The alveolate protozoan parasite Perkinsus olseni infects a range of marine bivalves inhabiting tidal flats and shallow subtidal zones, causing considerable damage to shellfish industries. Infection by P. olseni is typically assessed using Ray’s fluid thioglycollate medium (RFTM) assay, with gill tissue often employed as a diagnostic proxy for whole-body infection. However, the diagnostic reliability of gill-based assays across diverse ecological settings—particularly under low-infection conditions—remains uncertain. In this study, we investigated tissue-specific distribution and the diagnostic performance of the RFTM assay in detecting P. olseni in Manila clams (Ruditapes philippinarum) collected from 6 tidal flats along Korea’s west and south coast. The assay was applied to 6 different tissues, including gills and visceral mass. Infection prevalence reached 100% at most sites, except at Padori (90%). Whole-body infection intensity ranged from 0.1×104 to 3.7×106 cells per gram of tissue. The visceral mass consistently harbored the largest proportion of parasites (27.8%–49.0%), followed by the mantle (17.4%–30.6%) and gills (19.4%–25.2%). Gill infection levels correlated strongly with whole-body infection intensity (r²=0.6–0.95), supporting their diagnostic value in high-infection areas. However, at Padori—where infection levels were lowest—the efficacy of the gill assay dropped to 56%, resulting in a 44% false negative rate. These results underscore the limitations of relying solely on gill tissue in low-infection environments and highlight the need for a context-dependent diagnostic approach. A dual-tissue strategy incorporating both gill and whole-body samples is recommended to improve diagnostic accuracy in P. olseni surveillance of Manila clam populations.
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Acanthoparyphium shinanense n. sp. (Digenea: Echinostomatidae) is described from chicks experimentally infected with the metacercariae encysted in 2 brackish water clam species, Ruditapes philippinarum and Coecella chinensis, in the Republic of Korea. The metacercariae were round to oval, armed with 23 collar spines, and 0.216 (0.203-0.226) mm in diameter. From 5 chicks experimentally infected each with 200 metacercariae, 34 juvenile (5-day-old worms) and 104 adult flukes (7-day-old worms) were harvested from their small intestines, with the average worm recovery rate of 13.8%. The adult flukes were 3.18 (2.89-3.55) mm long and 0.68 (0.61-0.85) mm wide, with an elongated, posteriorly tapering body, and a prominent head collar armed with 23 collar spines arranged in a single uninterrupted row. The posterior testis of A. shinanense was longitudinally elongated, which is similar to Acanthoparyphium spinulosum Johnston, 1917 but unique from the other closely related species, including Acanthoparyphium tyosenense Yamaguti, 1939, Acanthoparyphium kurogamo Yamaguti, 1939, and Acanthoparyphium marilae Yamaguti, 1934. The eggs of A. shinanense were larger than those of A. spinulosum, and the anterior extent of 2 lateral groups of vitellaria was slightly more limited in A. shinanense than in A. spinulosum. Molecular analysis of nuclear and mitochondrial genes revealed low homology with A. spinulosum from USA (96.1% in 5.8S rRNA) and Ukraine (97.9% in 28S rRNA), Acanthoparyphium n. sp. from USA (98.0% in 28S rRNA), and Acanthoparyphium sp. from Australia, Kuwait, and New Zealand. Biological characteristics, including its first intermediate host and natural definitive hosts, as well as its zoonotic capability, should be elucidated.
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Life cycle stages, including daughter sporocysts, cercariae, and metacercariae, of Parvatrema duboisi (Dollfus, 1923) Bartoli, 1974 (Digenea: Gymnophallidae) have been found in the Manila clam Ruditapes philippinarum from Aphaedo (Island), Shinan-gun, Jeollanam-do, Korea. The daughter sporocysts were elongated sac-like and 307-570 (av. 395) μm long and 101-213 (av. 157) μm wide. Most of the daughter sporocysts contained 15-20 furcocercous cercariae each. The cercariae measured 112-146 (av. 134) μm in total length and 35-46 (av. 40) μm in width, with 69-92 (av. 85) μm long body and 39-54 (av. 49) μm long tail. The metacercariae were 210-250 (av. 231) μm in length and 170-195 (av. 185) μm in width, and characterized by having a large oral sucker, genital pore some distance anterior to the ventral sucker, no ventral pit, and 1 compact or slightly lobed vitellarium, strongly suggesting P. duboisi. The metacercariae were experimentally infected to ICR mice, and adults were recovered at day 7 post-infection. The adult flukes were morphologically similar to the metacercariae except in the presence of up to 20 eggs in the uterus. The daughter sporocysts and metacercariae were molecularly (ITS1-5.8S rDNA-ITS2) analyzed to confirm the species, and the results showed 99.8-99.9% identity with P. duboisi reported from Kyushu, Japan and Gochang, Korea. These results confirmed the presence of various life cycle stages of P. duboisi in the Manila clam, R. philippinarum, playing the role of the first as well as the second intermediate host, on Aphae-do (Island), Shinan-gun, Korea.
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Gymnophallid metacercariae found in the Manila clam Ruditapes philippinarum (‘Banjirak’ in Korean) from Gochang-gun, Jeollabuk-do, Korea were morphologically and molecularly confirmed to be Parvatrema duboisi (Dollfus, 1923) Bartoli, 1974. The metacercariae were morphologically characterized by having a large oral sucker, small ventral sucker, genital pore some distance anterior to the ventral sucker, no ventral pit, and 1 compact or slightly lobed vitellarium, which were all compatible with P. duboisi. Some of the metacercariae were experimentally fed to mice, and adult flukes were recovered at day 7 post-infection. The morphology of the adult flukes was basically the same as that of the metacercariae except for the presence of uterine eggs; the uterus was filled with up to 40 eggs. The nucleotide sequences (1,193 bp) from ITS regions (ITS1, 5.8S rDNA, and ITS2) of the metacercariae showed 99.7% identity with P. duboisi and 75.7% identity with Gymnophalloides seoi deposited in GenBank. These results confirmed the presence of P. duboisi metacercariae in the Manila clam R. philippinarum in an estuary region of Gochang-gun, Korea.
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A survey was performed to know the recent infection status of digenetic trematode metacercariae in clams and oysters from 4 sites in western coastal regions of the Republic of Korea (=Korea). Four species of clams (Mactra veneriformis, Ruditapes philippinarum, Cyclina sinensis, and Saxidomus purpuratus) were collected from Taean-gun, Chungcheongnam-do (Province), Buan-gun (County) and Gochang-gun, Jeollabuk-do, and oysters, Crassostrea gigas, from Shinan-gun, Jeollanam-do were transferred to our laboratory on ice and examined by the artificial digestion method. The metacercariae of Himasthla alincia were detected in 3 species of clams, M. veneriformis, R. philippinarum, and C. sinensis from the 3 surveyed areas. The positive rate and the mean density per clam infected were 98.9% (30.8 metacercariae) in M. veneriformis, 60.0% (5.0) in R. philippinarum, and 96.0% (28.4) in C. sinensis. The positive rate (mean density) of Acanthoparyphium tyosenense metacercariae in M. veneriformis was 50.0% (2.1) from Taean-gun and 70.0% (2.8) from Gochang-gun. The metacercariae of Parvatrema spp. were detected in M. veneriformis and R. philippinarum from Taean-gun and Gochang-gun; the positive rate (mean density) was 63.3% (4,123) and 50.0% (19) in M. veneriformis, and 6.7% (126) and 100% (238) in R. philippinarum from the 2 regions, respectively. The metacercariae of Gymnophalloides seoi were detected in all 30 oysters from Shinan-gun, and their average density per oyster was 646. From the above results, it has been confirmed that more than 3 species of metacercariae are prevalent in clams from the western coastal regions, and G. seoi metacercariae are still prevalent in oysters from Shinan-gun, Jeollanam-do, Korea
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Metacercariae of Acanthoparyphium marilae Yamaguti, 1934 (Digenea: Echinostomatidae) were discovered in an intertidal clam, Mactra veneriformis, in a southwestern coastal area of the Republic of Korea. A total of 128 metacercariae were detected from 10 clams examined. They were round, 320 ?m in average diameter, with 23 collar spines. They were fed experimentally to chicks, and 10 days later adult flukes were obtained. The adults were morphologically characterized by the head collar with a single row of 23 dorsally uninterrupted spines, without special end group spines, a round ventral sucker, 2 round and tandem testes, and vitellaria extending at lateral fields from the posterior extremity not beyond the middle level of the posterior testis. The most characteristic feature of this species was the limited distribution of vitellaria, which differs from Acanthoparyphium tyosenense Yamaguti, 1939, the metacercariae of which are encysted in the same mollusk species. This is the first report in which the metacercariae of this species were detected, and the intertidal bivalve, M. veneriformis, has been identified as a second intermediate host for A. marilae.
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Parvatrema chaii n. sp. (Digenea: Gymnophallidae) is described using the worms recovered from experimentally infected mice in Korea. The metacercariae were collected from surf-clams, Mactra veneriformis, from a tidal flat in Sochon-gun, Chungchongnam-do. The metacercariae were elliptical (0.262 × 0.132 mm), and the genital pore had an anterior arch of 16-17 sensory papillae in scanning electron microscopic view. Adult worms were ovoid to foliate (0.275-0.303 by 0.140-0.150 mm), and their characteristic features included the presence of lateral lips, short esophagus, genital pore located some distance anterior to the ventral sucker, club-shaped seminal vesicle, a compact to slightly lobed vitellarium, elliptical eggs (0.018-0.020 by 0.010-0.013 mm), and absence of the ventral pit. This gymnophallid is classified as a member of the genus Parvatrema because of the location of the wide genital pore some distance from the ventral sucker, and the absence of the ventral pit. It differs from previously reported Parvatrema species, including the type species, P. borinquenae. In particular, the morphologies of the vitellarium and the genital pore with an anterior arch of 16-17 sensory papillae are unique features. Therefore, we propose it as a new species, Parvatrema chaii n. sp. (Digenea: Gymnophallidae).
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