Metagonimiasis is an endemic intestinal trematode infection in Korea with 0.3% egg positives in the general population (i.e. 130,000 infected people) (Chai et al., 1993; Yu et al., 1994; Ministry of Health and Welfare and Korea Association of Health, 1997). Three species of the genus Metagonimus are found in Korea: M. yokogawai (Katsurada, 1912), M. takahashii (Suzuki, 1930), and M. miyatai (Saito et al., 1997). These species can be differentiated not only by their morphologies, geographical distributions, and their intermediate hosts which are mainly fish species, but also by their polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) patterns of the genes for 18 S rRNA, 5.8 S rRNA, and internal transcribed spacer 1 (ITS1) and by their random amplification of polymorphic DNA patterns (Saito et al., 1997; Yu et al., 1997a, 1997b; Chai et al., 1998). Chromosomal studies on trematode parasites have been significantly useful in taxonomic studies (Terasaki et al., 1982; Hirai et al., 1985; Park et al., 1998). In 1959, Walton phylogenetically studied the chromosome numbers of more than 50 species of helminthic parasites and showed definite chromosome numbers at the genus level. However, there have been few studies on karyotypes of human intestinal trematodes. In the present study, we compared the karyotypes of 3 species of Metagonimus in order to evaluate the taxonomical positions.

Twenty adult specimens each of M. takahashii, M. miyatai, and M. yokogawai were obtained from rats (Sprague-Dawley, 4 to 6-week-old) at 7 days post-infection (PI). Metacercariae of each species were collected from Carassius carassius, Zacco platypus, and Plecoglossus altivelis, respectively (Saito et al., 1997; Yu et al., 1997b). After collecting flukes from the intestine of rats, they were cultivated in 1X liquid MEM-199 medium (GIBCO BRL Lab., Ontario, Canada, pH 7.4) containing 0.001% colchicine, at 37℃ for 4 hr. Flukes were then fixed with modified Carnoy's fluid (1 part glacial acetic acid, 3 parts of 95% ethanol). The testis of each specimen was gently removed with a dissecting pin and transferred to the fixative using a fine forcep. Pieces of other tissues were removed as well. The testes were then minced gently in 45% acetic acid to prepare cell suspensions. The cells left on the slide were stained with aceticorcein solution for 10 to 30 min. The overstained cells were rinsed briefly with 45% acetic acid, covered with a cover glass, and then squashed. The slides were heated briefly with an alcohol lamp to remove air bubbles and mounted in Canada balsam. Observations were done under a light microscope. The absolute length of chromosomes, the total chromosome length of the haploid set, and the positions of centromeres were compared by each species. The classification of chromosomes was adopted according to the methods of Levan et al. (1964).

The number of bivalents of M. takahashii was nine at the meiotic metaphase of the primary spermatocytes (Fig. 1). However, diploid chromosomes of dividing spermatogonia were not detected. The spermatogonial metaphase of M. miyatai was observed and the diploid number was counted as eighteen (2n=18). Counted chromosomes were consisted of one pair of large metacentric (m) chromosomes (No. 1 of Fig. 4), seven pairs of submetacentric (sm) chromosomes (Nos. 2-8 of Fig. 4), and one pair of telocentric (t) chromosomes (No. 9 of Fig. 4) (2n=18: n=1m+7sm+1t). The length of chromosome pairs of M. miyatai was 3.19 -23.0 µm and the mean total length of the haploid components was 29.25±0.24 µm (Table 1; Fig. 4). The haploid state of M. yokogawai has a chromosome number of sixteen (n=16). Diploid number of M. yokogawai was thirty-two (2n=32) (Figs. 5, 6). The karyotype consisted of two pairs of metacentric (m) chromosomes (Nos. 1-2 of Fig. 7), 11 pairs of submetacentric (sm) chromosomes (Nos. 3, 5, 7-11, 13-16 of Fig. 7), and three pairs of subtelocentric (st) chromosomes (Nos. 4, 6, 12 of Fig. 7) (2n=32:n=2m+11sm+3st). The length of chromosomes of M. yokogawai ranged from 1.27 to 8.07 µm and the mean value of the total length of haploid components was 39.81±0.73 µm (Table 2; Fig. 7).

Karyotype data of human intestinal trematodes are rare (Walton, 1959). The diploid chromosome number of Neodiplostomum seoulense has been described as twenty (2n=20) (Park et al., 1998), and that of Echinostoma hortense and Echinostoma cinetorchis were twenty (2n=20) and twenty-two (2n=22), respectively (Terasaki et al., 1982). The present study revealed that the diploid chromosome number of M. yokogawai was thirty-two (2n=32) and that of M. miyatai was eighteen (2n=18). Therefore, these results could be an additional supporting evidence for the validity of new species, M. miyatai, distinct from M. yokogawai (Saito et al., 1997). Meanwhile, the presence of 9 bivalents in the first meiotic division of the primary spermatocytes of M. takahashii signified the existence of 18 chromosomes in the organism. Each of 9 bivalents consisted of two homologous chromosomes in synapsis. Therefore, we attempted to observe diploid chromosomes from M. takahashii several times, but we were not successful in obtaining the result.

The karyological results of our study showed the same haploid chromosome number of nine (n=9) from both M. miyatai and M. takahashii; however, the PCR-RFLP patterns of three Metagonimus spp. with a ITS1 site of rRNA and the mitochondrial cytochrome c oxidase genes showed different results with the estimation that the genetic distance between M. yokogawai and M. takahashii was closer than that of the distance between M. miyatai and M. takahashii (Yu et al., 1997b). Other related genes could probably give more information about the phylogenic distance.

Sasada (1978) reported 7 bivalents in the primary oocytes and the primary spermatocytes of M. yokogawai from Japan (2n=14), which did not coincide with the result obtained from the present studies. It is quite uncertain whether or not the difference is due to the intraspecific polymorphism or due to the intercellular junction of the gonocyte. It is necessary to simultaneously compare chromosomes of M. yokogawai from Korea with those from Japan to further verify. Despite the fact that chromosome size and number will reflect the size of the whole genome, less attention has been paid to the length of the mitotic metaphase chromosomes or to the total length of the mitotic metaphase chromosomes. In this study, variations in the chromosome size occurred. The squash method has been reported to produce larger size of chromosome than the air-drying method. Sasaki (1961) reported that the process of chromatid condensation is not always uniform or synchronous in all complements at a given stage of the mitotic cycle. When using fresh gonadal tissue directly, as in our study, it is especially difficult to gain a large number of uniform chromosome complements. Also, there are still more problems to be solved before the karyotypes can be analyzed in detail. It is, however, possible to qualitatively analyze chromosome sets clearly.