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

Abstract

― 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.