PONE-D-21-38148Deciphering the mechanism of anhydrobiosis in the entomopathogenic nematode Heterorhabditis indica through comparative transcriptomicsPLOS ONE

Dear Dr. Mohan,

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.

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We look forward to receiving your revised manuscript.

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Anil Kumar Singh, Ph.D.

Academic Editor

PLOS ONE

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 “This is part of PhD research carried out by the first author Mr Manimaran Balakumaran  funded by the Post Graduate School, ICAR-IARI, New Delhi, India - 110012”

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5. 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 If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references.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 If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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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: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: N/A

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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: Yes

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: The manuscript “Deciphering the mechanism of anhydrobiosis in the entomopathogenic nematode Heterorhabditis indica through comparative transcriptomics” studied the comparative transcriptome analysis of unstressed and anhydrobiotic H. indica. The research paper comprehend the scientific information revealing the role of different metabolic pathways for survival of H. indica during anhydrobiosis. These kinds of novel work provide insight regarding anhydrobiotic survival strategies of H. indica which could be useful for designing novel EPN formulations of H. indica with enhanced and sustained shelf-life during field applications, enhancing its biological control potential. The manuscript is well written and has potential to be accepted in PLOS ONE with few points needs to be incorporated for improvement.

- The material and methods are well written, however what was the source of IJs of Heterorhabditis indica strain KX601067 and on what basis it was identified, is not mentioned.

- The authors have well documented the results and supported the discussion with relevant literature nicely. But, a concrete conclusion about application of this research is lacking. Add one or two lines of conclusion in light of future application of this research.

Reviewer #2: The authors have compared the relative transcript abundance by RNA-seq during anhydrobiosis in the entomopathogenic nematode Heterorhabditis indica. The infective juveniles of H.indica were subjected to drought stress and compared the transcriptome between the control and drought stressed stages. Since the dried formulations of EPN are used as an alternative to chemical pesticides, this study might actually help in designing longer lasting formulations. However, there are some concerns, which can be addressed by the authors so that the overall quality of the manuscript is better.

1. In line 86, authors have mentioned that the read length was between 30 and 81 bp. Authors need to elaborate more on it. The information on whether it was single end or paired end sequencing is also not provided in the Materials and methods section. It is highly unlikely that read lengths were between 30-81 bp. Such a small length will give a misassembled de novo transcriptome. Detailed information on the sequencing depth, number of reads, purity filtered reads and elimination criteria for shortlisting the purity filtered reads should be provided in the MM.

2. Though authors have randomised the experiment, there is no information on the number of biological replicates used in the study. In case a single replicate was used, authors should compare at least their assembly from control samples with the previously published transcriptome data (Somvanshi et al., 2016).

3. Since the draft genome of the H.indica has recently been published (Bhat et al., 2022), authors are encouraged to perform a reference based mapping and quantification to improve their data. Alternatively, they can also map the de novo assembled transcriptome on the genome and remove the false and chimeric assemblies to improve the overall quality.

4. The results are too short, whereas the discussion is too long. In fact discussion has portions of results. It is advised that results can be merged.

5. Some of the sections in MM can be merged, for e.g., 4.2.2.2 can be merged with 4.2.2.3 and 4.2.2.4. Similarly, 4.2.2.5 can be merged with 4.2.2.6

6. The data should be submitted to a public database and requisite information should be provided in the manuscript.

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Reviewer #1: No

Reviewer #2: No

[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.

PONE-D-21-38148R1Deciphering the mechanism of anhydrobiosis in the entomopathogenic nematode Heterorhabditis indica through comparative transcriptomicsPLOS ONE

Dear Dr. Mohan,

Thank you for submitting your manuscript to PLOS ONE. 

Unfortunately, the original reviewers did not agree to review the revised manuscript and I had to restore it to the newer reviewers who highlighted a few new changes. Please go through the comments and respond accordingly.  Please submit your revised manuscript by Aug 21 2022 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-emailutm_source=authorlettersutm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Pankaj Bhardwaj, 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.

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: (No Response)

Reviewer #3: (No Response)

Reviewer #4: 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 #3: Partly

Reviewer #4: Yes

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

Reviewer #1: Yes

Reviewer #3: No

Reviewer #4: Yes

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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 #3: Yes

Reviewer #4: (No Response)

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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 #3: No

Reviewer #4: Yes

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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: (No Response)

Reviewer #3: The manuscript titled "Deciphering the mechanism of anhydrobiosis in the entomopathogenic nematode Heterorhabditis indica through comparative transcriptomics" described the transcriptome comparison of anhydrobiotic and unstressed Heterorhabditis indica. For this purpose, the authors isolated total RNA for H. indica subjected to dehydration with a glycerine-water solution for 48 h. Then, a standard RNASeq pipeline was applied, and the data indiacted a upregulation of major, well known pathways linked to anhydrobiosis, including heat shock tolerance, polyamine biosynthesis, antioxidant defenses, among many others. Despite the importance of the subject of the manuscript, some major questions impair the manuscript publication in its current form.

Major points:

1. Despite the authors simulate a desiccation process by using a glycerine:water solution, the glycerol itself can be itself as a kosmotrope and chaotrope substance, which depends on the final concentration [Timson, David J. "The roles and applications of chaotropes and kosmotropes in industrial fermentation processes." World Journal of Microbiology and Biotechnology 36.6 (2020): 1-13]. In the experimental setup made by the authors, it is not clear if a ~10% glycerol solution could be inducing a dehydration response per se. Why not lowering the humidity of the chamber (below 60%) to induce a dehydration response?

2. The description of GOs and its graphical representations are below the standard (Figures 4 and 5). What are the statisticals related to GO analysis (number of mapped transcripts, p-value, FDR)? A table containg the GO data and all statistical analysis is preferable instead of pizza graphics.

3. The authors performed a clustering analysis of qPCR data (Figures 7 and 8)? If not, I strongly advise in order to better see the clusters of similar expressed genes.

Reviewer #4: (No Response)

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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 #3: No

Reviewer #4: No

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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.

Deciphering the mechanism of anhydrobiosis in the entomopathogenic nematode Heterorhabditis indica through comparative transcriptomics

PONE-D-21-38148R2

Dear Dr. Mohan,

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,

Pankaj Bhardwaj, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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 #3: 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 #3: Yes

**********

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

Reviewer #3: 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 #3: 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 #3: 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 #3: The revised manuscript can be accepted for publication. All questions were addressed by the authors.

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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 #3: No

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