Author Response:
The following is the authors’ response to the original reviews.
Public Reviews:
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.
We acknowledge that a direct head-to-head comparison was not performed and that microbiome composition can affect vector competence. However, both the tsetse flies used in Ngoune et al. (2025) and those in our study originated from the same colony and were maintained under comparable standard laboratory conditions. In both cases, flies were fed on sheep blood through identical silicon membrane systems, minimizing potential differences.
(2) Lacks in vivo feeding validation (e.g., infecting flies directly on parasitaemic mice) to strengthen ecological relevance.
Our study deliberately focused on controlling experimental variables through the use of an artificial feeding system, which allows for standardization of parasite dose and exposure conditions. This approach facilitates reproducibility and direct comparison with previous studies. Also, to us it appears questionable if feeding flies on infected laboratory mice really adds ecological relevance.
(3) Mechanistic inferences are largely correlative (although not requested, there is no functional validation of genes or pathways emerging from the transcriptomics).
Functional validation of individual genes or pathways was not undertaken in this study. Instead, the aim was to identify and compare transcriptional signatures associated with slender-to-procyclic versus stumpy-to-procyclic differentiation, and to directly address previous criticism of original finding that slender bloodstream forms are capable of infecting the tsetse fly.
(4) Reliance on a single parasite clone (AnTat 1.1) and one vector species limits external validity.
Incorporating additional pleomorphic T. brucei clones and alternative tsetse species would undoubtedly broaden our understanding of parasite-vector interactions, and studies using fresh field isolates and wild-caught tsetse flies would be even more informative. However, in order to directly address the specific concerns raised against our original study (Schuster et al., 2021), it was essential to employ the same parasite clone and vector species.
We further emphasize that the pleomorphic clone used here is a well-characterized and widely employed T. brucei strain that closely reflects parasites encountered under natural conditions. Likewise, Glossina morsitans represents the standard vector species used in the majority of tsetse laboratories, thereby ensuring reproducibility and facilitating comparison with existing work in the field.
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.
We appreciate the critical evaluation concerning the identity of slender forms and potential presence of intermediate forms displaying slender morphology yet exhibiting cell-cycle arrest, as proposed in Larcombe et al. (2023). Indeed, our original paper is entitled “Unexpected plasticity in the life cycle of Trypanosoma brucei.” It is precisely this phenotypic plasticity that enables slender parasites to transition directly into the procyclic insect stage. Notably, we have shown that even monomorphic trypanosome strains are capable of undergoing this transition in the fly, and such strains are not considered to represent “intermediate” or “half-stumpy” forms. Consequently, while the question “what constitutes a slender parasite?” may be of conceptual interest, it currently is, in our view, not central to the biological conclusions of this study.
Nevertheless, we now have included an additional section in our Discussion that compares the slender cells used in our study with the commitment classification introduced by Larcombe et al. Our infection experiments were conducted using cells that meet the Larcombe-criteria of “true slender cells”, characterized by the absence of PAD1 expression and the maintenance of a slender morphology (Supplementary Figure 3A, B, following FACS sorting). Moreover, these cells are not cell-cycle arrested but continue to proliferate (Supplementary Figure 3C). Accordingly, our experimental assumptions and parameters align those of previous studies, in which continuous cell division, lack of cell cycle arrest, lack of PAD1 expression, and slender morphology are still established markers defining the slender bloodstream form.
(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.
We thank the reviewer for this helpful suggestion. Details on the maintenance of T. brucei cultures and culture conditions, including cell density, are provided in our previous publication (Schuster et al., 2021). In the present study, cultures were routinely monitored prior to infection to ensure that the cells used were GFP-negative and exhibited the characteristic slender morphology.
For infections performed with higher cell numbers, fluorescence-activated cell sorting (FACS) was used to obtain a 100% GFP-negative population, thereby avoiding the need for daily monitoring of GFP fluorescence. This approach ensured that all infection experiments were initiated with a homogeneous population of slender bloodstream forms.
(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.
The enhancing effect of N-acetylglucosamine (NAG) on stumpy-form infections of T. brucei is well established and widely accepted in the field (e.g. Peacock et al., 2006, 2012). In the present Research Advance, our objective was to directly address the specific concerns raised in response to our previous publication (Schuster et al., 2021), in which NAG supplementation during stumpy infections was already included and shown to function as expected. Accordingly, the aim here was not to reiterate the established role of NAG in promoting stumpy infections, but rather to directly examine infections initiated by slender bloodstream forms in the absence of NAG, thereby approximating more natural conditions.
(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.
The experiments presented in Figures 1 and 2 were not performed simultaneously. Importantly, the comparison between teneral and non-teneral flies was not intended as a direct quantitative comparison across experiments, but rather to assess infection outcomes under distinct physiological states of the vector. It is well established that teneral flies are generally more susceptible to T. brucei infection than non-teneral flies, a phenomenon commonly referred to as the “teneral phenomenon.”
Our objective was to demonstrate that slender bloodstream forms are capable of establishing infections also in non-teneral flies, thereby directly addressing concerns in the comment to our original study (Schuster et al.) that the experimental set-up may have created an unnaturally permissive environment. The data presented here in fact support the conclusion that slender forms can contribute to disease transmission under more natural conditions.
A key determinant of the increased susceptibility of teneral flies is the incomplete maturation of the peritrophic matrix (PM) (Walshe et al., 2011; Haines, 2013). In Glossina morsitans morsitans, the PM reaches its full length along the midgut approximately 84 hours post-eclosion (Lehane and Msangi, 1991). In addition, teneral flies have not yet taken a bloodmeal prior to the infective one, a factor known to further increase susceptibility (Haines, 2013).
In the present paper, non-teneral flies were selected that had received two non-infectious bloodmeals prior to the infective challenge. At 6-7 days post-eclosion, these flies possessed a fully established PM, which is known to increase refractoriness to infection (Walshe et al., 2011), while still being sufficiently young to survive the time required for T. brucei to complete its developmental cycle. This is an important point, as our timing allowed robust interpretation of infection outcomes, without the substantial loss of flies (approximately 40%) that has been reported to occur prior to dissection in Ngoune et al., 2025.
(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.
Regarding the PCA plot (Figure 3A), we agree that slender form differentiation transiently exhibits transcriptomic similarities to stumpy form profiles. However, as discussed in the paper, this overlap specifically reflects shared early differentiation responses rather than the adoption of a full stumpy-like transcriptome. The overall trajectory and clustering pattern indicate that slender-derived parasites follow a distinct differentiation path that - as expected -ultimately converges with the procyclic stage, consistent with our interpretation.
For the DEG analysis (Figure 3C), paired conditions were selected based on biologically meaningful time points corresponding to key stages in the differentiation process, allowing for direct comparisons between slender- and stumpy-derived populations either for the same timepoints following addition of cis-aconitate (Supplementary Figure 5) or timepoints plotting close on the PCA (Supplementary Figure 6).
We also appreciate the recommendation to consider alternative computational approaches for assessing temporal relationships. While our current analysis provides robust insights into transcriptomic transitions, we agree that future studies employing different tools could further refine our observations.
Finally, we have included the expression dynamics of PAD1 and PAD2 in the Supplementary Data (Supplementary Figure 8). The expression profile for procyclic-specific genes can now be found in Supplementary Figure 9.
(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.
The original description of stumpy induction factor (SIF)-mediated quorum sensing in Trypanosoma brucei was performed by the Boshart laboratory using (a) the same cell line employed in the present study and (b) an identical HMI-9 medium supplemented with the same amount of methylcellulose (Reuner et al., 1997; Vassella et al., 1997). All relevant controls were comprehensively reported in those studies in the late 1990s. There is therefore no experimental or historical basis to suggest that methylcellulose sensitises parasites to stumpy differentiation. Moreover, the viscosity of HMI-9-methylcellulose remains well below the threshold required to impose a diffusion barrier for small molecules such as peptides. Consequently, accumulation of SIF as a result of increased medium viscosity can be excluded on physical grounds.
The present Research Advance was conducted with a focused objective, namely, to directly address the specific concerns raised in response to our original publication (Schuster et al., 2021). Expanding the study to include additional experimental conditions, such as systematic comparisons of cultures grown with and without methylcellulose, or analyses of parasites freshly isolated from mouse blood, would have extended the scope well beyond what is useful for a Research Advance and would have diluted the central purpose of this contribution.
Recommendations for authors:
Reviewer #1 (Recommendations for the authors):
Thank you for your perseverance in filling the gaps flagged by others - these data strengthen the story.
Reviewer #2 (Recommendations for the authors):
(1) Figure 1: The use of teneral flies is not mentioned in the text or the legend
Thank you: we added this to the main text and figure legend (lines 103 and 140).
(2) Figure 1 legend (line 2): Typo - "with or 60 nm" should read "with or without 60 nm."
Thank you: this has been corrected (line 141).
(3) Figure 2. Please provide the FACS gating strategy and cell numbers before and after sorting
The cell number before gating is 1x107 cells, and 1x106 cells were collected via FACS for infection experiments. This is stated in the Materials & Methods section (lines 473 and 478).
(4) Figure 3. RNAseq data presentation could be improved:
(a) Clarify which type of differentially expressed genes are shown in panels B and C (presumably those upregulated in slender forms and those upregulated in stumpy forms).
Thank you: the information has now been added to the figure legend (lines 279 and 282).
(b) The color code in panel A is inverted relative to panels B and C.
Thank you: this has been corrected (figure 3B and C).
(c) The GO-term analysis represents an important conclusion and should be moved to the main figure.
As a Research Advance, this paper is restricted in the number of figures and therefore the decision had to be made to move the GO-term analysis to the Supplements.
(d) Provide dataset quality control in the supplement (genes detected per sample, sample consistency, replicate correlations, etc.).
Sequencing analysis is now explained in detail in the Materials & Methods section (lines 515 - 528).
(5) Figure legends: Indicate how many times each experiment was performed and the number of independent biological replicates.
The number of replicates (and flies per replicate) is stated for both infection experiments in the respective figure legends (lines 143 and 203/04). For the RNA sequencing, it is stated in the main text, and we now have also added the information to the figure legend (lines 219 and 276/77).
(6) Discussion: Despite the ongoing debate about midgut pH, could the authors also comment on other evidence suggesting that stumpy forms are better adapted to the fly?
The pH of the midgut has been determined by the Acosta-Serrano laboratory. We have cited the paper (Liniger et al. 2003) in lines 328-330 of the discussion. Furthermore, we have discussed the developing mitochondria of stumpy forms as well as expression of Krebs cycle, and the proposed higher resistance to proteolytic stress (Vickerman, 1965; Brown et al., 1973; Hamm et al., 1990; Reuner et al., 1997, Nolan et al., 2000).
