Ultrastructural and Molecular Phylogenetic Identification of the Diplomonad Flagellate Spironucleus salmonis Infecting Hatchery-reared Salmonid Fishes in Hokkaido

Systemic infections caused by diplomonad flagellates belonging to the genus Spironucleus commonly result in heavy mortalities in salmonids. ​Diplomonad flagellates were found in the intestinal lumen of hatchery-reared juvenile chum Oncorhynchus keta and masu salmon O. masou in Hokkaido. ​We performed ultrastructural observation and molecular phylogenetic analysis using the small subunit ribosomal RNA gene (SSUrDNA) of the flagellates to clarify their taxonomy. ​The flagellates showed elongated and tapered nuclei, and sub-apical location of the kinetosomes relative to the nuclei: which are the distinguishing characters of the genus Spironucleus among three genera of parasitic diplomonads. ​In addition, the flagellates had electron dense plaques at their posterior end, a posterior sac of dense free ribosomes, an electron dense body, and bowl-shaped membranous structures, identifying them as S. salmonis among four Spironucleus species that affect fish. ​The ultrastructure of the flagellates showed no variations between the two host species of salmon. ​All the isolated flagellate SSUrDNAs had the same 1,031 bp sequence, and no difference in the sequence. ​The SSUrDNA showed the highest homology (99%) with S. salmonis among 11 diplomonad flagellates. ​Thus, this study successfully used ultrastructure diagnosis and molecular phylogenetic analysis to confirm S. salmonis as the diplomonad flagellate that has infected hatchery-reared chum and masu salmon in Hokkaido.