PONE-D-20-17817

Experimental methodologies can affect pathogenicity of Batrachochytrium salamandrivorans infections

PLOS ONE

Dear Dr. Gray,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The reviewers and I agree this revision is a great improvement on the first submission, and acceptable for publication. They also suggest some minor revisions to increase the impact of the paper. I recommend you address these suggestions prior to acceptance. (And, apologies to the authors for the delayed decision, this revision got to me in the middle of a move.)

Please submit your revised manuscript by Sep 13 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Wendy C. Turner

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I commend the authors for their thorough revisions and believe the paper is much improved. My only remaining concern is that the introduction still lacks context, and as the introduction stands now the reader has no way of knowing a) why these five experimental objectives are relevant or b) what the underlying hypotheses/expectations are. I understand that these details are present in the discussion, but that is read last. An important purpose of an introduction is to establish context for the reader to understand the questions and objectives in the study before diving into the methods and results. However, I will leave it to the editor to decide whether to request this revision.

Reviewer #2: This manuscript is much improved and with a few minor changes I think could be accepted for publication at PLOS ONE. I very much appreciate the addition of Table 1 and the additional details in the methods sections which serve to clarify the experimental design. I also think that changing the language from “tolerance/resistance” to pathogenicity is appropriate and addresses many of the initial reviewer comments. However, I do feel that some of the comments raised by reviewer 1 could be addressed more thoroughly in the text, and not simply in the rebuttal letter.

First, the supplemental figure provided for reviewer 1 could be provided as a supplemental figure to accompany the text and address many of the reviewer comments asking for more information from the Bsal qPCR results. However I am slightly confused by one comment in the rebuttal letter that states the following “Response: We did not process all the swab samples collected as part of this experiment due to limited funds and these data weren’t necessary for inferences on pathogenicity.” This fact is not reported in the paper at all. Rather the methods section implies that all swabs were tested. Which swabs were tested and which were not? How did you test for significant differences in pathogen loads if you did not test all the swabs? This point must be clarified and transparently reported in the text.

Additionally, this paper still fails to connect the findings to a broader significance beyond recommendations for Bsal infection experiments, which I think is a missed opportunity. First, this narrow framing is manifest in the introduction which – as initially stated by reviewer 1 – is underdeveloped. I think the second paragraph of the introduction should be a broader discussion of why some of the factors such as passage number, type of enclosure, and inoculum method would affect pathogenicity and how understanding these effects are relevant to natural systems/conservation. Then the authors can get more specific and discuss the examples they cite.

At line 257 the authors could take an additional step and comment on how the infection efficiency of Bsal zoospores in water is relevant in an ecological or conservation context. Additionally at Line 276 the same comment applies. Discuss the tradeoffs between being able to shed your skin/shed zoospores into the environment vs the risk of being exposed to the pathogen in the water. This study does have broader implications for understating Bsal dynamics in natural settings and I think the authors should try to make some of these connections.

Other specific notes:

The data availability should have provide a unique URL to the specific data.

Line 138 – It is unclear what you mean by “method” here. Can you specify?

Line 191 – I prefer listing the SD along with the mean.

Line 220 – Typo in this sentence “of” should be “or”

**********

6. 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 #1: No

Reviewer #2: Yes: Allison Q Byrne

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment 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. Registration is free. 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 PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Experimental methodologies can affect pathogenicity of Batrachochytrium salamandrivorans infections

PONE-D-20-17817R1

Dear Dr. Gray,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Wendy C. Turner

Academic Editor

PLOS ONE