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  1. Reviewer #4 (Public Review):

    This paper describes the transmission of Trypanosoma brucei by the Tsetse vector. As part of these studies, the authors discovered that (i) a single parasite is sufficient for transmission and (ii) two stages of the Trypanosoma brucei life cycle (slender and stumpy forms) can be efficiently transmitted by the Tsetse vector. This was unexpected (as mentioned in the title) because only stumpy forms were known to be adapted for transmission.

    The life cycles of parasites are text-book knowledge that researchers rely on and rarely question. It's the slide #2 of every talk in parasitology. In the mammalian host, the life cycle of Trypanosoma brucei comprises two stages: the dividing slender forms and the cell-cycle arrested stumpy-forms, which are pre-adapted to survive in the midgut of the next host (Tsetse fly). In this report, Schuster, Subota et al. show that slender forms are sufficient to establish an infection in the Tsetse fly and thus ensure transmission. The claims and conclusions are justified by the data presented.

  2. Reviewer #3 (Public Review):

    In this work, Schuster et al. have explored the requirement of the short stumpy morphological form of the African trypanosome, Trypanosoma brucei, for the completion of the parasite lifecycle. Heretofore, short stumpy form parasites, which have been proposed to be pre-adapted for life in the tsetse fly insect vector, were considered an essential stage in the transitions from mammalian blood forms to insect-infective stages. These parasites do not divide and are generated in a density-dependent manner from the rapidly dividing long slender blood form. The quiescent short stumpy forms have been shown in vitro to undergo differentiation into insect-infective forms in response to a diversity of environmental cues and stress, supporting their position as the lifecycle stage that initiates colonization of the fly midgut.

    The findings presented in this work call into question the longstanding notion that short stumpy parasites play a central role in the lifecycle. Notably, the authors have found that long slender forms are as competent as short stumpy parasites to infect flies. This observation may solve a major conundrum raised when short stumpy forms are considered essential intermediates in disease transmission. That is, how is the parasite successfully transmitted to tsetse flies when the flies only ingest very small bloodmeals from hosts with parasitemia too low to trigger density dependent stumpy form development?

    The authors perform an extensive analysis of parasites isolated from infected flies and compare fly infections established using different numbers of short stumpy and slender parasites. This effort includes dissection of a variety of fly tissues and scoring parasites for expression of key developmental markers. Interestingly, the data indicate that the long slender parasites activate pathways described from short stumpy parasites to complete differentiation; however, unlike the stumpy forms that are arrested in the cell cycle, the parasites continue to proliferate. Overall, the process of differentiation to the insect stage is not identical for the long slender and short stumpy forms, as expression of key markers (PAD1 and EP1) occurs more quickly when short stumpy forms are used in fly infection studies while, unlike the long slender forms, they are delayed in return to the normal cell cycle.

    The conclusions of the paper are supported by the presented data and the discussion further develops the case that long slender forms may be key to parasite transmission to the vector. The work is based on using the standard model African trypanosome subspecies that infects rodents and not a trypanosome species that infects humans. This does not, however, diminish the potential impact of the work, as the rodent parasites are the field standard (and molecular tools have primarily been developed in that background). In addition to finding that long slender forms are competent for lifecycle completion, which could ultimately require amendment of medical school textbook lifecycles, this work also raises important questions about the role of the short stumpy form in parasite biology. The authors speculate the short stumpy forms may serve to control population size in a quorum sensing-dependent-fashion. While this notion conflicts with observations presented from human infections where blood parasite levels are very low, it remains unresolved what cues environments like the skin and other tissues present to the parasite, and how these may influence short stumpy differentiation.

  3. Reviewer #2 (Public Review):

    Differentiation pathways for parasitic organisms are of considerable importance, as they are relevant to understanding transmission, mechanisms of host specificity as well as, in some cases, offering possible routes to control measures. The transition between mammalian host and insect vector for African trypanosomes has been widely addressed due to accessibility and tractability. However, one view has been dominant, despite, as the authors suggest, considerable counter evidence. The present work posits an alternate pathway, questioning the role of the so called stumpy stage. This is of considerable importance to the immediate field and possibly wider.

    The major strengths here are in the use of a good model, and a high number of individual infections. The weaknesses include some assumptions with which I have issue, and given that this work is seeking to overturn a dogma, which also has assumptions, one needs to tread very carefully, to avoid falling into an unscholarly dispute. The major things are for me the assumption that PAD-1 cells are stumpy - almost anything seems to be able to activate PAD-1 and the lack of any quantitative data are concerning. This is difficult really and Matthews also says that PAD-1 does not equal stumpy and morphology is also important. Further, simple expression of EP procyclin is not sufficient for designation as pr cyclic, and the salivary gland cells are assumed metacyclic without demonstration of VSG expression for example. While I accept that these interpretations are reasonable, this is an assumption and in all three cases leads me to feel a little underwhelmed. Perhaps most concerning are the lack of statistical calculations as well as any attempt at further analysis beyond counting. The result is very much phenomenology and lacks any mechanistic insight.

  4. Reviewer #1 (Public Review):

    The data in the paper are mostly convincing, but might be somewhat over-interpreted: statistical analysis of the Tables is required. Yes, long slender bloodstream forms can definitely differentiate to pro cyclic forms and infect Tsetse. However, they take longer to differentiate than stumpy forms do, and even though morphologically stumpy forms are not an obligatory intermediate, expression of at least one stumpy-form mRNA (and presumably, others in the pathway) is definitely required. This should be stated in the Abstract. The conclusion that there is no cell-cycle arrest at all is not really supported by the data.

  5. Evaluation Summary:

    This paper describes the transmission of Trypanosoma brucei by the Tsetse vector. As part of these studies, the authors discovered that (i) a single parasite is sufficient for transmission and (ii) two stages of the Trypanosoma brucei life cycle (slender and stumpy forms) can be transmitted by the Tsetse vector - although the stumpy form developed more rapidly into proliferative parasites in the Tsetse midgut. The results are unexpected because it was previously thought that only stumpy forms were important for transmission.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

  6. Excerpt

    A parasite voyage re-visited: Trypanosoma brucei transmission to the tsetse fly is successful upon infection with slender forms of the parasite.