PONE-D-23-37049Peptidoglycan recognition protein PGRP-5 is involved in immune defense and neuro-behavioral disorders in zebrafish embryosPLOS ONE

Dear Dr. Liao,

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Dharmendra Kumar Meena

Academic Editor

PLOS ONE

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Additional Editor Comments:

The article is recommended for major revision

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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: Partly

Reviewer #2: Yes

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

Reviewer #1: No

Reviewer #2: Yes

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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

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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: No

Reviewer #2: Yes

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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: Xiong et al present an interesting finding that the Peptidoglycan recognition protein (PGRP) PGRP-5 is involved with zebrafish inflammatory response and locomotion. In particular the authors build a case for PGRP-5 through the use of sequence analysis, tissue expression levels with additional characterization of function with bacterial challenge (Gram + & -) and embryo tracking. However, the manuscript as presented needs several additional points of clarification in order to evaluate the findings thoroughly, as this reviewer’s background is pathogenesis in mammalian systems.

Major:

1) In line 90 the authors mentioned DrPGRP5 was cloned in the present study, but information on cloning is lacking in the methods. Moreover, in Fig4 the authors are measuring the mRNA level of DrPGRP5 – is this the endogenous PGRP5 or the short fragment that was cloned in response to bacterial challenge?

2) The authors should include methods for bacterial challenge and cultivation, as only the 107 CFU/mL is not enough. As it reads, it seems bacteria were added to embryos and over a 3-day period only embryos were checked for expression. Moreover, the authors mention dead embryos and larvae were removed daily – how many embryos died? How many embryos to larva events occur? This survival data may support the author's claims.

3) The authors use PGRP5-KD in fig5 to determine mRNA levels of inflammatory genes, but there is no mention of the stimulus, or if this is baseline. In the same context, what is fig6 (in the KD fish) measuring? Did the authors test these levels during bacterial challenge for both experimental setups? In line 248, please provide the data showing knockdown and define “pre-experimental” results.

4) Several points the authors go between embryo and larvae for experiments. Clarity is needed here – particularly for the 2.4 methods, fig3 and fig6.

5) Lines 327-329 needs context – exposed to what? PRRs recognize components of microbes.

Minor:

Several sections of the manuscript need revision for the reader’s clarity such as in lines 17-19, or 69-71.

Statistics across methods and the figure legends needs to be clarified as the methods mention n=4 and SEM were used, but in in several figures it is stated biological replicates and SD. Authors should check that the correct statistical test has been employed for the data (ie: are they parametric? )

Fig1 and results: Additional information pertaining to the % homology of the PGRPs is needed. Figure legend should include information about the full protein perhaps, as only the C-terminal amidase is shown.

Fig6: has n=4 from three biological replicates stated. Does this mean 4 samples per biological for a total n=12? Please show the individual data points and clarify this figure legend.

Reviewer #2: Manuscript Number: PONE-D-23-37049

Full Title: Peptidoglycan recognition protein PGRP-5 is involved in immune defense and neurobehavioral disorders in zebrafish embryos

Reviewer’s comments

A. Title (Line 1-2)

1. Appropriately written. Replace s with c in defence

B. Abstract –

1. Concise and reflect the content of the works done.

2. Line (27-28) – Cytokines are italicised here. It should be followed in the entire manuscript.

C. Keywords

1. Selection of keywords reflect the content completely.

D. Introduction

1. Line 50-51- Write immune response against instead of of .

2. Line 51 – Write full form of IMD

3. Line 63 - and structure also indicates. Add its before structure

4. Line 66 - challenge of pathogenic. Write due to instead of of. Add further before The

5. Line-75 - I should be small in intracellular

6. Line-81 – development and behaviour, of whom?

7. Line-83 – microbiota-brain interactions, of whom?

8. Line-85 – development and behaviour, of whom?

9. Line-85-87 – In…sentence is not complete.

10. Line 93- remove further after we

11. Line-96- replace aquatic ecosystem with fish

E. Materials and Methods

1. Line-142 – Write complete name of E. coli and M. lutues and italicize it.

2. Line-143 - 1 x 107 CFU/mL. it should be superscripted.

3. Line-162 - -actin…??

4. Line-168&181 – 500 L…??

F. Results

1. Line – 233 – write potential instead of potentially

2. Line-241- write Gram - before positive

3. Line-257 – TNF- α

4. Line-281- locomotor not loco-motor

G. Discussions

1. Written well.

H. Conclusion

1. Written well

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Reviewer #1: No

Reviewer #2: No

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Attachments

Attachment

Submitted filename: Reviewers comment_PONE-D-23-37049_31 May 24 for Author.docx

PONE-D-23-37049R1Peptidoglycan recognition protein PGRP-5 is involved in immune defence and neuro-behavioral disorders in zebrafish embryosPLOS ONE

Dear Dr. Liao,

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.

Please submit your revised manuscript by Jan 10 2025 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: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Jisheng Liu, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

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.

Additional Editor Comments:

The manuscript has been revised accordingly. The authors have addressed all the comments from the reviewers. However, there are some details that the authors should pay attention to. Therefore, I invite the authors to revise the details before the manuscript is accepted.

1. Beside the biological replicates, the technical replicates should be mentioned in M&M and the captions, such as Fig. 3, Fig. 4, Fig. 5, Fig. 6.

2. In Fig. 3A, what does the green and pink boxes stand for? Please indicates in the text and captions. Besides, the PGRP domain should be boxed as well.

3. Please explain and discuss why AChE was up-regulated in PGRP5-KD?

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. 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

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. 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: Yes

**********

5. 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

**********

6. 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 appreciate the diligence of the authors to address my points in the previous submission, and now feel these works are ready for publication. I do have a few minor comments below:

Minor:

- Fig3 B the intestine x-axis line is not straight

- Line 146, suggest to revise “in a nutshell…” vernacular to something like “Briefly,”

- Fig4 B y-axis label revise – states “Percent \\ %”

- It would be interesting to challenge PGRP-5 KD fish vs scrambled with E coli or M luteus as a definitive demonstration that loss of PGRP-5 leads to even more killing and reduced rate of hatching in fig4

- In 3.3 and throughout the manuscript be consistent with stating “Gram positive” / “Gram negative” or “Gram-positive” / “Gram-negative”

Reviewer #2: Dear Authors,

You have made necessary changes in revised manuscript satisfactorily which is technically sound, and data support the conclusions, data has been anaysed correctly and well written in standard English.

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7. 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: RAJU BAITHA

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[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.

Peptidoglycan recognition protein PGRP-5 is involved in immune defence and neuro-behavioral disorders in zebrafish embryos

PONE-D-23-37049R2

Dear Dr. Liao,

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.

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Kind regards,

Jisheng Liu, Ph.D.

Academic Editor

PLOS ONE