Dear Drs A. Hehl and C. Ramakrishnan,

Thank you very much for submitting your manuscript "Dissection of Besnoitia besnoiti intermediate host life cycle stages: from morphology to gene expression" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by 3 independent reviewers. 

As part of the lifecycle of Besnoitia besnoiti remains puzzling, the editors and reviewers have appreciated your study representing a useful addition to the field. However, we found that the presentation of the data needs rearrangement and more accuracy in the gene names; the main purpose/message of the work is confusing; and the work will benefit from a better integration of the results with sexual stages of other Apicomplexa.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Isabelle Coppens

Associate Editor

PLOS Pathogens

Vern Carruthers

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: A review of genome and RNAseq data on Besnoitia besnoiti isolated from cattle. The analysis of the sequence data is reasonable and is correlated with data in T. gondii and other Apicomplexa. The data indicate that a sexual cycle exists and, to an extent, exclude an insect host (based on looking at Plasmodium homologs that might indicate an insect host). I do not think this is completely excluded by the data, but it is a reasonable suggestion. The light and EM images of the cysts are useful and add to the literature. In fact a more detailed series of images in the supplemental figures could be useful.

Reviewer #2: 1. Here the authors use microscopy, genome sequencing, and transcriptomics to characterize Besnoitia besnoiti in its in vitro tachyzoite and in vivo bradyzoite forms. Genomic comparisons to Toxoplasma gondii, Neospora caninum, Eimeria tenella, and Plasmodium falciparum are made as well as transcriptomic comparisons to T. gondii. In silico evidence for a definitive B. besnoiti host and different metabolic pathways are presented. Overall, a useful addition to the field. Some confusion about the purpose of the paper- is the main idea comparison of intermediate stages as title suggests, or is it more about presenting an argument for a sexual stage and definitive host? To strengthen definitive host argument, deeper analysis of intermediate stage orthologs between T. gondii and B. besnoiti should be performed. Figure confusion needs to be addressed. Recommend some rearrangement and condensation of figures.

Reviewer #3: Besnoitia besnoiti is a cyst-forming apicomplexan protozoan parasite closely related to Toxoplasma gondii that can infect livestock, especially cattle, as intermediate hosts. Bovine besnoitiosis is a disease that progresses in two phases: an acute phase and a chronic phase. The acute phase can be fatal or cause infertility or sterility in males. Symptoms range from fever and anorexia to vascular disorders. In the chronic phase, various skin lesions may occur. Unfortunately, there are no vaccines or therapeutics to date that could halt the progression of the disease. This study represents a milestone in the study of Besnoitia besnoiti biology and provides a wealth of "omics" data on two different stages of the intermediate host cattle. Transmission electron microscope (TEM) analysis revealed that organellar structures match well with those of T. gondii, but also remarkable differences in the cyst wall, which has probably adapted to its particular niche in the skin.

A significant part of the life cycle of this parasite remains unknown, and to date no definitive host has been assigned to it. This study provides unequivocal genomic evidence for the presence of genes in Besnoitia besnoiti that are homologous to those of Toxoplasma and known to be specific for the sexual cycle (development of microgametes and macrogametes) in the cat intestine and oocyst formation, supporting the original hypothesis of a definitive carnivorous/scavenger host for this parasite. Regarding the expression of its genome, B. besnoiti has conserved a fingerprint of conserved stage-specific markers, but is also characterized by a specific subtranscriptome. As in Toxoplasma, the differences between the tachyzoite and bradyzoite stages show very distinct signatures.

The entire life cycle of B. besnoiti remains to be elucidated, but this study provides a fairly comprehensive analysis of the biology of zoites responsible for acute and chronic disease.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: The data is clearly presented and appears complete. I do not see much need for additional validation of the RNAseq data using immunoblot or proteomic approaches; as one would expect a reasonably close correlation between the RNAseq data and results on protein levels for the genes discussed.

The author notes that the CST1 homolog in Besnoitia lacks a mucin domain and would not be a source of lectin staining; however, I did not see any mention of lectin staining of these cysts. It would be useful to provide this data (e.g show that cysts stain with Dolichus). If they are DBA positive then the issue is if this is still due to glycosylation of the CST1 homolog or if another gene exists with a mucin or similar domain that would be DBA positive if post transitionally modified.

Reviewer #2: Regarding in silico evidence for a definitive host: authors rely heavily on existence/number/conservation of B. besnoiti orthologs of Toxo sex genes. To strengthen this argument, why not perform a similar analysis on bradyzoite/cyst genes? Because intermediate lifestage data are available and reliable in both Toxo and B. besnoiti, that analysis should serve as a positive control for what you might expect to observe for sex genes. For example, of the B. besnoiti genes that are highly expressed in Bb cysts relative to tachyzoites, how many are orthologous to Toxo bradyzoite/cyst genes? Add this comparative analysis to figure 6.

Reviewer #3: (No Response)

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: I would suggest that Figure S1 on the TEM of the cyst be included in the figures in the paper rather than supplemental data. In addition, if more images exist of the TEM and SEM they could be provided as additional supplemental images.

In Figure 1 higher magnification images of the light microscopy with HE and MB/fuchsin should be provided.

Reviewer #2: Figure legends:

o please specify that images are of the cyst wall surface in Fig. 1D-G

o 7D description is repeated. difference between 7C and D is unclear in legend, figure, and text (more below)

The authors write as though the most interesting part of the paper is evidence for an as-yet unknown definitive host, which is interesting. For example, in the intro and results, evidence for a definitive host is discussed and presented first. Recommend discussing/presenting sexual stage last after discussing intermediate stages.

Page 9: authors note that 432 genes were expressed in neither tachys nor bradys. how does that fraction compare to T. gondii gene expression studies? ie what fraction of T. gondii genes are not expressed in tachys or bradys?

Page 10/figure 4a: recommend adding an intermediate color (or multiple) between blue and red. it is difficult to distinguish the shades of red and blue, blue in particular.

Page 12/figure 6c: there is no figure 6c

Page 12/figure 6: text doesn’t match figure. figure shows highest percentage of genes are EES1, text states highest percentage of genes are EES5 and oocyst

Page 14/figure 7: figures 7c and 7d are confusing. why are there opposite colorations for most genes if the only different between c and d is the scale? I am interpreting “difference to common mean” as only a change in scale, so if that is not correct, the difference between c and d needs to be better explained in text and figure legend.

Page 14/figures 7 and 8: as is, recommend combining and condensing figures 7 and 8. move 7a to supplement and replace with 8a, as they are quite similar. 7b is fine but doesn’t say much: could either go to supplement or have added annotation to describe which classes of genes are high and low in tachys vs. cysts. there is plenty of room to make the colored portion of the plot narrower to make room for labels. 7c/d are very confusing, perhaps remove one? the dot plots in 8c/d are ok, but could easily be communicated as heatmaps like in 7c/d, with one heatmap for Toxo & one for B. besnoiti, side-by-side. 7c/d seem very similar to 8 c/d, just different genes. Given available data for Toxo, recommend adding a Toxo gene expression heatmap next to the 7c B. besnoiti heatmap. So in summary for 7c/d and 8c/d, make all into heatmaps, each with maps of B. besnoiti alongside maps of Toxo.

Reviewer #3: The manuscript contains a large amount of data, some of which is unfortunately inadequately presented. Some rewording is necessary to guide the reader through concepts that may be difficult for those outside the field to understand.

Below are some points to improve the clarity and presentation of the data:

1- Differential expression of SRS genes, ROP, MIC, and GRA genes (including newly discovered/renamed genes) should be represented as in Figure 3 of Hehl et al. (BMC Genomics, 2015). A similar graphical representation could also highlight conservation with Toxoplasma and other CFAs.

2- The authors should better document the conservation of proteins responsible for interaction with host cells, especially the ROP and GRA proteins of B. besnoiti, by specifying their polymorphism, which has often been associated with their function (e.g. ROP16) or stage-specific expression (e.g. ROP18) or even interaction with their partner, e.g. KIM domains of GRA24 as drivers of host MAPK p38-alpha interaction and activation.

3- AP2 factors deserve special attention and should be presented in a scheme with their structural domain (AP2, ACDC) together with their putative Toxoplasma, but also Eimeria orthologs, indicating at which stage they are expressed in these parasites. Identification of MORC-associated AP2 orthologs would provide further evidence that epigenetic mechanisms exist in B. besnoiti to control sexual commitment.

4- To identify genes likely involved in the B. besnoiti sexual cycle, the authors looked for known homologs in Toxoplasma, but what about conservation in Eimeria? Are the 267 transcripts mentioned in the study unique to B. besnoiti or are they conserved in some CFAs?

5- Table S7 should list TgIST, TgNSM, and TEEGR as bona fide proteins that are presents in dense granule.

**********

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Reviewer #1: No

Reviewer #2: Yes: Laura Knoll

Reviewer #3: No

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

References:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Dear Dr. Adrian B. Hehl,

Thank you very much for submitting your manuscript "Dissection of Besnoitia besnoiti intermediate host life cycle stages: from morphology to gene expression" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

For the Editor:

Two rectifications:

1) The morphological identification of organelle: In Fig. 3: It is speculative to identify a vacuolar compartment serving as a Ca2+ storage and sodium transport activity (VAC) in the absence of marker for this organelle. Could sections of progeny. Replace VAC by VAC? The ap for apicoplast in panel C is erroneous. An apicoplast is visible just above the nucleus based on multiple membranes and global shape. The inner membrane complex beneath the plasma membrane can also be shown. The IMC is an organelle.

2) Regarding a cyst wall staining with DAB: the image with Toxoplasma cysts as positive controls must be shown in the manuscript as it has been done. Not as personal communication to the reviewer.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Isabelle Coppens

Associate Editor

PLOS Pathogens

Vern Carruthers

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************

For the Editor:

Two rectifications:

1) The morphological identification of organelle: In Fig. 3: It is speculative to identify a vacuolar compartment serving as a Ca2+ storage and sodium transport activity (VAC) in the absence of marker for this organelle. Could sections of progeny. Replace VAC by VAC? The ap for apicoplast in panel C is erroneous. An apicoplast is visible just above the nucleus based on multiple membranes and global shape. The inner membrane complex beneath the plasma membrane can also be shown. The IMC is an organelle.

2) Regarding a cyst wall staining with DAB: the image with Toxoplasma cysts as positive controls must be shown in the manuscript as it has been done. Not as personal communication to the reviewer.

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #2: The author have adequately addressed the reviewers concerns.

Reviewer #3: This is a timely and well done study. The authors have addressed all the points raised by the reviewer. The work has been greatly improved with the addition of new analysis and tables.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #2: (No Response)

Reviewer #3: (No Response)

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #2: (No Response)

Reviewer #3: (No Response)

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: Yes: Laura J. Knoll

Reviewer #3: Yes: HAKIMI Mohamed-Ali

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

References:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Dear Dr. Adrian Hehl,

We are pleased to inform you that your manuscript 'Dissection of Besnoitia besnoiti intermediate host life cycle stages: from morphology to gene expression' has been provisionally accepted for publication in PLOS Pathogens.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Isabelle Coppens

Associate Editor

PLOS Pathogens

Vern Carruthers

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************************************************

Reviewer Comments (if any, and for reference):