Stumpy forms are the predominant transmissible forms of Trypanosoma brucei

Reviewer #1 (Public Review):

Summary:
Ngoune et al. present compelling evidence that Slender cells are challenged to infect tsetse flies. They explore the experimental context of a recent important paper in the field, Schuster et al., that presents evidence suggesting the proliferative Slender bloodstream T.brucei can infect juvenile tsetse flies. Schuster et al. were disruptive to the widely accepted paradigm that the Stumpy bloodstream-form is solely responsible for tsetse infection and T.brucei transmission potential.

Evidence presented here shows that in all cases, Stumpy form parasites are exponentially more capable of infecting tsetse flies. They further show that Slender cells do not infect mature flies.

However, they raise questions of immature tsetse immunological potential and field transmission potential that their experiments do not address. Specifically, they do not show that teneral tsetse flies are immunocompromised, that tsetse flies must be immunocompromised for Slender infection nor that younger teneral tsetse infection is not pertinent to field transmission.

Strengths:
Experimental Design is precise and elegant, outcomes are convincing. Discussion is compelling and important to the field. This is a timely piece that adds important data to a critical discussion of host: parasite interactions, of relevance to all parasite transmission.

Weaknesses:
As above, the authors dispute the biological relevance of teneral tsetse infection in the wild, without offering evidence to the contrary. Statements need to be softened for claims regarding immunological competence or relevance to field transmission.

Reviewer #2 (Public Review):

Summary:
Contrary to findings recently reported by Schuster S et al., this short paper shows evidence that the stumpy form of T. brucei is probably the most pre-adapted form to progress with the life cycle of this parasite in the tsetse vector.

Strengths:
One of the most important pieces of experimental evidence is that they conduct all fly infection experiments in the absence of metabolites like GlcNAc or S-glutathione; by doing so, the infection rates in flies infected with slender trypanosomes seem very low or nonexistent. This, on its own, is a piece of important experimental evidence that the Schuster S et al findings may need to be revisited.

Weaknesses:
I consider that the authors should have included their own experiments demonstrating that the addition of these chemicals enhances the infection rates in flies receiving bloodmeals containing slender trypanosomes.

Reviewer #3 (Public Review):

The dogma in the Trypanosome field is that transmission by Tsetse flies is ensured by stumpy forms. This has been recently challenged by the Engstler lab (Schuster et al. ), which showed that slender forms can also be transmitted by teneral flies. In this work, the authors aimed to test whether transmission by slender forms is possible and frequent.

For this, the authors repeated Tsetse transmission experiments but with some key critical differences relative to Schuster et al. First, they infected teneral and adult flies. Second, their infective meals lacked two components (N-acetylglucosamine and glutathione), which could have boosted the infection rates in the Schuster et al. work. In these conditions, the authors observed that most stumpy form infections with teneral and adult flies were successful while only 1 out of 24 slender-form infections was successful. Adult flies showed a lower infection rate, which is probably because their immune system is more developed.

Given that in Tsetse-infested areas most transmission is likely ensured by adult flies, the authors conclude that the parasite stage that will have a significant epidemiologic impact on transmission is the stumpy form.

Strengths:
• This work tackles an important question in the field.
• The Rotureau laboratory has well-known expertise in Tsetse fly transmission experiments.
• Experimental setup is robust and data is solid.
• The paper is concise and clearly written.

Weaknesses:
• The reason(s) for why this work has lower infection rates with slender forms than Schuster et al. remain unknown. The authors suggested it could be because of the absence of N-acetylglucosamine and/or glutathione, but this was not formally tested. Could another source of variation be the clone of EATRO1125 AnTat1.1 (Paris versus Munich origin)? To reduce the workload, such additional experiments could be done with just one dose of parasites.
• The characterization of what is slender and stumpy is critical. The authors used PAD1 protein expression as the sole reporter. While this is a robust assay to confirm stumpy, an analysis of the cell cycle would have been helpful to confirm that slender forms have not initiated differentiation (Larcombe S et al. 2023, preprint).
• Statistical analysis is missing. Is the difference between adult and teneral infections statistically significant?