of tegumental spines different in indi­ vidual parts of body; three transversal rows of preoral spine localized on dorsal lip of oral sucker (Fig. 12), nine other circumferential rows localized near oral sucker, in area with slightly folded tegument (Figs. 15 and 19); spines in these rows distinctly larger than spines of following circles, beco­ ming less dense and fine posteriorly (Fig. 18) and reaching backwards up to posterior extremity. In addition to tegu­ mental folds and spines, entire body surface, particularly its posterior part, covered with very small, round globules (Fig. 18); long-ciliated sensory endings, 8-12 µm long, arranged in two lateral longitudinal rows, each formed by at least 5-6 cilia (Figs. 17, 18). Oral sucker slightly subterminal, 36-41 µm in diameter; ventral sucker underdeveloped, sphaerical, in form of small, protruded papilla-like elevation (Fig. 13). Tail, measuring 360-475 x 16-29 µm , equipped with wide dorsoventral finfold, dorsally beginning at the first third of tail length and ventrally terminating in middle of tail (Figs. 20, 21). Ante­ rior third of tail with transversaly striated tegument.

Rediae Mother redia very long (up to 0.8 mm), narrow, sac-like (Fig. 4); daughter redia spindle-shaped or elongate (Figs. 5, 8). Body surface covered with tegument forming circumferential folds, mostly in anterior and posterior parts of body. In addition, tegument of anterior part carpeted with dense microvilli (Figs. 3, 6); surface of middle region and hindbody covered with reticular structure, formed by long, fine tegumental lamellae, and irregularly scattered numerous globular projections (Fig 7). Lamellae surroun­ ding posterior extremity very dense, thus resembling in their appearance microvilli around oral aperture. Transverse sec­ tions through tegumental lamellae visible under transmis­ sion electron microscope (illustrated in Figs. 9 and 10). These photomicrographs also show structure and arrange­ ment of muscular layers (bundles). Oral aperture and anterior part of redial body encircled by several circles of numerous uniciliate sensory endings with short cillium. Inner circles (3-4 in number), close to oral aperture, formed by smaller endings; outer circles (at least 8 in number), consisting of somewhat larger shortciliated structures becoming less dense posteriorly (Fig. 6). Posterior end of body bearing invagination resembling in shape protrusible papilla and long-ciliated sensory endings grouped irregularly near posterior extremity. Daughter redia with partly released cercaria illustrated in Fig. 8.

M etacercaria Metacercaria, released from cyst, elongated, measuring 320-820 x 100-140 µm (Fig. 22), and covered with tegu­ ment formed by narrow transverse depressions (ridges) (Figs. 24, 25). Entire body, excluding area around suckers and posterior extremity, armed with spines of different size, shape and arrangement in individual body regions (Fig. 23). Forebody encircled by circumferential rows of dentated spines, about 1.0-1.5 µm long, equipped with bluntly ended teeth (mostly 3-4) (Fig. 24); spines becoming posteriorly smaller and less densely arranged. Body behind ventral sucker covered with simple, sharply-pointed spines, mea­ suring up to 1.7 µm, reaching towards posterior extremity, very dense around posterior margin of ventral sucker and becoming finer and less numerous further back. Large papilla-like tubercles scattered throughout body in irregular, longitudinal rows, reaching to level of ventral sucker (Figs. 22, 24). Suckers well developed, with oral sucker placed subterminally and measuring 48-80 X 4580 µm ; large ventral sucker (46-85 x 48-82 µm in size) lying slightly behind midline of body (Fig. 17). Lips of both suc­ kers encircled with papillae and papilla-like tubercles. Oral sucker possessing outer circle of papilla-like structures formed by six low swellings on outer margin of sucker lip; indistinct small papillae situated on inner edge of sucker 83

T. SCHOLZ, O. DITRICH, M. GIBODA

F igs. 1-8. — Opisthorchis viverrini (Poirier, 1886). Fig. 1. Egg-shell with musk-melon structure and well-developed operculum (magnifica­

tion x 4,000). Fig. 2. Egg-shell after hatching of miracidium (X 4,600). Fig. 3. Anterior end of mother redia ( x 1,500). Fig. 4. Total view of mother redia ( x 160). Fig. 5. Total view of daughter redia (x 180). Fig. 6. Oral aperture of daughter redia encircled with several rows of uniciliate papilla-like structures ( x 2,850). Fig. 7. Detail of body surface of daughter redia ( x 5,900). Fig. 8. Daughter redia releasing cercaria (c) (x 760).

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lip. Ventral sucker also equipped with two circles of papillalike structures (Fig. 23). Outer circle formed by six papillalike tubercles; papillae o f inner circle indistinct and their

number were not determined. In addition, two small papillae localized on anterior margin of lip of ventral sucker

(Fig. 23).

F igs. 9-13. — Opisthorchis viverrini (Poirier, 1886). Fig. 9. Sagittal section through outer part of redial body with tegumental lamellae (tl);

bm — basal membrane, lm — longitudinal muscles, n — nucleus, te — tegument (magnification x 18,750). Fig. 10. The same, enlarged; cm — circular muscles, lm — longitudinal muscles, n — nucleus, te — tegument, tl — tegumental lamellae (x 23,600). Fig. U . Sagittal section through superficial part of cercarial body. Note tegumental spines (sp), tegument (te), circular muscles (cm), and multified nuclei (n) ( x 8,800). Fig. 12. Anterior part of cercaria with cephalic spines (csp) (x 3,300). Fig. 13. Ventral side of cercarial body with elevated, protrusion-like ventral sucker (vs) ( x 2,700).

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F igs. 14-25. — Opisthorchis viverrini (Poirier, 1886). Fig. 14. Total view of cercaria ( x 240). Fig. 15. Anterior end of cercaria; dorsally

(X 4,200). Fig. 16. Posterior end of cercaria ( x 1,650). Fig. 17. Lateral margin of cercaria with cilium-like structures ( x 1,200). Fig. 18. Detail o f long cilia on cercarial body (x 4,500). Fig. 19. Spines encircling anterior extremity (x 4,600). Fig. 20. Middle part of cercarial tail (x 850). Fig. 21. Posterior part of cercarial tail (x 1,850). Fig. 22. Total view of metacercaria (x 410). Fig. 23. Ventral sucker of metacercaria with anteriorly situated papillae (arrows) (x 1,500). Fig. 24. Scale-like tegumental spines of anterior part of metacercarial body (x 4,900). Fig. 25. Single-pointed tegumental spines of postacetabular region of metacercarial body (x 4,750).

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T. SCHOLZ, O. DITRICH, M. GIBODA

Adult worm Body lanceolate, flattened, tapering anteriorly and its length being about 2.0-4.5 mm. Body surface smooth, spi­ neless, covered with tegument forming narrow, irregularly arranged transverse depressions (folds) (Fig. 26). Surface of tegument formed by small, knob- or beadlike protube­ rances of rather similar shape and size (Fig. 30). In addi­ tion, body covered with button-like, randomly scattered papillae (Fig. 31) which were doubled or provided with short cilia. Oral sucker subterminal or nearly terminal; ventral sucker oval or nearly round (Fig. 27), lying between first and second thirds of body. Both suckers encircled by nume­ rous button-like papillae or papilla-like tubercles. Oral sucker surrounded by numerous papillae of different size and types—simple papillae with or without cilia; small, collar-likes structures provided with short and wide ending; or low swellings. Outer edge of sucker lip equipped with several papilla-like swellings; its inner margin possessing sharply pointed simple papillae, some of them being grouped together, forming pairs (Fig. 28). Small pores (ope­ nings of glands?) visible on anterior margin of oral sucker. Region around ventral sucker also encircled with nume­ rous, irregularly arranged papillae (Fig. 27). Outer margin of sucker’s lip surrounded with slightly elevated papilla­ like tubercles; inner margin with five indistinct small papillae. Genital pore, lying closely to anterior margin of ventral sucker, equipped with group of finger-like projections (Fig. 29).

DISCUSSION

Electron microscopical studies on the tegumental struc­ tures of digenetic trematodes have been widely used to pro­ vide valuable data about their morphology. This data can facilitate the identification of some fluke species or their developmental stages, and is necessary during field studies on the occurrence and epidemiology of medically impor­ tant flukes. For example, the morphological differences bet­ ween the metacercariae of Paragonimus westermani (Her­ bert) and P. miyazakii Kamo, Nishida, Hatsushika et

Tomimura, observed by Higo and Ishii (1984) in SEM, were suggested as suitable examples for observing the dis­ tinctions. However, stereoscan studies describing the external mor­ phology of some medically important trematodes are rather scarce. This is in spite of the fact that there are a lot of papers dealing with this problem in other trematodes. The scarcity of stereoscan observations concerns also the fluke, Opisthorchis viverrini, a frequent parasite of humans in South East Asia. Consequently, electron microscopy has been used for the first time to discern the surface morpho­ logy of some of the developmental stages (egg, rediae, cercaria, metacercaria, adult) of this fluke for this present paper. The current study revealed that the structure (which is musk-melon) covering O. viverrini eggs resembled that of the O. felineus flukes originated from the eastern parts of the distribution area of this species (Beer et al., 1990b). The surface structure of the eggs of all hitherto studied opisthorchiid trematodes is considered to play an impor­ tant role for the hatching or miracidium after digestion of eggs by snails from the family Bithyniidae (Beer et al., 1990a), which are the first intermediate hosts. As far as we know, there is no stereoscan study on the surface morphology of the rediae of trematodes from the family Opisthorchiidae; only Wykoff et al. (1965) gave some limited data about the gross morphology of the O. viverrini redia (i. e. the size of the body possessing the pharynx, the absence of an annulus, thickness of the tegu­ ment, etc.). The structure of the tegument of the O. viver­ rini rediae—which consists of circumferential tegumental folds carpeted with microvilli and microvillus-like projec­ tions or fine tegumental lamellae—somewhat resembles that of the rediae of the Cryptocotyle lingua (Creplin), Fas­ ciola hepatica L. and Mesorchis denticulatus (Rud.) as des­ cribed by Krupa et al. (1967), KØie et al. (1977) and KØie (1987). The arrangement of the tegumental microvilli and lamellae covering the surface of the O. viverrini rediae was clearly visible in TEM photomicrographs and corres­ ponded to the structure of the tegumental layer described by Krupa et al. (1967) in the rediae of C. lingua. The rediae of all the above mentioned trematodes also possess sen­ sory structures with ciliae similar to those of O. viverrini redia. However, it clearly differs from those of F. hepa-

Figs. 26-31. — Opisthorchis viverrini (Poirier, 1886). Fig. 26. Total view of adult worm (x 55). Fig. 27. Ventral sucker of adult with papillae (arrows) and genital pore (X 430). Fig. 28. Oral sucker of adult with papillae (arrow) inside sucker’s lip (X 980). Fig. 29. Genital pore of adult with finger-like projections and papilla-like swelling on inner edge of ventral sucker’s lip (arrow) (x 1,200). Fig. 30. Detail of body surface of adult with furrowed tegument and doubled papilla-like tubercles (x 1,050). Fig. 31. Enlarged view of body surface with single tubercle (x 4,800). (All photomicrographs were made at the Laboratory o f Electron Microscopy, Czechoslovak Academy o f Sciences, České Budĕjovice).

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SURFACE M O RPH OLO GY OF THE DEVELOPM ENTAL STAGES OF OPISTHORCHIS

tica and M. denticulatus because their bodies lack locomotory appendages or collars covered with knobs. The gross morphology of the O. viverrini cercaria has already been described in detail by Wykoff et al. (1965) and by Ditrich et al. (1992). The present stereoscan study on the surface structure of the O. viverrini cercaria fully confirms data given by the above mentioned authors. In addition, this study revealed that the tegument of the O. viverrini cercaria was folded into structures of two types—circumferential (transverse) striations (ridges) or panel-shaped structures. The ventral sucker, under the light microscope, was practically indistinguishable (see Wykoff et al., 1965; Ditrich et al., 1992), whilst under the SEM, it was observed as a small, elevated structure (protuberance). Wykoff et al. (1965) observed the presence of minute tegumental spines covering the surface of the O. viverrini cercaria under the light microscope. The present study has enabled us to describe more precisely their shape and arran­ gement, including those of the cephalic spines. Wykoff et al. (1965) reported these spines as small, toothlike structures. Cephalic spine similar to those of the O. viverrini cercariae are also common in the cercariae from members of other trematode families, e. g., Heterophyidae, Diplostomidae, Sanguinicolidae (KØie, 1977; Simon-Martin and Gomez-Bautista, 1986; Niewiadomska and Našincová, 1990; Ditrich et al., 1992). The arrangement, shape and number of these spines, probably acting as a « rasp » to break down the fish skin (KØie, 1977), serve as an important differential characteristic for species identification of some representatives of medically important flukes (Martin, 1958). Vajrasthira et al. (1965), studying the morphology of the O. viverrini metacercariae under the light microscope, mentioned the presence of small, scaly spines covering their surface and becoming finer and fewer posteriorly. However, the shape of these spines was not accurately described by the above-mentioned authors. The present electron micros­ copical observations revealed that dentated spines covering the tegument of the forebody of the O. viverrini metacercaria, somewhat resembled those covering the posterior part of the body of some heterophyid flukes (KØie, 1977; Scholz et al., 1991). However, the spination of the hindbody of the O. viverrini metacercariae distinctly differs from that of the heterophyids being formed by simple, single-pointed spines.

The presence of different type of tegumental spines on the metacercarial body seems to be a typical feature of opisthorchiid flukes because a similar arrangement and shape of these spines were observed in newly excysted juve­ niles from the Clonorchis sinensis (Fujino et al., 1979; Li

clearly ventrolateral. Vajrasthira et al. (1965) also found six papillae surrounding the oral opening, which tallies with our observations. These authors reported nine papillae encir­ cling the ventral sucker; six forming the outer circle and three the inner one. This fact could not be confirmed in the present study because the precise number of these indis­ tinct papillae could not be determined. However, Fujino et al. (1979) and Li et al. (1988), in accordance with results of Vajrasthira et al. (1965), found six round swellings on the outer edge of the lip and some (number not counted) ciliated papillae inside the lip of the C. sinensis excysted juveniles. The presence of six large and low swellings sur­ rounding the lip of the ventral sucker were also found in newly excysted Paragonimus juveniles (Higo and Ishii, 1984). The surface of the O. viverrini adult, visible under the scanning electron microscope with low magnification, seems to be smooth, spineless, thus generally resembling that of the Dicrocoelium dendriticum (Rud.) (Cifrian and GarciaCorrales, 1988). However, the fine structure of the tegu­ ment is quite different because the O. viverrini completely lacks the tegumental vesicles typical of the D. dendriticum (Cifrian and Garcia-Corrales, 1988). The surface topography of the O. viverrini adult corresponds to that of the C. sinensis, as described by Fujino et al. (1979), including the structure of the tegument (forming knob-like protube­ rances) and arrangement of papillae around the suckers. Nevertheless, we did not observe finger-shaped or filiform projections extended from the surface of the tegumental knobs of the C. sinensis (Fujino et al., 1979). On the other hand, the above mentioned authors did not describe the finger-like projections surrounding the genital pore. The data about the surface morphology of O. viverrini, presented here for the first time and obtained during elec­ tron microscopical study of developmental stages of this medically important parasite, may contribute to current knowledge about the comparative morphology of digenetic trematodes and can be used in the evaluation of their phylogenic relationships. Acknowledgements. — The authors are indebted to the staff of the Institute of Malaria and Parasitic Diseases, Vientiane, and the authorities of the water plant station Nam Ngum, in Ban Thinkeo, for their valuable help during field observations which enabled us in our studies. Thanks are also due to Dr. J. G u t virth , Regional Hospital, České Budĕjovice, Dr. V. NAšincová and Dr. Z. Ž darskÁ, both Institute of Parasitology, and to Mr. M. Tůma and Mrs. V. N ovÁkov Á, the staff of the Laboratory of Electron Microscopy, Czechoslovak Academy of Sciences, České Budĕjovice, for valuable help and advice.

et al., 1988).

REFERENCES

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Study on the surface morphology of the developmental stages of the liver fluke, Opisthorchis viverrini (Trematoda: Opisthorchiidae).

The external morphology of some developmental stages of the fluke Opisthorchis viverrini (Trematoda: Opisthorchiidae), parasitizing humans in Southeas...
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