Multilocus Genotyping of Sympatric Hepatozoon Species Infecting the Blood of Ontario Ranid Frogs Reinforces Species Differentiation and Identifies an Unnamed Hepatozoon Species.

Intraerythrocytic gamonts of at least 2 named Hepatozoon species have been reported to infect the erythrocytes of ranid frogs in Ontario, Canada. Although gamonts of both species are morphometrically similar, the cytopathological changes that 1 of these species, Hepatozoon clamatae, causes to host erythrocytes, manifested by nuclear fragmentation, was used historically to distinguish this parasite from Hepatozoon catesbianae. Molecular characterization of these 2 Hepatozoon species has been equivocal in correlating genotype with gamont morphotype. Amplification and sequencing of multiple potential genotyping loci within the nuclear (18S ribosomal deoxyribonucleic acid [rDNA]; internal transcribed spacer 1), apicoplast (23S rDNA), and mitochondrial genomes (complete genomes, cytochrome c oxidase subunits I and III [COI and COIII], and cytochrome b) were conducted on Hepatozoon species that infect ranid frogs in Ontario. Sequence data were then used to evaluate the diversity of parasites present in these amphibian hosts and to assign genotypes to gamont morphotypes, if possible. Three distinct genotypes were identified at all loci; the data permitted the discovery of a third, formerly unrecognized Hepatozoon species in ranid frogs from Ontario. Although all genetic loci demonstrated differences between Hepatozoon species, mitochondrial COIII sequences were most suitable for genotypic differentiation of these parasites of frogs. Linking genotypes to gamont morphotypes proved impossible; genotypes identified as H. catesbianae and H. clamatae were found in infections with or without nuclear fragmentation of their host erythrocytes. This suggests that differentiating these species must rely on suitable genotyping methods for identification in the blood of their amphibian intermediate hosts.