PONE-D-23-11181Ultrastructure of the nebenkern during spermatogenesis in the praying mantid ​Hierodula membranaceaPLOS ONE

Dear Dr. Fabig,

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PLOS ONE

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   "We would like to thank the members of the Core Facility Cellular Imaging (CFCI, Faculty of Medicine Carl Gustav Carus, TU Dresden) for help with light microscopy and the EM facility at MPI-CBG, Dresden for technical assistance with electron tomography. We would also thank various members of the slack channel “EMofCellsTissuesOrganisms” for help with identifying structures. We are also grateful to Dr. Daniel Baum (Zuse Institut Berlin) for support in using ZIB Amira. This work was supported by grants from the National Science Foundation (NSF, RUI1715157 to L.P.) and the Deutsche Forschungsgemeinschaft (DFG, MU1423/10-1 to T.M-R.)."

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Additional Editor Comments:

The current version of this manuscript cannot be considered for publication in PLOS One.  It needs a major revision. The following concerns should be addressed:

1. The abstract is very simple, the main conclusion is not included in the manuscript.

2. Improve the quality of some of the figures, refer to the reviewer's comments.

3. What is the main function of “nebenkern”?

4. Novelty of this study should be pointed in the "Conclusion" part.

All the two reviewers' concerns must be answered if you agree to revise your manuscript. If you decide to revise this manuscript, a list of changes should be uploaded along with the 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: Yes

Reviewer #2: Yes

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

Reviewer #1: N/A

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: Yes

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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: In this manuscript, the authors applied high-pressure freezing in combination with transmission electron microscopy to study the ultrastructure of sperm development in subadult males of the praying mantid Hierodula membranacea. They revealed some features of cellular structures during spermatogenesis, including the structure of the chromosomes in prophase I and the distribution of organelles in spermatids. And they focused on the structure of the nebenkern in spermatids, revealing that the nebenkern of this species has two zipper-like membrane structures at opposing positions. However, there are some major issues with this paper.

1. The Abstract of this manuscript is too sample to attract readers. And the main point of this manuscript is not summarized in the Abstract.

2. Although the new methods were used to study the ultrastructure of sperm development, the topic of this manuscript was insufficient of innovation. Besides, the quality of all figures was not high, and several of them were blurry.

3. The function of the nebenkern during spermatogenesis was not mentioned in this manuscript, so the scientific value and research significance of this study was not outstanding.

4. Most of the refs cited in this paper are too old. More recent studies should be cited. And the format of the refs should be checked and revised carefully.

Reviewer #2: Peer review report on“Ultrastructure of the nebenkern during spermatogenesis in the praying mantid Hierodula membranacea”

This paper used state-of-the-art sample preparation techniques in combination with three-dimensional (3D) imaging such as high-pressure freezing and serial-section electron tomography, showing a survey of the steps of spermatogenesis in praying mantid Hierodula membranacea. The authors mainly focused on a structure of “nebenkern”, which was found in multiple insects during spermatogenesis. They observed the ultrastructure of nebenkern through three dimensions, in addition, they certified that the special organelle was composed of two interwoven segments that were connected by a zipper-like structure at opposing positions. This paper provided the detailed information of nebenkern for other in-depth study.

Comments to Author:

Minor comments:

1. In the manuscript of review versions, you don’t number the line in the whole paper. I hope you add the line number if you submit your manuscript in the future.

2. In the section of “Movie legends -Movie 4”, “Stitched” should be changed into “Stitched”.

3. In the section of “Figure legends”, there are many abbreviations of proper nouns, the full names appear just need appear once, you can use these abbreviations after the second.

4. It will be better to change your alpha code form lowercase to capital letter in your figures, because there are both capital letter to code your figures in “Figure legends” and manuscript.

5. Why you used the development stage of subadult male praying mantid as your object in this research, but not male praying mantid in other development stage? What advantages of the subadult mantid?

6. In your article, you focused on the “spermatids at different stage of development”, but I don’t know clearly the staging criteria. In addition, I think there is a little chaos in your “spermatocyte” and “spermatid”, you should distinguish the two stages more clearly.

7. Can you explain the differences of “axonemal microtubules” with “microtubules” for me?

8. I want to know more about the function of nebenkern during spermatogenesis in insects, I mean you should supply more information of nebenkern in your “Introduction” section. For example, its formation process, composition, function in reproduction and so on. This part is a little poor in your manuscript now.

9. In Figure 3, you mentioned that you get seven serial semi-thick (300 nm) sections, but there are just four sections you show?

10. Why you want to examine the microtubules in the 3D structure of nebenkern? In your opinion, if the cytoskeleton participates in the formation of nebenkern, I think it is necessary to examine the dynamical changes of microfilament during this process.

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Reviewer #1: No

Reviewer #2: No

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Ultrastructure of the nebenkern during spermatogenesis in the praying mantid ​Hierodula membranacea

PONE-D-23-11181R1

Dear Dr. Fabig,

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.

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Kind regards,

Wan-Xi Yang, 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 #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: N/A

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

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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 #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: (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 #2: No

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