Replicative slender bloodstream forms complete transmission of Trypanosoma brucei without prior differentiation into stumpy forms

We have previously shown that the slender form of Trypanosoma (T.) brucei is able to infect teneral tsetse flies, develop to the first fly form, which is the procyclic form, and complete the life cycle in the insect vector (Schuster et al., 2021). Further, analysis of the transmission index (TI; defined as the number of salivary gland infections relative to the number of midgut infections) revealed a higher TI for slender as compared to stumpy forms under laboratory conditions, which included the addition of N -acetylglucosamine (NAG) to the infective bloodmeal. Here, we show that slender trypanosomes can establish infections in both male and female tsetse flies and in both teneral and non-teneral flies without requiring supplements in the bloodmeal. Additionally, an RNA sequencing time course was performed on both slender and stumpy cells during their transition into procyclic forms. This analysis revealed that slender- and stumpy-form trypanosomes remain transcriptionally distinct throughout differentiation into the procyclic form. Furthermore, while the protein associated with differentiation 1 (PAD1) remains essential for the transition, slender cells do not require expression of other hallmark stumpy-form traits, such as cell-cycle arrest or the shortening of their flagella or microtubule corset. Instead, slender trypanosomes are able to transition directly into procyclic forms. Taken together, these findings demonstrate that slender cells of T. brucei can follow a distinct transcriptional trajectory towards the procyclic form and can establish infections in teneral and non-teneral tsetse flies, thereby contributing to the transmission and spread of these African parasites.