We examined for the presence of trypanosomes in blood samples from 259 anurans (47 species from 8 families), the majority of which were from the Brazilian Amazonia, Atlantic Forest and Pantanal biomes. Trypanosomes were detected by a combination of microhaematocrit and haemoculture methods in 45% of the anurans, and 87 cultures were obtained: 44 from Hylidae, 22 from Leptodactylidae, 15 from Bufonidae, 5 from Leiuperidae and 1 from an unidentified anuran. High morphological diversity (11 morphotypes) was observed among blood trypanosomes from anurans of different species and of the same species as well as among trypanosomes from the same individual. Conversely, morphologically similar trypanosomes were found in anurans from distinct species and biomes. ITS and SSU rDNA polymorphisms revealed high diversity among the 82 isolates examined. Twenty-nine genotypes could be distinguished, the majority distributed in 11 groups. Phylogenetic relationships based on rDNA sequences indicated that isolates from more phylogenetically related anurans are more closely related. Comparison of anuran trypanosomes from Brazil and other countries revealed several new species among the isolates examined in this study. Phylogenetic relationships suggest that host restriction, host switching and overall ecogeographical structure may have played a role in the evolution of the anuran trypanosomes.
BackgroundBat trypanosomes have been implicated in the evolutionary history of the T. cruzi clade, which comprises species from a wide geographic and host range in South America, Africa and Europe, including bat-restricted species and the generalist agents of human American trypanosomosis T. cruzi and T. rangeli.MethodsTrypanosomes from bats (Rhinolophus landeri and Hipposideros caffer) captured in Mozambique, southeast Africa, were isolated by hemoculture. Barcoding was carried out through the V7V8 region of Small Subunit (SSU) rRNA and Fluorescent Fragment Length barcoding (FFLB). Phylogenetic inferences were based on SSU rRNA, glyceraldehyde phosphate dehydrogenase (gGAPDH) and Spliced Leader (SL) genes. Morphological characterization included light, scanning and transmission electron microscopy.ResultsNew trypanosomes from bats clustered together forming a clade basal to a larger assemblage called the T. cruzi clade. Barcoding, phylogenetic analyses and genetic distances based on SSU rRNA and gGAPDH supported these trypanosomes as a new species, which we named Trypanosoma livingstonei n. sp. The large and highly polymorphic SL gene repeats of this species showed a copy of the 5S ribosomal RNA into the intergenic region. Unique morphological (large and broad blood trypomastigotes compatible to species of the subgenus Megatrypanum and cultures showing highly pleomorphic epimastigotes and long and slender trypomastigotes) and ultrastructural (cytostome and reservosomes) features and growth behaviour (when co-cultivated with HeLa cells at 37°C differentiated into trypomastigotes resembling the blood forms and do not invaded the cells) complemented the description of this species.ConclusionPhylogenetic inferences supported the hypothesis that Trypanosoma livingstonei n. sp. diverged from a common ancestral bat trypanosome that evolved exclusively in Chiroptera or switched at independent opportunities to mammals of several orders forming the clade T. cruzi, hence, providing further support for the bat seeding hypothesis to explain the origin of T. cruzi and T. rangeli.
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