Beyond Leishmania: hidden trypanosomatid diversity reveals complex parasite-sand fly networks in southeastern Brazil

Sand flies (Diptera: Psychodidae) are well-known vectors of Leishmania, yet their associations with other trypanosomatids remain poorly understood. Expanding knowledge on these interactions is essential to elucidate the ecological diversity of parasites circulating in natural and periurban environments.

To characterise sand fly species composition and assess the diversity of trypanosomatids naturally infecting sand flies in the Serra do Cipó district, Minas Gerais, Brazil.

Sand flies were collected between 2023 and 2025 in the Mata da Tapera and nine surrounding peridomestic sites using Centre for Disease Control light trap (CDC-LT) and a Shannon trap. Females were examined by midgut dissection and screened individually for trypanosomatids using nested polymerase chain reaction (PCR) targeting the 18S rRNA V7-V8 region, followed by sequencing and phylogenetic analyses.

A total of 1,460 sand flies representing 21 species were collected, with Pintomyia pessoai (35.5%) being the most abundant. No flagellates were observed in 105 dissected females. Molecular screening of 730 females revealed 12 positives (overall positivity = 1.6%), including Leishmania infantum in Pi. pessoai, Pi. christenseni, and Pa. barretoi; L. braziliensis in Pi. monticola; and non-Leishmania taxa such as Herpetomonas samuelpessoai, Novymonas esmeraldas, a representative of Strigomonadinae, Trypanosoma sp. (Anura clade), and a lineage related to Sergeia.

These findings confirm the circulation of Leishmania in Serra do Cipó while revealing hidden trypanosomatid diversity spanning at least five genera. The results suggest that sand flies may act as ecological "hubs", transiently interacting with multiple parasite lineages beyond the classical Leishmania cycle, highlighting the need to broaden the ecological perspective in sand fly-parasite studies.