Review 2: "The Plasmodium Transmission-Blocking Symbiont, Microsporidia MB, is Vertically 1 Transmitted through Anopheles Arabiensis Germline Stem Cells"

RR:C19 Evidence Scale rating by reviewer:

• Reliable. The main study claims are generally justified by its methods and data. The results and conclusions are likely to be similar to the hypothetical ideal study. There are some minor caveats or limitations, but they would/do not change the major claims of the study. The study provides sufficient strength of evidence on its own that its main claims should be considered actionable, with some room for future revision.

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Review: In their manuscript "The Plasmodium transmission-blocking symbiont, Microsporidia MB, is vertically transmitted through Anopheles arabiensis germline stem cells", Onchuru et al. claim that (1) Microsporidia MB is vertically transmitted via the germarium, (2) the microsporidia goes from being distributed throughout the primary follicle early in egg development to primarily in the oocyte later in eg development, (3) the microsporidia exist at different sizes and undergo proliferation in the ovariole, and (4) microsporidia is lost during pre-imaginal stages of development in ~1/3 of offspring. 

The authors' claims are generally supported, however there are some uncertainties that must be addressed.

For claim 1, this reviewer agrees that the microsporidia is clearly in the germarium, however, do not share the authors' confidence in identification of the GSC and cystocyte nuclei in Figure 1D, especially given the lack of Click-IT edu staining in Figue 1D. Given that the authors used this stain to identify the cystocytes in Fgure 1C, why was it not included in Figure 1D? How did the authors confidently identify the GSCs, cystocytes, and boundary of the secondary follicle in Figure 1D, E, F? 

For claim 2, this reviewer agrees that the microsporidia are distributed throughout the primary follicle then later aggregated in the oocyte. However, staining for the microsporidia in the mature egg appears very faint to me (especially compared to staining in earlier stages). Are the authors confident they can rule out autofluoresence in Figure 2A Stage VII? It would be helpful to know what an uninfected control looks like compared to the potentially infected eggs at this mature oocyte stage to rule out the possibility of autofluorescence being mistaken for true signal. This is because it seems possible given these images that the microsporidia are being lost during the transition from Stage VI to Stage VII rather than during later development.

For claim 3, this reviewer agrees that the data support this claim.

For claim 4, the data generally support this claim. However, if the authors cannot provide additional support to their claim that microsporidia are found in high prevalene in Stage VII eggs, then they can’t conclusively state the microsporidia are lost during pre-imaginal stages of development. It seems equally plausible in this case that the microsporidia are lost during the final stage of egg maturation.