Research advance: Unexpected plasticity in the life cycle of Trypanosoma brucei

Reviewer #1 (Public review):

Summary:

This work provides evidence that slender T. brucei can initiate and complete cyclical development in Glossina morsitans without GlcNAc supplementation, in both sexes, and importantly in non-teneral flies, including salivary-gland infections.

Comparative transcriptomics show early divergence between slender- and stumpy-initiated differentiation (distinct GO enrichments), with convergence by ~72 h, supporting an alternative pathway into the procyclic differentiation program.

The work addresses key methodological criticisms of earlier studies and supports the hypothesis that slender forms may contribute to transmission at low parasitaemia.

Strengths:

(1) Directly tackles prior concerns (no GlcNAc, both sexes, non-teneral flies) with positive infections through to the salivary glands.

(2) Transcriptomic time course adds some mechanistic depth.

(3) Clear relevance to the "transmission paradox"; advances an important debate in the field.

Weaknesses:

(1) Discrepancy with Ngoune et al. (2025) remains unresolved; no head-to-head control for colony/blood source or microbiome differences that could influence vector competence.

(2) Lacks in vivo feeding validation (e.g., infecting flies directly on parasitaemic mice) to strengthen ecological relevance.

(3) Mechanistic inferences are largely correlative (although not requested, there is no functional validation of genes or pathways emerging from the transcriptomics).

(4) Reliance on a single parasite clone (AnTat 1.1) and one vector species limits external validity.

Reviewer #2 (Public review):

Summary:

This paper is an exciting follow-up to two recent publications in eLife: one from the same lab, reporting that slender forms can successfully infect tsetse flies (Schuster, S et al., 2021), and another independent study claiming the opposite (Ngoune, TMJ et al., 2025). Here, the authors address four criticisms raised against their original work: the influence of N-acetyl-glucosamine (NAG), the use of teneral and male flies, and whether slender forms bypass the stumpy stage before becoming procyclic forms.

Strengths:

We applaud the authors' efforts in undertaking these experiments and contributing to a better understanding of the T. brucei life cycle. The paper is well-written and the figures are clear.

Weaknesses:

We identified several major points that deserve attention.

(1) What is a slender form? Slender-to-stumpy differentiation is a multi-step process, and most of these steps unfortunately lack molecular markers (Larcombe et al, 2023). In this paper, it is essential that the authors explicitly define slender forms. Which parameters were used? It is implicit that slender forms are replicative and GFP::PAD1-negative. Isn't it possible that some GFP::PAD1-negative cells were already transitioning toward stumpy forms, but not yet expressing the reporter? Transcriptomically, these would be early transitional cells that, upon exposure to "tsetse conditions" (in vitro or in vivo), could differentiate into PCF through an alternative pathway, potentially bypassing the stumpy stage (as suggested in Figure 4). Given the limited knowledge of early molecular signatures of differentiation, we cannot exclude the possibility that the slender forms used here included early differentiating cells. We suggest:

1.1 Testing the commitment of slender forms (e.g., using the plating assay in Larcombe et al., 2023), assessing cell-cycle profile, and other parameters that define slender forms.

1.2 In the Discussion, acknowledging the uncertainty of "what is a slender?" and being explicit about the parameters and assumptions.

1.3 Clarifying in the Materials and Methods how cultures were maintained in the 3-4 days prior to tsetse infections, including daily cell densities. Ideally, provide information on GFP expression, cell cycle, and morphology. While this will not fully resolve the concern, it will allow future reinterpretation of the data when early molecular events are better understood.

(2) Figure 1: This analysis lacks a positive control to confirm that NAG is working as expected. It would strengthen the paper if the authors showed that NAG improves stumpy infection. Once confirmed, the authors could discuss possible differences in the tsetse immune response to slender vs. stumpy forms to explain the absence of an effect on slender infections.

(3) Figure 2. To conclude that teneral flies are less infected than non-teneral flies, data from Figures 1 and 2 must be directly comparable. Were these experiments performed simultaneously? Please clarify in the figure legends. Moreover, the non-teneral flies here are still relatively young (6-7 days old), limiting comparisons with Ngoune, TMJ et al. 2025, where flies were 2-3 weeks old.

(4) Figure 3. The PCA plot (A) appears to suggest the opposite of the authors' interpretation: slender differentiation seems to proceed through a transcriptome closer to stumpy profiles. Plotting DEG numbers (panel C) is informative, but how were paired conditions selected? Besides, plotting of the number of DEGs between consecutive time points within and between parasite types is also necessary. There may also be better computational tools to assess temporal relationships. Finally, how does PAD1 transcript abundance change over time in both populations? It would also be important to depict the upregulation of procyclic-specific genes.

(5) Could methylcellulose in the medium sensitize parasites to QS-signal, leading to more frequent and/or earlier differentiation, despite low densities? If so, cultures with vs. without methylcellulose might yield different proportions of early-differentiating (yet GFP-negative) parasites. This could explain discrepancies between the Engstler and Rotureau labs despite using the same strain. The field would benefit from reciprocal testing of culture conditions. Alternatively, the authors could compare infectivity and transcriptomes of their slender forms under three conditions: (i) in vitro with methylcellulose, (ii) in vitro without methylcellulose, and (iii) directly from mouse blood.