Single-cell atlas of Leishmania major development in the sandfly vector reveals the heterogeneity of transmitted parasites and their role in infection

Leishmania development in the sandfly vector is predominantly characterized as a series of sequential promastigote morphotypes. Apart from isolated mammal-infective metacyclic promastigotes, the transcriptional programs and molecular markers for other stages co-inhabiting the midgut or co-egested by the sandfly have not been resolved. Here, using single-cell RNA-sequencing, we elucidate the transcriptomic complexity of L. major promastigotes colonizing Phlebotomus duboscqi at early- and late-infection, providing unique markers for three subpopulations found in sandflies with mature, transmissible infections. Contrary to prevailing models, our analyses indicate a non-linear developmental program, with bifurcation into either replicating early metacyclics or attached and detached haptomonads. Haptomonads constitute part of the transmitted dose, and along with highly motile, non-dividing late metacyclics, are infectious to and exacerbate the pathology in mice. Our single-cell analysis and novel validated markers will facilitate further studies on the Leishmania life cycle and its implications for host infections.