Metagenomics enables the first detection of

Introduction: Bats play important ecological roles but can also harbor a wide diversity of pathogens, including trypanosomatids. Knowledge about the circulation of Trypanosoma spp. in bat ectoparasites remains limited, particularly in peri-urban environments. Methods: In this study, we used shotgun metagenomic sequencing to investigate the presence of Trypanosoma spp. in streblid flies parasitizing Carollia perspicillata bats collected in a peri-urban fragment of the Atlantic Forest in São Paulo, Brazil. A small, preliminary set of pooled samples was analyzed, followed by phylogenetic reconstruction. Results: Trypanosoma sequences were detected in flies from the family Streblidae. Phylogenetic analysis showed that these sequences cluster within the Neobat 4 clade, which has previously been reported in Carollia spp. bats. This represents the first detection of Trypanosoma sp. in streblid flies parasitizing bats in São Paulo. Discussion: Although the vector competence of streblid flies for Trypanosoma transmission is still unknown, their close ecological association with bats suggests that they may serve as a non-invasive tool for pathogen surveillance when direct bat sampling is limited. This study expands the known geographic distribution of the Neobat 4 clade and contributes to understanding parasite circulation among bats and their ectoparasites.