Author Response
The following is the authors’ response to the original reviews.
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Public Reviews:
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Reviewer #1:
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Summary:
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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.
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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.
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Strengths:
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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.
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Thank you
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Weaknesses:
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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.
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We have modified the revised version to soften these claims (l.156 and l.159). Please, note that the limited immunocompetence of teneral flies has been extensively studied by the labs of S. Aksoy at Yale and M. Lehane at Liverpool. In the discussion, we provide key references from these two labs 18-21. Our comment on the relevance to field transmission is simply based on field observations of the fly
biology.
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Reviewer #2:
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Summary:
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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.
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Strengths:
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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 non-existent. This, on its own, is a piece of important experimental evidence that the Schuster S et al findings may need to be revisited.
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Thank you
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Weaknesses:
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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.
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The main purpose of this study is to assess the intrinsic infectivity of SL Vs. ST in teneral Vs. adult flies, not to reproduce the results obtained by Schuster et al.. We think that the suggested experiment is not necessary as L-Glutathion is well-known to enhance infection rates by reducing the fly immune response efficiency (Ref 24). Most of the experimental infections with procyclic or ST forms (even at low densities) published by our lab and others, especially for studying parasite stages in the salivary glands, were actually performed by complementing the infective meal with L-Glutathion for this reason.
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Reviewer #3:
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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.
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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.
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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.
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Strengths:
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• This work tackles an important question in the field.
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• The Rotureau laboratory has well-known expertise in Tsetse fly transmission experiments.
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• Experimental setup is robust and data is solid.
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• The paper is concise and clearly written.
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Thank you
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Weaknesses:
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• 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.
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Differences between the strain clones, the cell culture conditions and/or the fly colony maintenance conditions could indeed explain the differences in infection rates observed in the two studies. However, the main purpose of this study is to assess the intrinsic infectivity of SL Vs. ST in teneral Vs. adult flies. Our study was designed to stand alone for providing a clear answer to this question, not to reproduce the results obtained by Schuster et al.. Hence, we don’t think that any additional
experiments are required here.
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• 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).
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In this study, ST are indeed defined by their general morphology and by the expression of PAD1 proteins at the cell membrane as assessed by IFA. This is the simplest and most accurate ST proxy accessible by IFA. We do not think that monitoring in more details the cell cycle would provide key information here. If some SL forms had initiated differentiation in our experiments, then, the low infection rates observed
with SL would have reinforced the fact that mostly mature PAD1+ ST are infectious for flies .
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• Statistical analysis is missing. Is the difference between adult and teneral infections statistically significant?
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An ANOVA statistical analysis was performed and a dedicated section was added to the revised version.
For all conditions, MG infection rate comparisons between adult and teneral flies were statistically significant.
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Recommenda8ons for the authors:
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Reviewer #1:
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While some perceived outcomes pertaining to immunological competence and transmission relevance of teneral flies are overstated, the overall tone of the paper is inappropriately apologe7c. The authors obviously don't want to offend their colleagues but the current wri7ng style obscures meaning, making the paper a bit 'flowery' and difficult to read.
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Ngoune et al. have important outcomes that need to be stated more directly.
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Words such as 'unequivocally' are not appropriate to Schuster et al's outcomes. As your study shows, their findings are experimentally based, with inherent caveats, and are therefore sugges7ve, not demonstrated or proven.
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The word 'unequivocally' has been removed from the revision.
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Reviewer #3:
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The Engstler lab cul7vates AntTaT1.1 in methylcellulose (Munich clone, if I am not mistaken). The Rotureau lab uses the Paris AntTaT1.1 clone and uses no methylcellulose. Given that methylcellulose helps stumpy forma7on, it seems important to show that the results of this paper are reproducible with the Munich clone grown in the presence of methylcellulose.
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Differences between the strain clones and culture conditions could indeed explain the differences in infection rates observed in the two studies. However, the main purpose of this study is to assess the intrinsic infectivity of SL Vs. ST in teneral Vs. adult flies. Our study was designed to stand alone for providing a clear answer to this question, not to reproduce the results obtained by Schuster et al..
Hence, we don’t think that any additional experiments are required here.

Christine Clayton

Dominique Soldati-Favre

eLife assessment
For decades it has been accepted that only the growth-arrested "stumpy" form of Trypanosoma brucei can infect the arthropod vector, the Tsetse fly, but this was recently challenged by a demonstration that - under artificial conditions that are known to enhance infectivity - the proliferative "slender" form can also establish Tsetse infections. The infectiousness of the two forms is a fundamental question in trypanosome biology and epidemiology, concerning both infection dynamics and parasite differentiation. The authors of the current study provide compelling evidence that without artificial enhancement, the "stumpy" form is indeed much more infective for Tsetse than the slender form; they suggest that this is probably also true in the wild. Since the authors of this paper did not themselves test the effect of enhancing conditions, the precise reason for the discrepancy in results between the two laboratories has not been demonstrated conclusively.

anonymous

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. was 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: 
In contrast to the recent findings reported by Schuster S et al., this brief paper presents evidence suggesting that the stumpy form of T. brucei is likely the most pre-adapted form to progress through the life cycle of this parasite in the tsetse vector.
Strengths: 
One significant experimental point is that all fly infection experiments are conducted in the absence of "boosting" metabolites like GlcNAc or S-glutathione. As a result, flies infected with slender trypanosomes present very low or nonexistent infection rates. This provides important experimental evidence that the findings of Schuster S and colleagues may need to be revisited.
Weaknesses: 
However, I believe the authors should have included their own set of experiments demonstrating that the presence of these metabolites in the infectious bloodmeal enhances infection rates in flies receiving blood meals containing slender trypanosomes. Considering the well-known physiological variabilities among flies from different facilities, including infection rates, this would have strengthened the experimental evidence presented by the authors.

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. ), who 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. The authors observed that most stumpy forms infections with teneral and adult flies were successful while only 1 out of 24 slender form infections were successful.
In this revised version of the manuscript, the authors made some text changes and included statistical testing as a new section of the Materials and Methods. It seems the comparison of midgut infection in adult vs teneral flies was significant in most of the conditions. However, the critical comparison is still missing: within each type of fly (adult or teneral), was the MG infection significantly different between slender and stumpy forms?
Given no additional experiments were performed, it remains unknown why this work and Schuster et al. reached different conclusions. As a result it remains unclear in which conditions slender forms could be important for transmission. Several variables could explain differences between the two groups: the strain used, the presence or absence of N-acetylglucosamine and/or glutathione, how Tsetse colonies were maintained, thorough molecular and cellular characterisation of slender and stumpy forms (to avoid using intermediate forms as slender forms), comparison to recent field parasite strains.

Stumpy forms are the predominant transmissible forms of Trypanosoma brucei

Figures and data

Stumpy forms are the predominant transmissible forms.

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