Distinct gene expression patterns in vector-residing Leishmania infantum identify parasite stage-enriched markers

Background: Leishmaniasis is a vector-borne neglected disease. Inside the natural sand fly vector, the promastigote forms of Leishmania undergo a series of extracellular developmental stages to reach the infectious stage, the metacyclic promastigote. There is limited information regarding the expression profile of L. infantum developmental stages inside the sand fly vector, and molecular markers that can distinguish the different parasite stages are lacking. Methodology/Principal findings: We performed RNAseq on unaltered midguts of the sand fly Lutzomyia longipalpis after infection with L. infantum parasites. RNAseq was carried out at various time points throughout parasite development. Principal component analysis separated the transcripts corresponding to the different Leishmania promastigote stages, the procyclic, nectomonad, leptomonad and metacyclics. Importantly, there were a significant number of differentially expressed genes when comparing the sequential development of the various Leishmania stages in the sand fly. There were 836 differentially expressed (DE) genes between procyclic and long nectomonad promastigotes; 113 DE genes between nectomonad and leptomonad promastigotes; and 302 DE genes between leptomonad and metacyclic promastigotes. Most of the DE genes do not overlap across stages, highlighting the uniqueness of each Leishmania stage. Furthermore, the different stages of Leishmania parasites exhibited specific transcriptional enrichment across chromosomes. Using the transcriptional signatures exhibited by distinct Leishmania stages during their development in the sand fly midgut, we determined the genes predominantly enriched in each stage, identifying multiple potential stage-specific markers for L. infantum. Conclusions: Overall, these findings demonstrate the transcriptional plasticity of the Leishmania parasite inside the sand fly vector and provide a repertoire of potential stage-specific markers for further development as molecular tools for epidemiological studies. Author summary: One of the major gaps in Leishmania research is the lack of stage-specific markers to differentiate the Leishmania promastigote stages in the sand fly vector as well as in culture. In this study, we report the transcriptional boundaries amongst Leishmania infantum promastigote stages developing in the sand fly vector Lutzomyia longipalpis. Despite the policystronic nature of Leishmania mRNAs, we were able to detect 10- to 100 fold differences in transcriptional abundance between genes throught RNA sequencing. Significant number of genes were differentially expressed between subsequent stages, showing enrichment of stage specific up-regulated genes even across chromosomes. In light of such differences, we identified potential stage-specific markers for each promastigote stage of Leishmania with potential use in epidemiological studies as well as in further characterization the developmental differences amongst the promastigote stages in the vector.