Leishmania tarentolae and Trypanosoma platydactyli are reptile parasites that naturally share the same vertebrate hosts and are presumed to be transmitted by the sand fly Sergentomyia minuta. Despite their long-recognized association, the development of T. platydactyli in its most probable natural vector has remained largely unknown, and the interactions of both parasites during co-infection have not been investigated. Here, we experimentally infected laboratory-reared S. minuta by xenodiagnoses on naturally infected Tarentola mauritanica geckos carrying either single or mixed infections. Parasite species were distinguished using species-specific PCRs, and their localization, infection dynamics, and morphological development were examined by light microscopy and morphometric analyses. Leishmania tarentolae predominantly colonized the Malpighian tubules, whereas T. platydactyli exhibited a suprapylarian development, consistently colonizing the abdominal and thoracic midguts, and frequently reaching the cardia and the stomodeal valve. During development, T. platydactyli underwent a transition from epimastigotes to paramastigotes and trypomastigotes, with the latter becoming the predominant late-stage form that likely represents the transmissible stage. In contrast, L. tarentolae maintained the characteristic developmental pattern previously described for the subgenus Sauroleishmania, with short and long promastigotes predominating throughout development and metacyclic promastigotes appearing during late infections. Our findings provide the first comprehensive description of the development of T. platydactyli in S. minuta and demonstrate distinct tissue tropisms between the two trypanosomatids sharing the same vertebrate host and insect vector. These results support the hypothesis that long-term co-evolution has driven niche partitioning within the sand fly, allowing both parasites to coexist while potentially employing distinct transmission strategies.
