PONE-D-21-37709Verification of Hypsibius exemplaris Gąsiorek et al., 2018 (Eutardigrada; Hypsibiidae) application in anhydrobiosis researchPLOS ONE

Dear Dr. Kaczmarek,

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.

Dear Authors, two external reviewers have now assessed your manuscript "Verification of Hypsibius exemplaris Gąsiorek et al., 2018 (Eutardigrada; Hypsibiidae) application in anhydrobiosis research”, providing the comments that are reported below. As you can see, they both found identified a number of issues that would require careful revision before this paper is recommendable for acceptance.

Based on the reviewers' assessment, I'm thus here inviting you to take all of these comments into careful consideration and to modify your manuscript according to the provided constructive suggestions. I will then be happy to receive and further examine your revised version together with a point-by-point reply to each comment by myself and each reviewer, where you will need to explain any changes done to a particular piece of text, or include supported and convincing counterarguments to any points you may disagree with I'm confident you will find the present comments and suggestions relevant and useful to improve your work and I'm thus looking forward to hearing back form you by the due time.

Please submit your revised manuscript by Feb 24, 2022 . 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,

Marcos Rubal García, PhD

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.

2. Thank you for stating the following in the Acknowledgments Section of your manuscript: 

[These studies were supported by the research grant of National Science Centre, Poland, NCN 2016/21/B/NZ4/00131. The technical contribution of Kamil Janelt is highly appreciated.

Studies have been conducted in the framework of activities of BARg (Biodiversity and Astrobiology Research group).]

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. 

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: 

 [These studies were supported by the research grant of National Science Centre, Poland, NCN 2016/21/B/NZ4/00131.]

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

[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: Partly

Reviewer #2: No

**********

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

Reviewer #1: I Don't Know

Reviewer #2: No

**********

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: Yes

**********

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: This manuscript describes several alternative ways to dehydrate tardigrades of the species Hypsibius exemplaris. The alternative means used are interesting but the manuscript lacks precision in the motivation of these experimental choices. The quality of the images presented does not fully support the conclusions presented in the manuscript and needs to be improved.

Reviewer #2: General comments:

In this manuscript, authors used the tardigrade Hypsibius exemplaris and three dehydration protocols to study the shape of the tuns, the ultrastructure of the storage cells and the survival during rehydration.

After seven days, the three anhydrobiosis protocols used in this study showed variable efficiency in the formation of viable tun. The transmission electron microscopy analysis of tuns specimens showed storage cells degeneration status. Those status can be correlated with variable efficiency in the formation of viable tun. Propawa et al. concluded that only tuns without visible degeneration of storage cells are able to support a successful rehydration process.

This manuscript needs major revisions. Indeed, the poor quality of the pictures doesn’t allow to conclude anything.

Specific comments:

Text:

1. Line 84: There is no reference or accurate description of the experimental protocol for “environmental drying”. Why did authors choose these two substrates?

2. Line 96: Propawa et al. used "double-distilled water and Spring water" instead of the classical Chalkley medium (http://cshprotocols.cshlp.org/content/2018/11/pdb.prot102319.full). Please justify this choice.

3. Line 108: In this work, authors mentioned Boothby’s protocol (http://cshprotocols.cshlp.org/content/2018/11/pdb.prot102327.full.pdf). In this protocol, tardigrades are starved a day before dehydration. In the present work, it’s not described? Please specify if a starvation was conducted.

4. Line 125: Propawa et al. described a terrarium sand substrate.

Line 128: Authors described a substrate consisting of soil and decomposed plants.

I have the same remark as point 1. Why did they choose these two substrates?

5. Line 148 + Line 151: Authors mentioned twice the same fixation protocol with 2.5% glutaraldehyde. Only one is enough.

6. Line 150 to Line 153: Protocols A and B are totally different.

Tuns obtained by protocol A were photographed on agar plates using an Olympus SZ61 stereomicroscope.

Tuns obtained using protocol B were fixed in 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer, mounted in a drop of water on a slide, covered by coverslip, and photographed with the use of Olympus BX60 stereomicroscope and OLYMPUS DP50 camera.

It is impossible to compare both specimens.

7. Line 154: Authors indicated that 10 specimens were analyzed. Is it 10 specimens in total or 10 specimens per experimental condition? It is not clear. Indeed, line 131, 5 specimens are mentioned.

8. Lines 175 to 177: Propawa et al. describe Figure 1. As indicated in another comments (comment on figure 1 and point 6), the quality of these pictures is not sufficient to compare the two protocols and protocols are not comparable. A figure 1 modification is required.

9. Line 181: Same question as the point 7. Please clarify the number of tardigrades.

10. Line 186: TEM ultrastructural analysis of specimen integrity is based only on storage cells according to a previous study in another species (Czernekova, 2018). Is the analysis of a single cell type sufficient?

11. Line 192: Authors describe mitochondria in Figure 3A. But the quality of the picture needs to be improved. This figure doesn’t allow to observe the state of the mitochondria or mitochondria cristae. We can’t conclude anything on mitochondria with this figure.

12. Line 194: Authors describe for Figure 3B: "single vacuole and autophagosomes appeared". But, according to me, we can also observe a vacuole in stage O.

13. Line 198: Like above, a better image quality is required to allow conclusions.

14. Line 212: " ca” should be replaced by "can".

Figures:

Figure 1: The quality of the two tuns pictures is insufficient. Images are totally different and cannot be compared to each other. As described in the M&M section, two different stereomicroscopes are used. The exposure for the two pictures is also different. The picture for the protocol B doesn’t allow to correctly observe the tun. The resolution is not sufficient.

For the storage cells TEM pictures, we don’t have scale bar.

Figure 3: The quality of pictures needs to be improved. Status of mitochondria or cristae for exemple are not clearly distinguishable.

Figures captions:

Figure 1: A description of tuns in the middle of the figure is required. And it’s precisely pictures that cannot be compared.

Figure 2: Statistical t-test is necessary. The test is mentioned in the M&M section but we can’t find the result in the rest of the paper.

Figure 3: Scale bars are very different between pictures (0.38um, 0.53um, 0.42um,…). For an easy and good reading of images, it is necessary to homogenize the scale bars.

**********

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: 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.

Attachments

Attachment

Submitted filename: Propawa-et-al-Review-2021-1.docx

Verification of Hypsibius exemplaris Gąsiorek et al., 2018 (Eutardigrada; Hypsibiidae) application in anhydrobiosis research

PONE-D-21-37709R1

Dear Dr. Kaczmarek,

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,

Marcos Rubal García, PhD

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 #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

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: (No Response)

Reviewer #2: Authors have addressed all my comments and remarks. They increased explanations concerning their experimental choices. They also improved the quality of images as suggested. In its present form, the paper can be accepted for publication.

**********

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: No