Small subunit ribosomal metabarcoding reveals extraordinary trypanosomatid diversity in Brazilian bats

Background: Bats are a highly successful, globally dispersed order of mammals that occupy a wide array of ecological niches. They are also intensely parasitized and implicated in multiple viral, bacterial and parasitic zoonoses. Trypanosomes are thought to be especially abundant and diverse in bats. In this study, we used 18S ribosomal RNA metabarcoding to probe bat trypanosome diversity in unprecedented detail. Methodology/Principal Findings: Total DNA was extracted from the blood of 90 bat individuals (17 species) captured along Atlantic Forest fragments of Espírito Santo state, southeast Brazil. 18S ribosomal RNA was amplified by standard and/or nested PCR, then deep sequenced to recover and identify Operational Taxonomic Units (OTUs) for phylogenetic analysis. Blood samples from 34 bat individuals (13 species) tested positive for infection by 18S rRNA amplification. Amplicon sequences clustered to 14 OTUs, of which five were identified as Trypanosoma cruzi I, T. cruzi III/V, Trypanosoma cruzi marinkellei, Trypanosoma rangeli, and Trypanosoma dionisii, and seven were identified as novel genotypes monophyletic to basal T. cruzi clade types of the New World. Another OTU was identified as a trypanosome like those found in reptiles. Surprisingly, the remaining OTU was identified as Bodo saltans–closest non-parasitic relative of the trypanosomatid order. While three blood samples featured just one OTU (T. dionisii), all others resolved as mixed infections of up to eight OTUs. Conclusions/Significance: This study demonstrates the utility of next-generation barcoding methods to screen parasite diversity in mammalian reservoir hosts. We exposed high rates of local bat parasitism by multiple trypanosome species, some known to cause fatal human disease, others non-pathogenic, novel or yet little understood. Our results highlight bats as a long-standing nexus among host-parasite interactions of multiple niches, sustained in part by opportunistic and incidental infections of consequence to evolutionary theory as much as to public health. Author summary: Bats make up a mega-diverse, intensely parasitized order of volant mammals whose unique behavioural and physiological adaptations promote infection by a vast array of microorganisms. Trypanosomes stand out as ancient protozoan parasites of bats. As cryptic morphology, low parasitaemia and selective growth in culture have recurrently biased survey, we used 18S ribosomal RNA metabarcoding to resolve bat trypanosomatid diversity in Atlantic Forest fragments of southeast Brazil. Next to several unknown species, our deep sequence-based detection and assignment protocol recognized multiple known human-pathogenic trypanosomes, another linked to reptile hosts as well as a non-parasitic kinetoplastid in the blood of various phyllostomid bats. The striking permissivity exposed here, in a region where bat trypanosomes recently featured in a fatal case of Chagas disease, compels further research on bats’ role in the dispersal and spill-over of various microorganisms among humans and wildlife