Morphological and Molecular Characterization of Hepatozoon Species Infecting Frogs and Snakes across the Central and Eastern United States

The genus Hepatozoon Miller, 1908 (Apicomplexa: Adeleorina) includes vector-borne, intracellular blood parasites that infect a wide range of vertebrate hosts, including frogs and snakes. Species identification of bloodstream forms is difficult because there are usually not many morphological characters to distinguish species and gamonts of genetically different isolates. Traditionally, Hepatozoon spp. have been distinguished by characters such as pathology to host erythrocytes and/or developmental stages in the invertebrate vector. However, recent molecular studies are finding that these distinctions do not correlate with gene sequence data. Specifically, this is the case for 2 closely related Hepatozoon species that infect North American anurans, Hepatozoon catesbianae and Hepatozoon clamatae. Gamonts infecting erythrocytes of these 2 species are morphologically indistinguishable, and traditionally they have been differentiated based on whether they fragment the host erythrocyte nucleus; however, recent genetic studies indicate that this character does not correlate with specific genotypes. In this study, we sampled frogs and snakes from the east central United States for Hepatozoon spp. and compared their effects on the host erythrocytes with genotype. Hepatozoon spp. infections were morphologically characterized with microscopy and molecularly characterized with Sanger sequencing at 3 loci (COIII, ITS-1, and 18S rDNA). We found that individuals of 3 ranid species (Rana catesbeiana, Rana clamitans, and Rana sphenocephala) were infected with Hepatozoon species. Of those, only individuals of R. clamitans were infected with Hepatozoon spp. that fragmented the host erythrocyte nuclei. As previously reported, mixed infections, determined with both microscope observation of fragmentation characters and Sanger sequencing, were common and obfuscated species identification and the usefulness of the fragmentation character in differentiating H. catesbianae and H. clamatae infecting North American anurans. We recommend that future studies aim to establish better definite links between cellular fragmentation characteristics and DNA sequences to differentiate these 2 species. We also report and characterize Hepatozoon cf. sipedon from 3 snake species. Infected erythrocytes in all 3 snake species displayed variation in the extent of cytoplasm clearing. Sequences from these 3 snakes were identical at ITS-1 and 18S rDNA (COIII was sequenced from only 1 isolate). In our 18S rDNA phylogeny, Hepatozoon spp. infecting frogs were in a single clade, whereas Hepatozoon spp. infecting snakes were found in multiple clades with Hepatozoon spp. that infect other hosts, including lizards, small mammals, and frogs. This study adds to a growing number of studies that indicate snakes are capturing Hepatozoon spp. from their prey, and we discuss the implications of these host captures for the life cycle evolution of Hepatozoon spp. infecting snakes.