Molecular phylogeny, distribution, and pathogenicity of a novel thraustochytrid protist in the Manila clam, Ruditapes philippinarum, on the west and south coasts of Korea

Protozoa are major threats to wild and cultured marine bivalves. In this study, a protozoan pathogen was isolated from the hemolymph of the Manila clam, Ruditapes philippinarum, in the coastal region of Chungnam province, Republic of Korea. Based on an 18S rRNA gene sequence analysis, the species was likely a novel thraustochytrid and was designated as Manila clam parasite unknown (MCPX). Bioinformatic and phylogenetic analyses clearly indicated that MCPX is a well-supported lineage in the family Thraustochytriidae. The trophozoites of MCPX had a smooth, round surface without vacuoles, with a diameter of 6.42 ± 1.70 μm. Nuclei were located at the edge of the cell. In a national survey based on conventional PCR, MCPX was widely distributed in Manila clams in the western and southern coastal regions of Korea, while no infection was found on the eastern coast. Digital PCR analysis revealed that MCPX mostly occurred as co-infections with Perkinsus olseni at a low density. An in vivo challenge experiment indicated that Manila clams infected with MCPX had higher mortality rates than those of clams infected with P. olseni. In vitro, greater inhibition of phagocytosis and higher nitric oxide production were observed in the hemolymph of Manila clams upon challenge with the MCPX than in clams infected with P. olseni, suggesting that the species has high pathogenicity. These results provide detailed information on a newly identified parasite at the molecular and morphological levels as well as its pathogenicity in Manila clam for the first time in the Republic of Korea. Furthermore, our findings emphasize the importance of monitoring new pathogens of Manila clams in Korea.