ISSN (Print) ISSN (Online) ORIGINAL ARTICLE Korean J Parasitol Vol. 56, No. 4: , August

ISSN (Print) 0023-4001 ISSN (Online) 1738-0006 ORIGINAL ARTICLE Korean J Parasitol Vol. 56, No. 4: 341-349, August 2018 https://doi.org/10.3347/kjp.2018.56.4.341 Infections with Centrocestus armatus Metacercariae in Fishes from Water Systems of Major Rivers in Republic of Korea Woon-Mok Sohn 1, *, Byoung-Kuk Na 1, Shin-Hyeong Cho 2, Jung-Won Ju 2, Cheon-Hyeon Kim 3, Ki-Bok Yoon 4, Jai-Dong Kim 5, Dong Cheol Son 6, Soon-Won Lee 7 1 Department of Parasitology and Tropical Medicine, and Institute of Health Sciences, Gyeongsang National University College of Medicine, Jinju 52727, Korea; 2 Division of Vectors and Parasitic Diseases, Centers for Disease Control and Prevention, Osong 28159, Korea; 3 Division of Microorganism, Jeollabuk-do Institute of Health and Environment, Imsil 55928, Korea; 4 Division of Microbiology, Jeollanam-do Institute of Health and Environment, Muan 58568, Korea; 5 Infectious Disease Examination Section, Chungcheongnam-do Institute of Health and Environment, Hongseong 32254, Korea; 6 Infectious Disease Research Section, Gyeongsangbuk-do Institute of Health and Environment, Youngcheon 38874, Korea; 7 Infection Disease Intelligence Division, Gangwon Institute of Health and Environment, Chuncheon 24203, Korea Abstract: The infection status of Centrocestus armatus metacercariae (CaMc) was broadly surveyed in freshwater fishes from major river systems in the Republic of Korea (Korea) during 2008-2017. A total of 14,977 fishes was caught and examined by the artificial digestion method. CaMc were detected in 3,818 (97.1%) (2,114 Z. platypus: 96.1% and 1,704 Z. temminckii: 98.4%) out of 3,932 Zacco spp. examined and their density was 1,867 (2,109 in Z. platypus and 1,567 in Z. temminckii) per fish. The prevalences with CaMc were high, 93.7-100%, in Zacco spp. from all surveyed areas. However, their densities were more or less different by the surveyed areas and fish species. They were most high in Nakdong-gang in Gyeongsangnam-do (4,201 in average), and followed by Geum-gang (2,343), Nakdong-gang in Gyeongsangbuk-do (1,623), Han-gang (1,564), Tamjin-gang and Yeongsan-gang (1,540), streams in the east coast (1,028), Seomjin-gang (488) and Mangyeong-gang (170). In another species of rasborinid fish, Opsariichthys uncirostris amurensis, CaMc were detected in 222 (74.8%) out of 297 ones examined and their density was 278 (1-4,480) per fish. CaMc were also detected in total 41 fish species except for the rasborinid fish, Z. platypus, Z. temminckii and O. uncirostris amurensis. Conclusively, it was confirmed that among the 3 species of rasborinid fish, Z. platypus and Z. temminckii are highly prevalent and O. uncirostris amurensis is moderately prevalent with CaMc. Additionally, we could know that variety of fish species act as the second intermediate hosts of C. armatus in Korea. Key words: Centrocestus armatus, Zacco platypus, Zacco temminckii, Opsariichthys uncirostris amurensis, metacercaria, river INTRODUCTION Received 3 March 2018, revised 9 July 2018, accepted 12 July 2018. * Corresponding author (wmsohn@gnu.ac.kr) 2018, Korean Society for Parasitology and Tropical Medicine This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Centrocestus armatus (Digenea: Heterophyidae) is a small intestinal fluke bearing 42-48 circumoral spines, which mainly inhabits in the small intestines of birds and mammals. This fluke was first recovered from dogs, cats, rabbits, rats and mice experimentally fed with cyprinoid fish with C. armatus metacercariae (CaMc) in Japan [1]. Human infections were experimentally proven in Japan, and a natural human case was reported in the Republic of Korea (Korea) [1,2]. The freshwater snails, Semisulcospira spp., are known to be the first intermediate hosts in Japan and Korea [3,4]. Various species of freshwater fish, including Zacco platypus and Z. temminckii, were reported as the second intermediate hosts in Japan and Korea [5-13]. As the natural definitive hosts of this fluke, the large egret, Egretta alba modesta, and stray cat, Felis catus, were recorded in Korea [14-17]. This species of heterophyid fluke is not clinically important unlike Clonorchis sinensis and Metagonimus spp. as fishborne zoonotic trematodes. And so the epidemiological study on the metacercarial infections in fish hosts has been rarely done in Korea [6-8,10,11]. In Korea, Chun [6] described more than 11 species of digenetic trematode metacercariae (DTM) together with CaMc detected in 16 fish species from streams and ponds in adjacent areas of Nakdong-gang. Rhee et al. [8] detected CaMc in 7 out of 32 fish species from Mangyeong-gang and they also found them in 5 out of 33 fish species from Dongjin- 341

342 Korean J Parasitol Vol. 56, No. 4: 341-349, August 2018 gang [9]. In 1988, Hong et al. [10] investigated the infection status of CaMc in 2 species of chub, Z. platypus and Z. temminckii, collected from 19 sites in 6 major rivers of Korea. Recently, Cho et al. [12] reported the infection status of zoonotic trematode metacercariae including CaMc in fishes from the water systems in Gangwon-do. Sohn et al. [13] surveyed the infection status of DTM in fishes from the water systems of Hantan-gang and Imjin-gang. However, most of previous studies were performed in limited areas except for Hong et al. [10]. Even in Hong et al. [10], only 2 species of chub, Z. platypus and Z. temminckii, collected from 19 sites were examined. Therefore, the present study was performed to estimate the endemisities of CaMc in the freshwater fishes nationwidely and moreover, to expand the fauna on the fish hosts of C. armatus in Korea. MATERIALS AND METHODS Fish collected from water systems of Han-gang Fishes from Hantan-gang in Cheorwon-gun (2010: 177 fishes in 15 spp.; 2012: 255 fishes in 18 spp.; 2013: 200 fishes in 23 spp.; 2014: 400 fishes in 15 spp.), Suipcheon in Yanggugun (2009: 196 fishes in 14 spp.), Hongcheon-gang in Hongcheon-gun (2010: 186 fishes in 14 spp.), Pyeongchang-gang in Pyeongchang-gun (2013: 230 fishes in 15 spp.), Dong-gang in Yeongwol-gun (2009: 251 fishes in 19 spp.), Seom-gang in Hoengseong-gun (2011: 184 fishes in 20 spp.) and Choyanggang in Jeongseon-gun (2012: 196 fishes in 15 spp.), Gangwon-do, and Dalcheon in Goesan-gun (2011: 99 fishes in 12 spp.), Chungcheongbuk-do were examined. Fish collected from water systems of Geum-gang Fishes from Chogangcheon in Yeongdong-gun (2011: 132 fishes in 15 spp.), Chungcheongbuk-do, Cheonnae-gang in Geumsan-gun (2011: 140 fishes in 20 spp.; 2013: 146 fishes in 13 spp.; 2014: 161 fishes in 17 spp.; 2015: 79 fishes in 14 spp.), Yugucheon in Gongju-si (2013: 146 fishes in 11 spp.; 2015: 165 fishes in 11 spp.) and Jicheon in Cheongyang-gun (2014: 89 fishes in 13 spp.), Chungcheongnam-do, Jujacheon in Jinangun (2012: 208 fishes in 19 spp.) and Namdaecheon in Mujugun (2012: 123 fishes in 14 spp.), Jeollabuk-do were examined. Fish collected from water systems of Mangyeong-gang and Dongjin-gang Fishes from Soyangcheon in Wanju-gun (2012: 82 fishes in 8 spp.; 2013: 97 fishes in 12 spp.; 2014: 165 fishes in 9 spp.; 2015: 200 fishes in 12 spp.) and Jungeupcheon in Jungeup-si (2013: 88 fishes in 11 spp.), Jeollabuk-do were examined. Fish collected from water systems of Yeongsan-gang and Tamjin-gang Fishes from Hwangryong-gang in Jangseong-gun (2011: 107 fishes in 14 spp.), Jiseokcheon in Hwasun-gun (2011: 54 fishes in 8 spp.) and Naju-si (2013: 86 fishes in 9 spp.), Yeongamcheon in Yeongam-gun (2013: 45 fishes in 8 spp.;), Tamjingang in Jangheung-gun (2014: 167 fishes in 13 spp.; 2015: 243 fishes in 15 spp.; 2016: 302 fishes in 16 spp.; 2017: 420 fishes in 19 spp.) and Gangjin-gum (2014: 110 fishes in 12 spp.; 2017: 407 fishes in 15 spp.), Jeollanam-do were examined. Fish collected from water systems of Seomjin-gang Fishes from Osucheon in Imsil-gun (2011: 105 fishes in 10 spp.; 2012: 190 fishes in 10 spp.; 2013: 46 fishes in 5 spp.), Seomjin-gang in Sunchang-gun (2014: 69 fishes in 22 spp.; 2015: 241 fishes in 23 spp.) and Songdaecheon in Namwon-si (2012: 139 fishes in 12 spp.; 2013: 57 fishes in 7 spp.), Jeollabuk-do, Seomjin-gang in Gokseong-gun (2015: 146 fishes in 11 spp.) and in Gurye-gun (2014: 183 fishes in 21 spp.), Jeollanam-do, Hoengcheon (2014: 156 fishes in 13 spp.), Namsancheon (2015 and 2016: 234 fishes in 12 spp.) and Jugyocheon (2011: 54 fishes in 14 spp.) in Hadong-gun, Gyeongsangnamdo were examined. Fish collected from water systems of Nakdong-gang in Gyeongsangnam-do Fishes from Yangcheon in Sancheong-gun (2010: 350 fishes in 15 spp.; 2011: 201 fishes in 14 spp.; 2012: 221 fishes in 11 spp.; 2013: 644 fishes in 20 spp.; 2014: 291 fishes in 14 spp.; 2015: 183 fishes in 15 spp.; 2016: 253 fishes in 16 spp.; 2017: 408 fishes in 22 spp.), Jisucheon in Jinju-si (2014: 94 fishes in 11 spp.) and Hamancheon in Haman-gun (2014: 73 fishes in 9 spp.) were examined. Fish collected from water systems of Nakdong-gang in Gyeongsangbuk-do Fishes from Naeseongcheon in Bonghwa-gun (2008: 97 fishes in 12 spp.) and Yecheon-gun (2008: 131 fishes in 18 spp.; 2014: 103 fishes in 15 spp.), Nakdong-gang in Andong-si (2008: 118 fishes in 12 spp.; 2009: 56 fishes in 11 spp.), Yeong-gang in Mungyeong-si (2009: 108 fishes in 15 spp.), Banbyeoncheon in Yeongyang-gun (2008: 86 fishes in 12 spp.; 2015: 161 fishes

Sohn et al.: Centrocestus armatus metacercaria in fish from Korea 343 OS VS EB A B Fig. 1. Metacercariae of Centrocestus armatus encysted in the viscera of a pale chub, Zacco platypus (A) and an isolated one (B). They are characterized by long elliptical shape, 0.20-0.25 0.10-0.12 mm in size, providing 42 circumoral spines around oral sucker (OS) arranged 2 rows, a ventral sucker (VS) in the median and X-shaped excretory bladder (EB). Scale bar is 0.1 mm. in 12 spp.), Namdaecheon in Uiseong-gun (2009: 83 fishes in 9 spp.), Wicheon in Gunwi-gun (2008: 136 fishes in 16 spp.; 2011: 105 fishes in 13 spp.; 2013: 107 fishes in 12 spp.; 2014: 338 fishes in 24 spp.; 2015: 245 fishes in 19 spp.; 2016: 279 fishes in 24 spp.; 2017: 199 fishes in 17 spp.), Bukcheon in Sangju-si (2008: 45 fishes in 11 spp.), Hoecheon in Goryeonggun (2013: 165 fishes in 13 spp.), Nakdong-gang in Dalseonggun (2009: 63 fishes in 5 spp.) and Baekcheon in Seongju-gun (2009: 71 fishes in 12 spp.) were examined. Fish collected from water systems in the east coast of Korea Fishes from Namdaecheon in Yangyang-gun (2009: 174 fishes in 11 spp.; 2015: 140 fishes in 13 spp.), Osipcheon (2009: 155 fishes in 11 spp.; 2015: 143 fishes in 15 spp.) and Gagokcheon (2009: 109 fishes in 8 spp.) in Samcheok-si, Gangwon-do, Osipcheon in Yeongdeok-gun (2008: 104 fishes in 9 spp.; 2015: 122 fishes in 11 spp.), Wangpicheon in Uljin-gun (2015: 239 fishes in 13 spp.) and Hyeongsan-gang in Gyeongju-si (2015: 111 fishes in 12 spp.), Gyeongsangbuk-do, Cheokgwacheon (2015: 221 fishes in 10 spp.) and Taehwa-gang (2015: 89 fishes in 14 spp.) in Ulju-gun, Ulsan Metropolitan City were examined. Examination methods All collected fishes with ice were transferred to the laboratory of the Department of Parasitology and Tropical Medicine, Gyeongsang National University College of Medicine, Jinju, Korea. After the identification of fish species, they were individually ground with a mortar or grinder. Each ground fish meat was mixed with artificial gastric juice and the mixture was incubated at 36 C for 2 hr. The digested material was filtered through 1 1 mm of mesh, and washed with 0.85% saline untill the supernatant is clear. The sediment was carefully examined under a stereomicroscope. CaMc were separately collected by the general feature [11] (Fig. 1), counted and calculated for infection rates (%) and densities (No. of CaMc per fish ) by fish species. RESULTS Infection status with CaMc in Zacco spp. from Han-gang The metacercariae of C. armatus (CaMc) were detected in 553 (99.3%) Zacco spp. (253 Z. platypus: 99.6% and 300 Z. temminckii: 99.0%) and their average density was 1,564 (1,904 in Z. platypus and 1,278 in Z. temminckii) per fish. The infection status with CaMc by the surveyed areas was detailed in Table 1.

344 Korean J Parasitol Vol. 56, No. 4: 341-349, August 2018 Table 1. Infection status with Centrocestus armatus metacercariae in rasborinid fish, Zacco platypus and Z. temminckii, from the water system of Han-gang and Geum-gang (River), Korea Zacco platypus Z. temminckii Total No. (%) (range) a No. (%) (range) a No. (%) (range) a Locality (year examined) Han-gang 253 (99.6) 1,904 (1-25,850) 300 (99.0) 1,278 (2-11,470) 553 (99.3) 1,564 (1-25,850) Hantan-gang (2010 b, 2012 b, 2013 b, 2014) 100 (100) 1,300 (8-6,620) 141 (100) 637 (17-3,890) 241 (100) 912 (8-6,620) Suipcheon (2009) b 25 (96.2) 158 (1-887) 22 (95.7) 329 (77-1,035) 47 (95.9) 238 (1-1,035) Hongcheon-gang (2010) b 20 (100) 6,158 (780-13,650) 16 (100) 3,343 (42-8,041) 36 (100) 4,907 (42-13,650) Pyeongchang-gang (2013) b 20 (100) 1,821 (240-8,080) 34 (100) 4,376 (96-11,470) 54 (100) 3,429 (96-11,470) Dong-gang (2009) b 30 (100) 357 (54-1,006) 30 (100) 206 (31-663) 60 (100) 281 (31-1,006) Seom-gang (2011) b 23 (100) 1,809 (21-8,720) 3 (60.0) 585 (2-1,385) 26 (92.9) 1,667 (2-8,720) Choyang-gang (2012) b 20 (100) 318 (53-978) 39 (100) 1,039 (163-3,250) 59 (100) 795 (53-3,250) Dalcheon (2011) 15 (100) 8,635 (2,710-25,850) 15 (100) 2,372 (333-6,680) 30 (100) 5,503 (333-25,850) Geum-gang 133 (89.3) 4,253 (1-43,340) 127 (100) 343 (7-6,920) 260 (94.2) 2,343 (1-43,340) Cheonnae-gang (2011, 2013, 2014, 2015) 43 (100) 3,266 (101-11,805) 44 (100) 463 (24-3,706) 87 (100) 1,848 (24-11,805) Chogangcheon (2011) 3 (100) 1,040 (22-3,046) 3 (100) 2,695 (145-6,920) 6 (100) 1,868 (22-6,920) Jujacheon (2012) 24 (60.0) 206 (1-1,823) 40 (100) 246 (26-1,336) 64 (80.0) 231 (1-1,823) Jicheon (2014) 4 (100) 754 (128-1,637) 29 (100) 99 (7-509) 33 (100) 178 (7-1,637) Yugucheon (2013, 2015) 59 (100) 7,019 (2,000-43,340) - - 59 (100) 7,019 (2,000-43,340) Namdaecheon in Muju (2012) - - 11 (100) 225 (13-506) 11 (100) 225 (13-506) a No. of CaMc per fish. b Already reported in Cho et al. (2014) [12]. Infection status with CaMc in Zacco spp. from Geum-gang CaMc were detected in 260 (94.2%) Zacco spp. (133 Z. platypus: 89.3% and 127 Z. temminckii: 100%) and their average density was 2,343 (4,253 in Z. platypus and 343 in Z. temminckii) per fish. The infection status with CaMc by the surveyed areas was detailedly revealed in Table 1. Infection status with CaMc in Zacco spp. from Mangyeonggang and Dongjin-gang CaMc were detected in 136 (98.6%) Zacco spp. (56 Z. platypus: 96.6% and 80 Z. temminckii: 100%) and their average density was 170 (137 in Z. platypus and 193 in Z. temminckii) per fish. The infection status with CaMc by the surveyed areas was detailed in Table 2. Infection status with CaMc in Zacco spp. from Yeongsangang CaMc were detected in 84 (100%) Zacco spp. (80 Z. platypus and 4 Z. temminckii) and their average density was 1,493 (1,403 in Z. platypus and 3,290 in Z. temminckii) per fish. The infection status with CaMc by the surveyed areas was detailedly revealed in Table 2. Infection status with CaMc in Zacco spp. from Tamjin-gang CaMc were detected in 391 (100%) Zacco spp. (213 Z. platypus and 178 Z. temminckii) and their average density was 1,550 (1,970 in Z. platypus and 1,048 in Z. temminckii) per fish. The infection status with CaMc by the surveyed areas was detailed in Table 2. Infection status with CaMc in Zacco spp. from Seomjin-gang CaMc were detected in 531 (99.4%) Zacco spp. (311 Z. platypus: 99.7% and 220 Z. temminckii: 99.1%) and their average density was 488 (596 in Z. platypus and 336 in Z. temminckii) per fish. The infection status with CaMc by the surveyed areas was detailedly shown in Table 2. Infection status with CaMc in Zacco spp. from Nakdonggang in Gyeongsangnam-do CaMc were detected in 713 (100%) Zacco spp. (323 Z. platypus and 390 Z. temminckii) and their average density was 4,201 (4,383 in Z. platypus and 4,050 in Z. temminckii) per fish. The infection status with CaMc by the surveyed areas was detailed in Table 3.

Sohn et al.: Centrocestus armatus metacercaria in fish from Korea 345 Table 2. Infection status with CaMc in rasborinid fish, Zacco platypus and Z. temminckii, from the water systems in the western and southern regions of Korea Locality (year examined) No. (%) Zacco platypus Z. temminckii Total No. (%) (range) a No. (%) (range) a (range) a Mangyeong-gang b 56 (96.6) 137 (1-1,148) 80 (100) 193 (1-2, 536) 136 (98.6) 170 (1-2,536) Soyangcheon (2012-2015) 28 (100) 259 (13-1,148) 80 (100) 193 (1-2, 536) 108 (100) 210 (1-2,536) Jungeupcheon (2013) b 28 (93.3) 16 (1-85) - - 28 (93.3) 16 (1-85) Yeongsan-gang 80 (100) 1,403 (5-19,200) 4 (100) 3,290 (123-7,200) 84 (100) 1,493 (5-19,200) Hwangryong-gang (2011) 15 (100) 289 (34-814) - - 15 (100) 289 (34-814) Jiseokcheon (2011, 2013) 50 (100) 45 (5-399) - - 50 (100) 45 (5-399) Yeongamcheon (2013) 15 (100) 7,042 (420-19,200) 4 (100) 3,290 (123-7,200) 19 (100) 6,252 (123-19,200) Tamjin-gang 213 (100) 1,970 (11-13,420) 178 (100) 1,048 (12-7,120) 391 (100) 1,550 (11-13,420) In Jangheung (2014-2017) 138 (100) 2,711 (11-13,420) 152 (100) 1,113 (12-7,120) 290 (100) 1,873 (11-13,420) In Gangjin (2014, 2017) 75 (100) 607 (42-2,661) 26 (100) 665 (16-2,787) 101 (100) 622 (16-2,787) Seomjin-gang 311 (99.7) 596 (6-7,440) 220 (99.1) 336 (1-4,300) 531 (99.4) 488 (1-7,440) Osucheon (2011, 2012, 2013) 99 (99.0) 213 (17-2,125) - - 99 (99.0) 213 (17-2,125) In Sunchang (2014, 2015) 51 (100) 798 (14-4,480) 15 (100) 17 (4-48) 66 (100) 621 (4-4,480) Songdaecheon (2012, 2013) 46 (100) 336 (6-3,620) 57 (100) 716 (3-4, 300) 103 (100) 547 (3-4,300) In Gokseong (2015) 16 (100) 814 (99-1,985) 37 (97.4) 244 (1-2, 540) 53 (98.2) 416 (1-2,540) In Gurye (2014) 24 (100) 1,024 (37-5,429) 13 (92.9) 77 (2-710) 37 (97.4) 691 (2-5,429) Hoengcheon (2014) 14 (100) 736 (20-2,615) 25 (100) 137 (4-394) 39 (100) 352 (4-2,615) Namsancheon (2015, 2016) 41 (100) 1,072 (83-7,440) 68 (100) 277 (5-3, 005) 109 (100) 576 (5-7,440) Jugyocheon (2011) 20 (100) 804 (123-2,245) 5 (100) 123 (11-205) 25 (100) 668 (11-2,245) a No. of CaMc per fish. b + Dongjin-gang. Table 3. Infection status with CaMc in rasborinid fish, Zacco platypus and Z. temminckii, from the water system of Nakdong-gang (River) in Korea Locality (year examined) No. (%) Zacco platypus Z. temminckii Total (range) a No. (%) (range) a No. (%) (range) a Nakdong-gang b 323 (100) 4,383 (2-30,500) 390 (100) 4,050 (55-41,460) 713 (100) 4,201 (2-41,460) Yangcheon (2010-2017) 279 (100) 4,669 (6-30,500) 360 (100) 4,262 (55-41,460) 639 (100) 4,440 (6-41,460) Jisucheon (2014) 35 (100) 3,186 (25-12,400) 5 (100) 3,527 (560-13,260) 40 (100) 3,228 (25-13,260) Hamancheon (2014) 9 (100) 156 (2-318) 25 (100) 1,108 (295-3,376) 34 (100) 856 (2-3,376) Nakdong-gang c 542 (91.1) 2,026 (1-24,190) 236 (95.6) 699 (1-10,501) 778 (92.4) 1,623 (1-24,190) Wicheon (2008, 2011, 2013-2017) 329 (100) 2,961 (15-24,190) 163 (99.4) 901 (7-10,501) 492 (99.8) 2,279 (7-24,190) Naeseongcheon in Bonghwa (2008) 4 (36.4) 14 (1-50) 6 (85.7) 16 (1-44) 10 (55.6) 15 (1-50) Banbyeoncheon (2008, 2015) 58 (100) 1,793 (3-6,290) 37 (97.4) 408 (10-1,170) 95 (99.0) 1,254 (3-6,290) In Andong (2008, 2009) 44 (95.7) 305 (3-1,985) 24 (96.0) 122 (16-352) 68 (95.8) 240 (3-1,985) Hoecheon (2013) 21 (60.0) 4 (1-10) 6 (46.2) 6 (1-20) 27 (56.3) 4 (1-20) Naeseongcheon in Yecheon (2008, 2014) 45 (88.2) 117 (1-786) - - 45 (88.2) 117 (1-786) Yeong-gang (2009) 19 (95.0) 38 (3-123) - - 19 (95.0) 38 (3-123) Namdaecheon in Uiseong (2009) 15 (100) 17 (8-33) - - 15 (100) 17 (8-33) Baekcheon (2009) 5 (33.3) 14 (1-61) - - 5 (33.3) 14 (1-61) In Dalseong (2009) 2 (13.3) 3 (1-5) - - 2 (13.3) 3 (1-5) a No. of CaMc per fish. b in Gyeongsangnam-do. c in Gyeongsangbuk-do. Infection status with CaMc in Zacco spp. from Nakdonggang in Gyeongsangbuk-do CaMc were detected in 778 (92.4%) Zacco spp. (542 Z. platypus: 91.1% and 236 Z. temminckii: 95.6%) and their average density was 1,623 (2,026 in Z. platypus and 699 in Z. temminckii) per fish. The infection status with CaMc by the surveyed areas was detailedly revealed in Table 3.

346 Korean J Parasitol Vol. 56, No. 4: 341-349, August 2018 Table 4. Infection status with CaMc in rasborinid fish, Zacco platypus and Z. temminckii, from the water systems in the east coast of Korea Locality (year examined) No. (%) Zacco platypus Z. temminckii Total No. (%) (range) a No. (%) (range) a (range) a Namdaecheon in Yangyang (2009 b, 2015) 28 (84.8) 207 (2-1,561) 56 (86.2) 1,530 (1-14,419) 84 (85.7) 1.089 (1-14,419) Osipcheon in Samcheok (2009 b, 2015) 37 (97.4) 454 (9-4,925) - - 37 (97.4) 454 (9-4,925) Gagokcheon (2009) b 18 (94.7) 469 (1-1,880) 12 (80.0) 202 (1-1,518) 30 (88.2) 362 (1-1,880) Wangpicheon (2015) 30 (100) 1,412 (46-5,300) 15 (100) 3,079 (371-6,904) 45 (100) 1,967 (46-6,904) Osipcheon in Yeongdeok (2008, 2015) 58 (98.3) 1,121 (2-5,320) 2 (100) 848 (310-1,385) 60 (98.4) 1,112 (2-5,320) Hyeongsan-gang (2015) 8 (100) 2,613 (195-7,650) 33 (100) 806 (88-5,833) 41 (100) 1,158 (88-7,650) Cheokgwacheon (2015) 15 (100) 895 (76-2,670) 50 (100) 940 (57-4,236) 65 (100) 929 (57-4,236) Taehwa-gang (2015) 9 (64.3) 5 (1-31) 1 (100) 72 10 (66.7) 12 (1-72) Total 203 (94.0) 851 (1-7,650) 169 (93.4) 1,240 (1-14,419) 372 (93.7) 1,028 (1-14,419) a No. of CaMc per fish. b Already reported in Cho et al. (2014) [12]. Table 5. Overall infection status with CaMc in rasborinid fish, Zacco platypus and Z. temminckii, by the water systems of Korea Locality (river) No. (%) Zacco platypus a Z. temminckii a Total (range) e No. (%) (range) e No. (%) (range) e Han-gang 253 (99.6) 1,904 (1-25,850) 300 (99.0) 1,278 (2-11,470) 553 (99.3) 1,564 (1-25,850) Geum-gang 133 (89.3) 4,253 (1-43,340) 127 (100) 343 (7-6,920) 260 (94.2) 2,343 (1-43,340) Mangyeong-gang b 56 (96.6) 137 (1-1,148) 80 (100) 193 (1-2,536) 136 (98.6) 170 (1-2,536) Yeongsan-gang 80 (100) 1,403 (5-19,200) 4 (100) 3,290 (123-7,200) 84 (100) 1,493 (5-19,200) Tamjin-gan 213 (100) 1,970 (11-13,420) 178 (100) 1,048 (12-7,120) 391 (100) 1,550 (11-13,420) Seomjin-gang 311 (99.7) 596 (6-7,440) 220 (99.1) 336 (1-4,300) 531 (99.4) 488 (1-7,440) Nakdong-gang c 323 (100) 4,383 (2-30,500) 390 (100) 4,050 (55-41,460) 713 (100) 4,201 (2-41,460) Nakdong-gang d 542 (91.1) 2,026 (1-24,190) 236 (95.6) 699 (1-10,501) 778 (92.4) 1,623 (1-24,190) Streams in the east coast 203 (94.0) 851 (1-7,650) 169 (93.4) 1,240 (1-14,419) 372 (93.7) 1,028 (1-14,419) Total 2,114 (96.1) 2,109 (1-43,340) 1,704 (98.4) 1,567 (1-41,460) 3,818 (97.1) 1,867 (1-43,340) a Total 2,200 Z. platypus and 1,732 Z. temminckii were examined. b Soyangcheon and Jungeupcheon (a stream of Dongjingang). c in Gyeongsangnam-do. d in Gyeongsangbuk-do. e No. of CaMc per fish. Infection status with CaMc in Zacco spp. from the streams in the east coast of Korea CaMc were detected in 372 (93.7%) Zacco spp. (203 Z. platypus: 94.0% and 169 Z. temminckii: 93.4%) and their average density was 1,028 (851 in Z. platypus and 1,240 in Z. temminckii) per fish. The infection status with CaMc by the surveyed areas was detailed in Table 4. Overall infection status with CaMc in Zacco spp. by the water systems of Korea CaMc were detected in 3,818 (97.1%) (2,114 Z. platypus: 96.1% and 1,704 Z. temminckii: 98.4%) out of 3,932 Zacco spp. examined and their density was 1,867 (2,109 in Z. platypus and 1,567 in Z. temminckii) per fish. The prevalences with CaMc were high, 93.7-100%, in Zacco spp. from all surveyed areas. Their densities were more or less different by the surveyed areas and fish species. The infection status with CaMc by the surveyed areas was detailedly revealed in Table 5. Infection status with CaMc in Opsariichthys uncirostris amurensis from the water systems of Korea CaMc were detected in 222 (74.8%) out of 297 O. uncirostris amurensis examined and their density was 1-4,480 (278 in average) per fish. The infection status with CaMc by the surveyed areas was detailedly shown in Supplementary Table S1.

Sohn et al.: Centrocestus armatus metacercaria in fish from Korea 347 Fish species with CaMc except for the rasborinid fish CaMc were also detected in total 41 fish species except for the rasborinid fish, Z. platypus, Z. temminckii and O. uncirostris amurensis. Fish species with CaMc by the surveyed areas (year examined) were detailedly designated in Supplementary Table S2. DISCUSSION In the present study, we investigated the infection status with CaMc nationwidely in the freshwater fishes from the water systems of major rivers in Korea. Total 89 times in 50 sites (Han-gang: 11 times in 8 sites; Geum-gang: 10 times in 6 sites; Mangyeong-gang and Dongjin-gang: 5 times in 2 sites; Yeongsan-gang and Tamjin-gang: 10 times in 5 sites; Seomjin-gang: 13 times in 8 sites; Nakdong-gang: 29 times in 13 sites; streams in the east coast: 11 times in 8 sites) were surveyed. However, the data from the 12 times surveys performed in 10 sites (Hangang: 9 times in 7 sites; streams in the east coast: 3 times in 3 sites) were already reported in Cho et al. [12]. At any rate, total 14,977 fish including 2,200 Z. platypus, 1,732 Z. temminckii and 297 O. uncirostris amurensis were examined in this study. The proportion of Zacco spp. is more or less different by the surveyed sites (19.8-33.0%: 26.3% in average) and fish species (Z. platypus: 9.2-22.1%; Z. temminckii: 9.1-14.4%). These differences are due to the fish ecology in surveyed rivers. The prevalences with CaMc were very high, 93.7-100% (97.1% in average), in Zacco spp. from all surveyed sites. However, their densities were more or less different by the surveyed areas, and slightly higher in Z. platypus (2,109 in average) than in Z. temminckii (1,567). They were most high in Nakdong-gang in Gyeongsangnam-do, (4,201 in average), and followed by Geum-gang (2,343), Nakdong-gang in Gyeongsangbuk-do (1,623), Hangang (1,564), Tamjin-gang (1,550), Yeongsan-gang (1,493), streams in the east coast (1,028), Seomjin-gang (488) and Mangyeong-gang and Dongjin-gang (170) (Table 5). The prevalence in another species of rasborinid fish, O. uncirostris amurensis, was 74.8% (222 out of 297 fish examined) and their density was 278 per fish. On the other hand, Hong et al. [10] reported 86.8% and 78.5% prevalences in Z. platypus (164 out of 189 fish examined) and Z. temminckii (142 out of 181 fish examined), from 19 sites in the 6 major rivers of Korea. They also reported that densities with CaMc were more higher in Z. platypus (224 in average) than in Z. temminckii (131) [10]. In Hong et al. [10], the prevalences with CaMc in Zacco spp. were more or less different by the surveyed rivers, 60.6-100% (82.7% in average), and their densities were most high in Nakdonggang (347 in average) and followed by Seomjin-gang (120), Tamjin-gang (59), Yeongsan-gang (44), Han-gang (29) and Geum-gang (27). The endemicities of CaMc in Zacco spp. in our study is much higher when we compared with those of Hong et al. [10]. However, the infection trend by fish species, more higher endemicity in Z. platypus than in Z. temminckii, is the same in Hong et al. [10]. Therefore, it appears that the endemicity of CaMc is currently much higher than in the past. It s reason why is obscure, but we can only suppose with general knowledges in the Parasitology textbook. Recently, the ecological conditions for this heterophyid fluke will be much better than in the past in Korea. Chun [6] detected CaMc in 9 fish species, i.e., Z. platypus, Z. temminckii, P. altivelis, P. esocinus, P. parva, P. herzi, T. hakonensis, S. gracilis maejimae, S. japonicus coreanus, from the water systems of Nakdong-gang in Gyeongsangnam-do. Rhee et al. [8] found CaMc in 7 fish species, i.e., Aphyocypris chinensis, C. auratus, C. splendidus, Microphysogobio yaluensis, Rhodeus uyekii, P. parva and Z. platypus, from Mangyeong-gang and they also detected them in 5 fish species, i.e., Gnathopogon similis (=Squalidus intermedius), Pseudobagrus fulvidraco, P. parva, Rhodeus ocellatus ocellatus and Z. platypus, from Dongjin-gang [9]. Cho et al. [12] reported 15 fish species, i.e., A. rhombeus, A. signifier, C. herzi, G. brevibarba, H. longirostris, H. mylodon, K. rotundicaudata, L. andersoni, M. longidorsalis, O. uncirostris amurensis, Orthrias toni, P. esocinus, P. herzi, R. oxycephalus, and T. hakonensis, from the water systems of Gangwon-do as the new second intermediate hosts of C. armatus. Recently, Sohn et al. [13] detected CaMc in 5 fish species, Z. platypus, Z. temminckii, A. rhombeus, A. yamatsutae, and A. macropterus from the water systems of Hantangang and Imjin-gang. In this study, we found CaMc in total 44 fish species including 3 rasborinid fish, Z. platypus, Z. temminckii and O. uncirostris amurensis. Among them, 25 species were already reported in the previous studies [6-13]. Remain 19 species, i.e., A. springeri, A. gracilis, A. koreensis, A. majusculus, C. urotaenia, C. lutheri, C. kawamebari, G. strigatus, H. labeo, H. eigenmanni, M. salmoides, O. platycephala, O. masou masou, P. phoxinus, S. nigripinis morii, S. variegatus wakiyae and S. asotus, are to be the new second intermediate hosts of C. armatus in Korea. Accordingly, total 50 fish species (33 genera) are listed as the second intermediate hosts of C. armatus in Korea. In the genus Centrocestus, the number of circumoral spines is considered to be the most reliable character in the species identification. C. armatus, having 42-48 circumoral spines, was

348 Korean J Parasitol Vol. 56, No. 4: 341-349, August 2018 first described by Tanabe [1] in Japan. Kobayashi [18] classified members in the genus Centrocestus into 4 groups on the basis of the number of circumoral spines. The group of C. yokogawai has less than 30 circumoral spines (26-30), C. formosanus has 32-40 ones, C. armatus has 42-48 ones, and C. polyspinosus has more than 50 ones (50-60) respectively. Among Centrocestus species with 42-48 circumoral spines, C. armatus is differentiated by characteristic morphological features include a small number of intrauterine eggs, the median location of the ovary, and the side by side location of the 2 testes [2,20]. On the other hand, Chai et al. [21] proposed to consider 6 species, i.e., C. cuspidatus, C. armatus, C. formosanus, C. kurokawai, C. polyspinosus, and C. asadai, in the genus Centrocestus as the valid ones. In Korea, 3 species of Centrocestus fluke, i.e., C. armatus, C. asadai and C. formosanus, were reported in the literatures [2,4, 6,22,23]. Choi et al. [22] described C. asadai adults, which were recovered in the small intestines of mouse experimentally with the metacercariae from Tribolodon taczanowskii. Cho et al. [23] reported only the presence of C. formosanus cercariae in Korea. They found these cercariae from Semisulcospira libertina snails collected around the Jinyang-ho (ho means lake) in Gyeongsangnam-do, Korea. In regard to C. armatus, a lot of findings on the intermediate and definitive hosts were revealed by many Korean workers [2,4,6-17]. Adult worms were recovered from a human, the large egret (Egretta alba modesta) and stray cats (Felis catus) naturally [2,14-17]. Cercariae of this fluke were detected from Semisulcospira snails collected in Judongcheon in Dalseong-gun, Gyeongsangbuk-do [4]. Metacercarial infections were verified in the surveys of freshwater fish including Z. platypus and Z. temminckii [5-13]. Accordingly, we can know that C. armatus is predominant species in Korea and the presence of another 2 species, C. asadai and C. formosanus, is obscure, so there are no reports about those flukes since the first records [22,23]. Conclusively, it is confirmed that rasborinid fish, Z. platypus, Z. temminckii, and O. uncirostris amurensis, are highly and heavily with CaMc and the densities with CaMc are more or less different by the surveyed areas and fish species. Additionally, 19 fish species are to be the new second intermediate hosts of C. armatus in Korea. ACKNOWLEDGMENTS This study was supported by an anti-communicable diseases control program, 2013-0171 (Studies on the biological resources of human infecting trematodes and their larval infections in intermediate hosts), 2014-E54002-00 (Investigation of fishborne parasites and acquisition of their biological resources in the southern regions of Korea) and 2015-E54001-00 (Investigation of fish-borne parasites and acquisition of their biological resources in the easthern regions of Korea) of National Institute of Health (NIH), Korea Centers for Disease Control and Prevention (KCDCP). This study was partly supported by a grant of Korea Association of Health Promotion (2017). We thank Jung-A Kim and Hee-Joo Kim (Department of Parasitology and Tropical Medicine, Gyeongsang National University College of Medicine, Jinju, Korea), for their help in the examination of fish. CONFLICT OF INTEREST The authors have no conflicts of interest concerning the work reported in this paper. REFERENCES 1. 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Supplementary Table S1. Infection status of CaMc in Opsariichthys uncirostris amurensis from the water systems of Korea Locality (year) No. of fish examined No. (%) of fish Range No. of CaMc detected Suipcheon (2009) 5 1 (20.0) - 3 Hongcheon-gang (2010) 2 2 (100) 1,460-2,290 1,875 Cheonnae-gang (2013-2015) 4 4 (100) 12-562 255 Yugucheon (2013, 2015) 18 18 (100) 182-5,240 871 Nonsancheon (2013) 7 2 (28.6) 1-2 2 Soyangcheon (2013) 1 1 (100) - 2 Osucheon (2012) 21 16 (76.2) 3-35 11 Seomjin-gang in Sunchang (2014, 2015) 14 14 (100) 8-976 153 Seomjin-gang in Gurye (2014) 13 13 (100) 103-631 301 Naeseongcheon in Yecheon (2008, 2013) 19 14 (74.0) 1-203 32 Yeonggang (2009) 7 7 (100) 12-50 30 Nakdong-gang in Andong (2009) 8 8 (100) 2-973 130 Namdaecheon in Uiseong (2009) 10 9 (90.0) 2-34 12 Wicheon (2008, 2015, 2016) 10 10 (100) 24-748 305 Hoecheon (2013) 10 1 (10.0) - 2 Jisucheon (2014) 4 4 (100) 64-728 352 Hamancheon (2014) 23 13 (56.5) 1-26 4 Hyeongsan-gang (2015) 3 3 (100) 41-446 222 Taehwa-gang (2015) 10 2 (20.0) 3-7 5 Total 292 217 (74.3) 1-4, 480 284

Supplementary Table S2. Fish species with CaMc except for the rasborinid fish in the present study Fish species Locality (year examined) Abbottina springeri Seomjin-gang in Sunchang (2014) Acanthorhodeus gracilis Naeseongcheon in Yecheon (2008); Osucheon (2012); Seomjin-gang in Gurye (2014) & Sunchang (2014, 2015); Cheokgwacheon (2015); Cheonnae-gang (2015); Soyangcheon (2015); Wicheon (2017); Yangcheon (2017) Acanthorhodeus macropterus Yangcheon (2010); Hoecheon (2013); Hantan-gang (2014); Hyeongsan-gang (2015); Soyangcheon (2015) Acheilognathus koreensis Hantan-gang (2014); Hoengcheon (2014); Wicheon (2014, 2015); Jicheon (2014); Seomjin-gang in Sunchang (2015) Acheilognathus lanceolatus Tamjin-gang (2014): Yugucheon (2015) Acheilognathus majusculus Yangcheon (2014) Acheilognathus rhombeus Yangcheon (2010, 2017); Hantan-gang (2012, 2014); Wicheon (2014, 2015); Seomjin-gang in Gurye (2014); Osipcheon in Yeongduk (2015); Cheokgwacheon (2015); Yugucheon (2015); Seomjin-gang in Sunchang (2015); Tamjin-gang (2015, 2017) Acheilognathus signifier Hantan-gang (2010) Acheilognathus yamatsutae Wicheon (2013, 2014, 2015); Yangcheon (2017) Carassius auratus Hantan-gang (2010); Wicheon (2015); Cheokgwacheon (2015); Yangcheon (2017) Chaenogobius urotaenia Osipcheon in Samcheok (2015) Cobitis lutheri Wicheon (2017) Coreoleuciscus splendidus Hongcheon-gang (2010); Pyeongchang-gang (2013); Banbyeoncheon (2015) Coreoperca herzi Hongcheon-gang (2010); Yangcheon (2010, 2015); Choyang-gang (2012); Wicheon (2014); Seomjin-gang in Gurye (2014) & Gokseong (2015) Coreoperca kawamebari Tamjin-gang (2015, 2017) Gnathopogon strigatus Songdaecheon (2012); Seomjin-gang in Sunchang (2014, 2015); Gobiobotia brevibarba Hantan-gang (2013) Hemibarbus labeo Wicheon (2016) Hemibarbus longirostris Hongcheon-gang (2010); Yangcheon (2010, 2015); Hantan-gang (2012, 2013); Naeseongcheon in Yecheon (2014); Cheonna-gang (2014); Soyangcheon (2014, 2015); Yugucheon (2015); Seomjin-gang in Sunchang (2015); Tamjingang (2017) Hemibarbus mylodon Pyeongchang-gang (2013) Hemiculter eigenmanni Tamjin-gang (2014) Koreocobitis rotundicaudata Choyang-gang (2012) Ladislabia taczanowskii Osipcheon in Samcheok (2015) Liobagrus andersoni Choyang-gang (2012) Microphysogobio longidorsalis Hantan-gang (2012, 2013); Pyeongchang-gang (2013) Micropterus salmoides Tamjin-gang (2014): Taehwa-gang (2015); Yangcheon (2017) Odontobutis platycephala Yangcheon (2010, 2013); Wicheon (2014, 2016); Hyeongsan-gang (2015); Soyangcheon (2015) Onchorhynchus masou masou Namdaecheon in Yangyang (2015); Osipcheon in Samcheok (2015) Phoxinus phoxinus Osipcheon in Samcheok (2015) Plecoglossus altivelis Namdaecheon in Yangyang (2015); Osipcheon in Samcheok (2015) Pseudogobio esocinus Wicheon (2008); Yeong-gang (2009); Yangcheon (2010); Hantan-gang (2012, 2014); Choyang-gang (2012); Pyeongchang-gang (2013); Yugucheon (2013); Yangcheon (2013, 2017); Jisucheon (2014); Jicheon (2014); Seomjin-gang in Gurye (2014) & Sunchang (2015); Cheonnae-gang (2015); Soyangcheon (2015); Namsancheon (2016) Pseudorasbora parva Seomjin-gang in Gurye (2014) Pungtungia herzi Bukcheon in Sangju (2008); Hongcheon-gang (2010); Yangcheon (2013, 2016, 2017); Hantan-gang (2014); Namdaecheon in Yangyang (2015); Wangpicheon (2015); Wicheon (2017) Rhodeus ocellatus Hantan-gang (2013); Osucheon (2013) Rhynchocypris oxycephalus Osipcheon in Samcheok (2009, 2015); Namdaecheon in Yangyang (2015); Wicheon (2015); Cheokgwacheon (2015); Taehwa-gang (2015) Sarcocheilichthys nigripinis Wicheon (2017) Sarcocheilichthys variegatus Soyangcheon (2013); Seomjin-gang in Sunchang (2015) & Gokseong (2015) Silurus asotus Baekcheon (2009) Squalidus gracilis maejimae Osipcheon in Yeongdeok (2008); Seomjin-gang in Sunchang (2015) Squalidus japonicus coreanus Yangcheon (2013, 2017); Seomjin-gang in Sunchang (2015) & Gokseong (2015); Wicheon (2017) Tribolodon hakonensis Namdaecheon in Yangyang (2009, 2015); Osipcheon in Samcheok (2015); Wangpicheon (2015)