Developmental adaptations of trypanosome motility to the tsetse fly host environments unravel a multifaceted in vivo microswimmer system

Essential revisions:

1) The Introduction and the Discussion are heavily skewed to making a case that the importance of the work is to provide a platform or model system for computational biology of microswimmers. The microenvironments and the complex morphology of this system do not readily lend themselves to the simplification that is typically needed for mathematical modeling. This is not considered as a system of choice given the numerous variables that difficult to control and hard to simulate. The authors would do better to focus both introduction and discussion on the novel findings of their own report, which are considerable.

We agree with the reviewers criticism of overly promoting the model system for computational biology in parts of Introduction and Discussion. Therefore, we have now removed the redundant mentions of the model character of our work and focus more on the biological impact. Nevertheless, the intention of the study was primarily to introduce the tsetse vector as an enclosed and tractable system as a whole, for studying the natural complexity of microswimmer interactions with/in highly intricate environments. It is true that today the system is by far too little understood to allow mathematical modelling in its entirety. But we can learn from the fly. For example, we have been able to model the swimming behaviour of the different morphotypes, and we have identified in vivo motion patterns that can be modelled, e.g., the near-wall swimming of mesocyclic parasites along the peritrophic membrane (see also our reply to comment 4).

2) The study revealed interesting patterns of solitary and collective motion and reports rapid switching between synchronized and "chaotic" motion of cells in the ectoperitrophic space of the midgut. On this latter point, the authors raise the question whether synchronization of cell behaviour was driven by chemical cues or by hydrodynamic self-organisation. The data presented in Figure 10 is interpreted as "unambiguous" (subsection “Self-organisation of parasites by hydrodynamic interaction”, fourth paragraph) evidence for the latter. The basis for this conclusion is not entirely clear. The authors say a chemical process would "generally be slower" (first paragraph of the aforementioned subsection) and it would be helpful if they could be more precise about the difference in time scales so that the cell behaviour in Video 10 can be assessed against these expectations.

Indeed, the text was imprecise; “unambiguous” was referring to the fact that there is no evidence of cell attachment and that hydrodynamic interactions are most likely. We have rephrased and clarified the paragraph, including the direct comparison to the very limited data there is available regarding time scales of potential chemotactic behaviour in trypanosomes:

“It should be noted, that the timescale of a few seconds observed here for collective organisational status change is significantly smaller than the shortest known timescale of potential chemotactic behaviour in trypanosomes, which is at least several minutes (Oberholzer et al., 2010). Although chemical signals will undoubtedly be relevant for rapid fluctuations of flagellar beating, an adaptive reaction system required for directed behaviour of parasite swarms is unlikely to be responsible for establishing the switching behaviour observed in our experiments.”

3) Despite the stunning quality of the images and videos produced the study does not actually reveal anything new about the tsetse-trypanosome interaction. Even the "astonishing degree of convolution" of the peritrophic matrix (PM) (subsection “Multicolour light sheet fluorescence microscopy reveals the complex three-dimensional architecture of the microswimmer habitats in the tsetse vector”, third paragraph and Discussion, third paragraph) has been described previously, including data on its dimensions, as it collapses when water from the bloodmeal is excreted. The claim for priority is unfounded: "This was a surprising finding, which allowed a first realistic view into an important environmental compartment for the procyclic and mesocyclic stages of T. brucei." Entomologists in the last century were well aware of this e.g. Wigglesworth (1929). The authors are referring to cartoons of the PM: "a simple hollow sleeve as mostly depicted in the literature". Incidentally, the description of the PM as "a chitinous sleeve" is also inaccurate, as it contains a lot of protein as well as chitin. A comprehensive coverage of the literature is a prerequisite.

We appreciate this important comment, especially the Wigglesworth reference, which we have now cited. Furthermore, we have screened the literature for more information on the convolution of the PM, however, could not find better data than provided by Wigglesworth (1929) and maybe Yorke (1933). In fact, most publications describe the PM only as a continuous, hollow sleeve or tube. We have added a paragraph in the Discussion with the most relevant citations:

“This distension of the PM upon feeding and the extensive folding after excretion of liquid has been described elegantly in early work on Glossina (Wigglesworth, 1929). […] Studies were undertaken to establish the composition and the role of the PM in trypanosome infection, especially addressing the question how and where the parasites manage to cross this barrier, but data was focussed on detail regions of the matrix, mostly displaying single folds and necessarily in 2D (e.g. Hoare, 1931; Yorke et al., 1933; Willett, 1966; Moloo et al., 1970; Ellis and Evans, 1977; Gibson and Bailey, 2003).”

We have rephrased "a chitinous sleeve" to:

“non-cellular, glycosaminoglycan, glycoprotein and chitin containing, cylindrical sleeve”

Our work provides, among other things, a detailed 3D-topological map of the geometry of the convoluted PM. We show the distribution of the parasites in the folds and we reveal the motion pattern of the trypanosomes in their natural environment. We would like to believe these aspects and the combination thereof are of considerable novelty.

4) The authors justify the study as a prelude to investigating "what physical and biological constraints the versatile trypanosome microswimmers experience in the vast and multifaceted environment of their insect host", but it is not clear what hypotheses they would test in future studies. They claim that the tsetse microenvironment offers a tractable model system for studying microswimmers, but the tsetse milieu is not static, changing both in terms of its own movements – peristalsis of the alimentary tract and salivary glands – and in terms of the chemical environment as the infected bloodmeal is digested and further bloodmeals are taken. Comparison with controlled microfluidic environments may be more informative, e.g. to explore why trypanosomes change from synchronized to chaotic swimming (Video 10).

As stated above our study marks a starting point. It provides a static blueprint of the tsetse topology and the distribution of the different trypanosome morphotypes. In the next step, we e.g. explore fluid flow at different scales, ranging from rather slow microflows caused by the trypanosomes itself to massive flows produced by peristalsis. In fact, our current and future work is nature-inspired, i.e. we measure the real environments in the fly and the behaviour of the trypanosomes therein. Then, we abstract from nature and construct microfluidic environments that allow the control of parameters. The results from these experiments could provide hints that might help to explain the in vivo behaviour of the trypanosome microswimmers. We now provide more information of this workflow in the Discussion:

“In fact, such analyses with tsetse fly trypanosomes directly follow already mentioned experimental setups used to assess the reaction of mammalian trypanosomes to mechanical forces (Heddergott et al., 2012; Bargul et al., 2016). […] Using the combination of flexible microfluidic devices and reversible microfluidic pump systems, even the analysis of peristaltic effects seems in reach.”