Invertebrate-Derived DNA (iDNA) to Identify Sand Flies’ Bloodmeal: A Molecular Approach to Identifying Hosts in Blood-Feeding Vectors of Leishmaniasis

DNA metabarcoding data obtained by next generation sequencing (NGS) has been used to identify species in mixed biological samples, such as DNA from the gut content of invertebrates that feed on vertebrates (invertebrate-derived DNA, iDNA). This investigation employed DNA metabarcoding approach to determine vertebrate hosts of female phlebotomine sand flies, blood-feeding leishmaniasis vectors. We evaluated performance across three mitochondrial markers: a mammal-specific mini-barcode (16S rRNA), a pan-vertebrate mini-barcode (12S rRNA), and a standard CytB barcode region. Phlebotomine sand flies collections occurred in the Cacao Region of Southeastern Bahia, Brazil, an American Tegumentary Leishmaniasis (ATL) endemic zone. Our analysis examined iDNA from forty female specimens pooled in thirteen samples of seven sand fly species, including confirmed ATL vectors. Metabarcoding-derived operational taxonomic units (OTUs) underwent taxonomic assignment through comparison with GenBank NCBI^® reference databases. Results identified twenty vertebrate OTUs: primates (four OTUs), rodents (four), ungulates (five), marsupials (one), plus a domestic dog and a chicken. Notably, non-mammalian taxa, including reptiles (one OTU) and amphibians (three), were detected. The iDNA metabarcoding approach allowed us to accurately sample the diversity of phlebotomine sandflies' bloodmeals in a single specimen of a non-engorged female sand fly with mixed feeding.