Novel clade of Rickettsia spp. from leeches

Abstract

Intracellular rickettsia-like structures were found in the tissues of a glossiphoniid leech, Torix tagoi, by transmission electron microscopy. Diagnostic PCR analysis using specific primers suggested that of the nine glossiphoniid species examined, two species, T. tagoi and Hemicrepsis marginata, harbored bacteria of the genus Rickettsia. A 1.5-kb eubacterial 16S rRNA gene segment obtained from each of these species was amplified by PCR, cloned, and sequenced. Phylogenetic analysis of the 16S rRNA gene demonstrated that the Rickettsia species found in the leeches constituted a novel clade that is distinct from the clade of arthropod-associated Rickettsia species. In natural populations, 97.7% (43 of 44) of T. tagoi leeches and 100% (9 of 9) of H. marginata leeches carried Rickettsia, suggesting that infection with Rickettsia is prevalent in these leeches. This is the first report of Rickettsia found in annelids.

FIG. 1.

Transmission electron microscopic images of the epidermal tissue of T. tagoi. (A) Intracellular rickettsia-like structures. Bar, 500 nm. (B) Enlarged image of the intracellular rickettsia-like structure. Bar, 100 nm. Abbreviations: R, rickettsia-like structure; M, mitochondrion.

FIG. 2.

PCR detection of Rickettsia spp. in glossiphoniid leeches. (A) Detection of mitochondrial cytochrome oxidase I gene of leeches with primers LCO1490 and HCO2198; (B) detection of eubacterial 16S rRNA gene with primers 16SA1 and 16SB1; (C) specific detection of citrate synthase gene of Rickettsia spp. with primers CS1 and CS2; (D) specific detection of 16S rRNA gene of Rickettsia spp. with primers 16SA1 and Rick16SR; (E) specific detection of the ftsZ gene of Wolbachia spp. with primers ftsF and ftsR. Lane M, DNA size markers (1,500, 1,000, 900, 800, 700, 600, 500, 400, 300, and 200 bp from top to bottom); lane 1, Helobdella stagnalis; lane 2, Placobdelloides sp.; lane 3, Ancyrobdella smaragdina; lane 4, H. marginata; lane 5, Glossiphonia complanata; lane 6, Glossiphonia sp.; lane 7, Alboglossiphonia lata; lane 8, T. tukubana; lane 9, T. tagoi; lane 10, a bruchid beetle, Kytorhinus sharpianus, harboring a Rickettsia sp.; lane 11, a bruchid beetle, Callosobruchus chinensis, harboring a Wolbachia sp.; lane 12, no template control. Arrows indicate specific PCR products. Other faint bands are due to nonspecific amplifications.

FIG. 3.

Phylogenetic positions of the Rickettsia spp. from T. tagoi and H. marginata based on the 16S rRNA gene sequences in the α subdivision of the Proteobacteria. (A) An NJ tree; (B) an MP tree. The bootstrap values that were higher than 70%, obtained by 1,000 resamplings for NJ analysis and 100 resamplings for MP analysis, are shown at the nodes. For Rickettsia and Orientia species, the host organisms are given in parentheses. The numbers in brackets are accession numbers.