INTRODUCTION
After long debates on the species of the genus Metagonimus (Takabayashi, 1953; Ito, 1964; Saito, 1984), Metagonimus miyatai was reported as a new species (Saito et al., 1997). The validity of M. miyatai, however, should be studied further until it is fully recognized. In this connection, a genetic study using polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) analysis revealed that Metagonimus Miyata type (= M. miyatai), together with M. takahashii, are genetically distinct species from the type species, M. yokogawai (Yu et al., 1997).
Surface ultrastructure of trematodes has been studied to understand not only their morphological and functional characteristics but also taxonomic significance of each species; Cryptocotyle lingua (Køie, 1977), Heterophyes aegualis (Taraschewski, 1984), M. yokogawai (Lee et al., 1984), Neodiplostomum seoulense (Lee et al., 1985), Heterophyopsis continua (Hong et al., 1991) and Heterophyes nocens (Chai et al., 1992). These studies have revealed that their surface ultrastructures vary by species especially in terms of size, shape, number and distribution of tegumental spines and sensory papillae, and differentiation patterns of cytoplasmic processes according to developmental stages.
Surface ultrastructure of M. miyatai, however, has not been reported. The present study was performed to observe the surface ultrastructure of M. miyatai metacercariae and adults, and results were compared with those reported for M. yokogawai (Lee et al., 1984).
MATERIALS AND METHODS
Metacercariae of M. miyatai were collected from the scale of the pale chub (Zacco platypus) caught from Umsong-gun, Chungchongbuk-do, by artificial digestion technique. Two hundred metacercariae were fed orally to each of 5 Sprague-Dawley rats. The rats were killed at 1, 2 and 4 weeks post-infection and adult flukes were recovered. Metacercariae were excysted in trypsin solution (Tris buffer, pH 8.0) at 38℃. Excysted metacercariae and adults were washed several times with 0.2 M cacodylate buffer (pH 7.3) and fixed in 2.5% glutaraldehyde solution at 4℃. After washing with phosphate-buffered saline, they were dehydrated through a graded series of ethanol (60%, 70%, 80%, 90%, 95% and 100%), dried in a critical point dryer, and mounted on aluminum stubs. They were coated with gold using an ion sputtering coater (IB-3, Giko Engineering Co., Japan) and observed with a scanning electron microscope (ISI DS-130C, Korea) at an accelerating voltage of 10 kV.
RESULTS
1. Excysted metacercariae
Excysted metacercariae was leaf-like, ventrally concave, ovoid or pyriform in shape (Fig. 1). The oral sucker was big, locating near the anterior end of the body (Fig. 2). The ventral sucker was positioned in the right middle field of the body, facing toward the oral sucker. The whole body surface was covered with flat cytoplasmic processes, numerous tegumental spines (Figs. 1-3) and sensory papillae. The lip of the oral sucker was devoid of spines (Fig. 2) and had single or grouped type I sensory papillae (= ciliated knob-like swellings) and 7 type II sensory papillae (= round swellings of the tegument). Several type I papillae were also seen on the lip of the ventral sucker (Fig. 4). Tegumental spines anterior to the ventral sucker were dense and digitated into 5-7 points (Fig. 3). However, the number of points decreased laterally and posteriorly (Fig. 5). Near the posterior end of the body excretory pore was opened (Fig. 6), spines were sparsely distributed and digitated only into 2-3 points.
The dorsal surface was also densely and regularly covered with tegumental spines. Spines were, however, short, blunt, and 5-6 pointed anteriorly, and 6-8 pointed in the middle portion of the body. Toward the posterior half of the body, spines were converted into single-pointed peg-like structures, and the distance between spines was increased. Type I single or clumped (groups of 2-3) papillae were sparsely distributed.
2. Adult flukes
One-week old worms: The body was similar to metacercariae in shape, but became much enlarged, and covered with knob- or cobblestone-like cytoplasmic processes, and tegumental spines (Figs. 7-12). On the lip of the oral sucker 7 type II sensory papillae were seen (Fig. 7), and at the inner side of the lip one pair of large and two pairs of small type I sensory papillae were seen on each side. The ventral sucker became thicker than in metacercariae, and located at the right side of the anterior body. Around the ventral sucker (Fig. 8), type I papillae, 3-4 in number, were distributed.
The distribution of spines was very similar to that of metacercariae, but the space between spines became a little wider (Figs. 9-11) than in metacercariae. The anterior part of the ventral surface was covered densely with large and 9-11 pointed spines, and with many type I sensory papillae (Figs. 9-10). On the posterior surface, spines were sparse, with 2-6 points.
On the dorsal surface, spines were well differentiated into 10-11 points anteriorly, but became less pointed posteriorly. The Laurer's canal was seen with a sperm entering into the canal (Fig. 11). The opening of the Laurer's canal was surrounded by several layers of sphincter-like structures. The distribution of spines around the Laurer's canal was moderate. The excretory pore was anus-shape (Fig. 12), and slightly depressed. The pore was completely devoid of spines, and at a little remote areas a few spines were seen. The surface around the excretory pore was wrinkled irregularly.
Two- and four-week old worms: The general feature of the worm (Fig. 13) was not different from that of one-week old adults. Around the lip of the oral sucker, 7 large type II sensory papillae were observed, and at inner side of the lip one pair of large and two pairs of small type I sensory papillae were characteristically seen on each side (Fig. 14).
Tegumental spines (Fig. 15) were distributed over the whole body surface except areas of oral and ventral suckers. The ventral sucker was located on the right side of the anterior part of the body, with many wrinkles on its lip. Sperms were seen escaping from the genital pore (Fig. 16). Around the ventral sucker, numerous 9-11 pointed spines (Fig. 15), and several type I sensory papillae were observed. On the surface near the posterior end, spines were sparsely distributed, and pointed simply.
On the dorsal surface, the degree of spine differentiation was nearly the same as on the ventral surface. The Laurer's canal was clearly recognized, and many sperms were seen entering into the Laurer's canal (Fig. 17). By cracking of the mid-dorsal surface, intrauterine eggs were visualized (Fig. 18); they were ovoid to ellipsoid in shape, with an operculum and relatively smooth shell surface.
DISCUSSION
As is a common feature of all kinds of trematodes, the whole tegument of M. miyatai, metacercariae and adults, was covered with cytoplasmic processes, together with tegumental spines and sensory papillae. According to their development, the body size became bigger, flat cytoplasmic processes in metacercariae differentiated into knob- or cobblestone-like projections in adults, digitations of tegumental spines increased, and interspace between spines became wider.
The surface ultrastructure of M. miyatai was generally similar to that reported for M. yokogawai (Lee et al., 1984). However, a few differential features were observed in the morphology and distribution of tegumental spines and sensory papillae. In metacercariae of M. yokogawai, tegumental spines on the ventral surface of the anterior body had 7-8 pointed tips, but in M. miyatai, the number of tips was only 5-7, a less differentiated feature. In adults of M. yokogawai, however, digitations of well differentiated tegumental spines were 7-9 points (Lee et al., 1984), whereas in M. miyatai adults, 9-11 pointed spines were popularly seen. Also the dense distribution of spines was limited from anterior to middle surfaces of the body in M. yokogawai (Lee et al., 1984), but extended more posteriorly in M. miyatai. The type III papillae (round swelling of cytoplasmic ridges) observed at inner side of the lip of the oral sucker in 4-week old adults of M. yokogawai (Lee et al., 1984) was not seen in one- to four-week old adults of M. miyatai. Instead, one pair of large and two pairs of small type I papillae were characteristically seen at inner side of the lip of the oral sucker in M. miyatai adults.
The tegumental surface of trematodes is known to have physiological or biochemical functions essential for their existence and thriving (Lumsden, 1975). In larval trematodes, the tegument is generally flat or cobblestone-like, but as they mature, it is differentiated into a more fine and velvety one (Lee et al., 1985; Hong et al., 1991). This modification of the surface structure is regarded as a consequence to suffice the increase of nutrient requirements by parasites in the host body (Bennett and Threadgold, 1975; Fujino et al., 1979; Font and Wittrock, 1980; Lee et al., 1984).
The size, shape and distribution of tegumental spines are different by species of parasites as well as developmental stages, habitats, and migratory behaviors (Fujino et al., 1979; Bennett and Treadgold, 1980; Font and Wittrock, 1980; Lee et al., 1989). In the case of Clonorchis sinensis, it was reported that larval flukes which excysted in the duodenum have double or triple-pointed tegumental spines at anterior half of the body, but spines gradually disappear as the worms grow to be adults, and full-grown adults become compeletely devoid of spines (Fujino et al., 1979; Lee et al., 1982). On the contrary, it was shown in larval worms of Fasciola hepatica that tegumental spines of single-pointed form metamorphose into multipointed ones just prior to the entry into the bile duct (Bennett and Threadgold, 1975). Conversion of simple spines into serrated ones during developmental stages is also known in Neodiplostomum seoulense (Lee et al., 1985) and Paragonimus iloktsuenensis (Lee et al., 1989). These developmental changes were thought to help flukes adapt to the changing environment in the host (Bennett and Threadgold, 1975).
The function of ciliated type I papillae has been suggested to be tango- and/or rheoreceptive, when their morphology and distribution in various trematodes were considered (Erasmus, 1967; Morris and Threadgold, 1967; Bennett and Threadgold, 1975; Fujino et al., 1979; Lee et al., 1984; Seo et al., 1984). On the other hand, Ip and Desser (1984) suggested that ciliated sensory papillae might be either chemo- and/or mechanoreceptive because this type sensory papillae extended through the whole thickness of the tegument and had a free sensory cilium.
Spines were lacking on the lip of the oral sucker in excysted metacercariae and adults of M. miyatai. In the cercarial stage of Metagonimus spp., however, it was reported that about 30 hook-like spines were present on the oral sucker (Fujino et al., 1976). The disappearance of oral spines seems to be due to their loss after attachment at and penetration into the fish intermediate host.
The presence of sawtooth or brush-shaped tegumental spines seem to be one of characteristic features of the family Heterophyidae. Tegumental spines of H. nocens, for example, were in most cases multidigitated with 12-17 points (Chai et al., 1992). Similar features were reported in C. lingua (Køie, 1977), H. aequalis (Taraschewski, 1984), M. yokogawai (Lee et al., 1984) and H. continua (Hong et al., 1991). However, the number of points and its changing patterns according to worm development appear to be different by species of parasites. In the case of H. continua, for example, the number of points was increased from 10-14 in metacercariae to 15-17 in adults (Hong et al., 1991), and from 5-7 (metacercariae) to 9-11 (adults) in M. miyatai (this study), whereas the number of points remained nearly unchanged between metacercariae and adults in M. yokogawai (Lee et al., 1984).
It was interesting to note in this study that the function of the Laurer's canal opened on the dorsal surface should be an important route to receive sperms in M. miyatai, as many sperms were seen entering into the Laurer's canal. Similarly in C. sinensis, the Laurer's canal was suggested to be a copulatory organ receiving sperms (Jeong, 1983). Also interesting was to see the morphology of intrauterine eggs of M. miyatai, by cracking the dorsal surface of adult flukes, which were oval to ellipsoid in shape, with an operculum, and relatively smooth shell surface showing less prominent muskmelon patterns.
Conclusively, the surface ultrastructure of M. miyatai was generally similar to that of M. yokogawai (Lee et al., 1984), however, differences were observed in the digitation and differentiation patterns of tegumental spines and distribution of sensory papillae in metacercariae and adults, which may be of taxonomic significance.
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