Several species of freshwater fishes, i.e., Misgurnus anguillicaudatus, Misgurnus mizolepis, Odontobutis obscura interrupta (O. interrupta), Moroco oxycephalus (Rhynchocypris oxycephalus), Coreoperca kawamebari and Squalidus coreanus (S. japonicus coreanus), yellowfin goby, Acanthogobius flavimanus, and tadpoles of Rana nigromaculata were reported as the second intermediate hosts and/or the source of human infection in Korea. Most of these reports were performed in limited areas and with small number of animals [29–35]. Therefore, we examined the dark sleepers, Odontobutis spp., broadly collected from 13 sites in 5 rivers, i.e., Mangyeong-gang, Geum-gang, Tamjin-gang, Seomjin- gang and Nakdong-gang, of Korea.

Total 237 Odontobutis spp. were collected from 13 sites in 5 rivers, i.e., Mangyeong-gang (n=49), Geum-gang (n=32), Tamjin-gang (n=73), Seomjin-gang (n=13) and Nakdong-gang (n=70), and examined all with the artificial digestion method for 5 years (2013–2017) (Fig. 1). The number of fish examined by the surveyed sites was detailedly revealed in Table 1.

The metacercariae of I. hortensis (IhMc) were globular or elliptical, 143–165 (154) by 128–158 (144) μm in size, and have a double layered cyst wall, 27 collar spines including 4 end group ones in each side of the head crown, a ventral sucker transversely elliptical, 2-fold as larger as the oral sucker, and granules in the excretory bladder arranged in 2 rows of tube (Fig. 2).

A total of 137 (57.8%) Odontobutis spp. were infected with 14.8 IhMc in average. The prevalence was most high in Nakdong- gang areas (62.9%) and followed by it in Mangyeong-gang (57.1%), Geum-gang (56.3%), Tamjin-gang (54.8%) and Seomjin-gang (53.9%) areas. Metacercarial densities were 28.1 in Geum-gang, 13.9 in Mangyeong-gang, 13.3 in Nakdong-gang, 13.1 in Tamjin-gang and 2.3 per fish infected in Seomjin-gang respectively. Especially, in case of Yugucheon, a branch of Geumgang, IhMc were detected in all fish (100%) examined and their density was about 48 per fish. The infection status with IhMc in fish from 13 surveyed sites was detailedly designated in Table 1.

In the present study, it was confirmed that 2 species of dark sleepers, O. platycephala, and O. interrupta, are suitable fish hosts of I. hortensis although the infection status of IhMc is more or less different by the surveyed areas. Moreover, the prevalences and metacercarial densities of this study were much higher than those of previous studies. Ahn et al. [30] detected total 32 IhMc in 10 (27.8%) out of 36 dark sleepers, O. interrupta, from Seom-gang, a branch of Namhan-gang, in Wonseong-gun (gun=county), Gangwon-do (do=province). Ahn and Ryang [22] also detected IhMc in 4 (22.2%) out of 18 dark sleepers from Namhan-gang. Lee et al. [23] found IhMc in only 1 (2.3%) out of 44 dark sleepers from Cheongsong- gun, Gyeongsangbuk-do. Ryang [20] detected IhMc in 11 (20.3%) out of 54 dark sleepers from Chungju-ho (ho means lake) and the upper streams of Namhan-gang. In case of suitable fish host, muddy loach (Misgurnus anguillicaudatus), Chai et al. [29] reported that total 64 (41.6%) out of 154 loaches were infected with 1–29 (8.1 in average) IhMc per fish infected. Ahn and Ryang [22] detected IhMc in 106 (34.1%) out of 311 loaches from Namhan-gang. Lee et al. [23] found IhMc in 2 (10.5%) out of 19 loaches from Cheongsong-gun, Gyeongsangbuk- do. Ryang [20] detected IhMc in 30 (40.5%) out of 74 loaches from Chungju-ho and the upper streams of Namhan- gang. In another fish hosts of I. hortensis, M. mizolepis, R. oxycephalus, C. kawamebari, S. japonicus coreanus and A. flavimanus, small number of IhMc were detected in Korea [29–35].

Some endemic foci of I. hortensis infections have been reported in Korea [20–23]. Especially in the villages of Cheongsong- gun in Gyeongsangbuk-do, 59 (22.4%) out of 263 residents were positive with echinostome eggs and 1–649 (51 in average) adult worms of I. hortensis were collected from 35 egg-positive cases in the worm recovery after praziquantel treatment and MgSO₄ purgation. As the source of human infections, the dark sleeper is most convincing among the second intermediate hosts in these endemic areas. It has been known that residents in inland of Korea frequently eat raw flesh of freshwater fishes such as Mandarin fish (Siniperca scherzeri), Korean aucha perch (Coreoperca herzi) and dark sleepers (O. platycephala and O. interrupta) [20–23,30]. These fishes are powerful predators in the ecosystems of freshwater and presently eaten in raw by residents living in riverside areas of Korea. Therefore, Korean peoples pay attention to the raw consumption of dark sleepers, Odontobutis spp., to prevent the infections with this species of echinostomid fluke.