Peer Review History

Original SubmissionJanuary 30, 2020
Decision Letter - Blossom Damania, Editor, Robert F. Kalejta, Editor

Dear Dr. Li,

Thank you very much for submitting your manuscript "Exosome-mediated apoptosis pathway during WSSV infection in crustacean mud crab" 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. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

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Sincerely,

Robert F. Kalejta

Associate Editor

PLOS Pathogens

Blossom Damania

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

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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: In this manuscript, Gong and collaborators revealed the involvement of exosomal miRNAs in antiviral immune response of crustacean mud crab. In invertebrates, the exosome relevant researches are quite limited. This manuscript firstly identifies the exosomes in crustaceans. In general, this is a valuable contribution to our understanding of exosome-mediated immune pathway of crustaceans and the host defense against the invading virus. Overall, the results described in the manuscript looks very convincing and some results sound interesting to the readers in the relevant fields. The authors are very proficient at the experimental techniques and relatively reasonable at experimental design. However, some issues need to be addressed.

Reviewer #2: The current manuscript titled “Exosome-mediated apoptosis pathway during WSSV infection in crustacean mud crab” describes how exosomes released from WSSV-injected mud crabs could suppress viral invasion by inducing apoptosis of hemocytes. Using WB analysis of exosome markers (CD9 and TSG101) and cytoplasmic marker (calnexin), the authors have found that Caspase 3/7 activity was significantly increased in the exosome-WSSV and WSSV co-injected group compared to the control group. Here, the controls included the apoptosis inducer cycloheximide and apoptosis inhibitor Z-VAD-FMK for their effects on WSSV proliferation in hemocytes of mud crab. Results showed lower WSSV copy number in the cycloheximide and WSSV-injected group, but significantly higher WSSV copy number in the Z-VAD-FMK and WSSV-injected group. Finally, in an attempt to reveal the roles of miR-137 and miR-7847 in apoptosis, mud crabs were injected with AMO-miR-137 and AMO-miR-7847, and Flow analysis revealed that both AMO-miR-137 and AMO-miR-7847 induced higher apoptosis compared to the controls.

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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: No.

Reviewer #2: Overall, this is an interesting manuscript and there are some novel findings that is validated in an animal model. The concerns include:

1. It is not clear what is the ratio of EVs to Cells, i.e., Is it 1:1010, 1:1000, 1:10000 to score for an effect?

2. Data in Figure 2D requires at least multiple WBs to validate the results with confidence

3. For mir137 to work in cells, they require high levels of miRNA machinery components. What are the levels of Ago2, Dicer, TRBP, etc in the recipient cells?

4. What percent of HSP70 binds to AIF vs Bax?

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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: 1) The manuscript contains some ill sentences and grammar errors. Addressing these concerns would help them to improve their manuscript. For example, the Latin name spelling should be Scylla paramamosain, not Scally paramamosain.

2) In references section, article titles should only be capitalized on the first word, not the first letter of each word.

3) The protocols of exosomes isolation should be described in detail. At present, there is no mature instruction for the exosomes isolation in crustaceans.

4) The authors found that two exosomal miRNAs (miR-137 and miR-7847) were both involved in antiviral immune regulation in mud crab. It is necessary to explore whether there exist primary or secondary relationships during the regulatory process.

5) The authors only detect the interactions between miRNAs and AIF, while the relationship between exosomes and AIF is not studied. Thus, the effect of exosomes challenge on AIF expression should also be investigated.

6) It is found from the data that AIF could interact with HSP70, while HSP70 could interact with Bax. Thus, whether AIF could directly interact with Bax should be clarified in detail accordingly.

Reviewer #2: None

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Reviewer #1: No

Reviewer #2: No

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Revision 1

Attachments
Attachment
Submitted filename: Response to the decision letter.doc
Decision Letter - Blossom Damania, Editor, Robert F. Kalejta, Editor

Dear Dr. Li,

We are pleased to inform you that your manuscript 'Exosome-mediated apoptosis pathway during WSSV infection in crustacean mud crab' 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,

Robert F. Kalejta

Associate Editor

PLOS Pathogens

Blossom Damania

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):

Formally Accepted
Acceptance Letter - Blossom Damania, Editor, Robert F. Kalejta, Editor

Dear Dr. Li,

We are delighted to inform you that your manuscript, "Exosome-mediated apoptosis pathway during WSSV infection in crustacean mud crab," has been formally accepted for publication in PLOS Pathogens.

We have now passed your article onto the PLOS Production Department who will complete the rest of the pre-publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Pearls, Reviews, Opinions, etc...) are generated on a different schedule and may not be made available as quickly.

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Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

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

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