PONE-D-24-02112Genetic sex-determining mechanism in Hyla eximia treefrog inferred from H-Y antigenPLOS ONE

Dear Dr. L,

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PLOS ONE

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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

Reviewer #3: Partly

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

Reviewer #1: N/A

Reviewer #2: Yes

Reviewer #3: 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

Reviewer #3: 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

Reviewer #3: 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: Ruiz and colleagues reported the study of treefrog sex determination by H-Y antigen, presenting some interesting data, while I have some concerns about the experimental design and data interpretation.

Major concerns:

1. I am confused about the cloning and structural analysis of Actb, what is the relationship of these data with H-Y antigen?

2. H-Y antigen is 33 bp, while the amplicon is 276 bp. I would suggest to show the sequence and primer location, in order to facilitate the readers’ understanding.

3. As shown in Figure 5, H-Y antigen was actually also expressed in male, I wonder if the conclusion could be supported or not.

Minor concerns:

1. In Figure 2C, I suggest to remove Bufo gargarizans and Bufo bufo in upper part

2. Line 58, the sentence “which causes ZZ male to female sex reversal” should be revised. In fish, there are more cases that ZW individuals develop into male under high temperature.

3. Grammatic improvement is required. For example, Line 161, qPCR or quantitative q-PCR, should be streamlined throughout the manuscript.

Reviewer #2: Authors present their work on identification and quantification of the HY-antigen gene in Hyla eximia tree frog. It is a solid piece of work and I enjoyed reading it. There are some major points I would like to raise.

- Title can be toned down a little as authors haven't fully uncovered the sex-determination mechanism. They have identified potential sex chromosomes.

- I found discussion section to be inadequate. Some sections belonged in introduction and results must not be repeated. It is not contextualised broadly in terms of why this study matters to the field. Please rewrite discussion. Discussion should contain information that you could not write before the study was conducted.

One pointer is:

- What are the similarities and differences compared to other species with HY expression in the context of breeding season. Be careful that antigen requires antibodies. So it is antibodies that are relevant and discuss implications from that angle too.

- You can discuss Actb gene and how this can be used as a resource.

Please provide all karyotype images as supplementary information.

Other minor points are listed below:

Abstract:

Line 32: “We also obtained Actb…” – state why it is important. Seems disconnected. Perhaps say that sequence can be used as control for other molecular studies.

Line 34: change qPCR to RT-PCR

Introduction:

Line 49: Please remove “or polygenic”. See https://doi.org/10.1016/j.tig.2022.12.002/ for details.

Line 58-60: Argument for GSD is incorrect. Sry gene can be on autosome and it is sufficient to produce XX males. change it to the gene specific nature and indicate that the sex determining gene generally lies on the sex chromosomes. As per sex-chromosome evolution, first the sex-determining factor evolves and then the sex chromosome forms around it.

Line 63: “Ruiz-Garcia …. commented on” - change the framing of the sentence to “there is a lack of understanding [citation].”

Line 65 and 66: “they commented ……” – This is not true. It is an allele at a locus, generally on the sex chromosome, determine GSD. Process is well understood but not easy to study due to technological limitations. Please correct the framing of the sentence.

Line 82: “SMCY/SMCX genes diverged….” – This is specific to eutherian mammals. Best to be specific when divergence time is mentioned.

Methods:

Line 118: How were cardiacal lymphocytes collected?

There were several instances of 1000 xg. Please change “x” to mathematical symbol.

Primer pairs for HY-antigen in expression profiles were different to the isolation of and expression of H-Y antigen section. Please correct if this is a mistake. If this is not a mistake then justify why primer pairs were different.

Results: Line 210: “morpholically undifferentiated” – change undifferentiated to differentiated.

Line 223: “this observation …” – change to “This implies females are heterogametic sex.”

Line 227: “Despite these results,” is not required here.

Line 229: “Also,” is not required. Please remove that word.

Line 248: “Statistically significant difference…) – Please mention the test, p-value and fold-change in the sentence.

Line 249: “male/female tissues” – change to “male/female non-gonadal tissues” for clarity

Figure 5: what is the unit of measurement for the y-axis. Please state.

Reviewer #3: This manuscript deals with sex determination in the treefrog Hyla eximia.

The work is scientifically sound and concerns a topic of broad scientific interest, however the authors should deal with some issues before it can be accepted for publication.

In particular, it does not seem to me that sex chromosomes were identified in the study species. Differential antigen expression appears to support the hypothesis of a homomorphic sex chromosome system, but the sex chromosome themselves should still be identified (e.g. pair number) cytogenetically and or molecularly. This should be clearly specified in the manuscript.

The introduction needs more information on the study species (e.g. geographic range, phylogeny, previous cytogenetic data etc.) and on sex determination systems in general (ESD, TSD with simple and multiple sex chromosome systems, see below).

Some of the methods should also be better described (e.g. calculation of the centromeric index and chromosome staining and classification, see below).

Here below are some section-specific points that should be carefully addressed.

Introduction

Is this the first karyotype description of the species? Otherwise, the authors should probably add some information in the Introduction on what is currently known on the karyotype of the species and add the relevant citations

Lines 56-60: Also please specify that the cited case of ZZ male to female sex reversal concerns Dragon lizards. That can hardly be generalised for reptiles. In order to avoid confusion, it is probably worth to better describe genetic and environmental sex determination as well as known cases of sex reversal in fish, reptile and amphibians

Lines 56-60: Given the research topic it would be very important to mention that multiple sex chromosome systems (X1X2Y and Z1Z2W) are known in mammals (Saunders and Veyrunes 2021 Genes 8, 12, 1770) reptiles (with either oviparous and viviparous clades) (Petraccioli et al. 2019 Cytogenetic and Genome research, 157, 65-76) and amphibians (Miura et al. 2021 Cells. 10, 661).

Line 68: 95% of described species or 95% of the species with a known karyotype? Please, be more specific.

Line 82: I would invert this sentence in something like “they are probably homologous and likely diverged”

Methods

Line 98: change “from” with “of”

Line 101: The authors should probably add some citations on the breeding season of the species (e.g. Hernández-Salinas et al. 2018, PeerJ., 6, e5897).

Line 107: please describe RNA isolation methods or at least add relevant citations.

Line 131: Please add some information on karyotyping, including chromosome staining techniques, number of images collected per studied sample and if chromosomes were classified based on the centromeric index following Levan et al. (1964) Hereditas 1964, 52, 201–220. Then possibly add calculation of the centromeric index.

Line 134: mention of RNA isolation is repeated in different paragraphs. I suggest to mention and describe it only once in the methods.

Results

Lines 211-212. It does not seem to me that sex chromosomes were identified, for example using C-banding, molecular cytogenetics etc. The authors should specify in the manuscript that the occurrence of homomorphic sex chromosomes is a likely hypothesis based on what is known from other species, but sex chromosome were not cytogenetically identified.

Discussion

Line 285: sex determination is a characteristic of species, not antigens.

Fig 1 caption: this caption should be reworked. Please change “similarities” with something like “the same chromosome number”. Furthermore, 2n = 24 is simply the diploid number, it does not indicate a sex determination system.

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Reviewer #1: No

Reviewer #2: Yes: Hardip Patel, John Curtin School of Medical Research, Australian National University

Reviewer #3: No

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PONE-D-24-02112R1Understanding the genetic sex-determining mechanism in Hyla eximia treefrog inferred from H-Y antigenPLOS ONE

Dear Dr. Ramos L,

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.

==============================

ACADEMIC EDITOR: Dear authors, I have received the reminders of the reviews, please review each question point by point.. 

==============================

Please submit your revised manuscript by Jun 10 2024 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,

Mozaniel Santana de Oliveira, 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.

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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 #2: All comments have been addressed

Reviewer #3: (No Response)

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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 #2: Yes

Reviewer #3: Partly

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

Reviewer #2: Yes

Reviewer #3: (No Response)

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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 #2: Yes

Reviewer #3: 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 #2: No

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 #2: There are many cases where semicolon is used. These can generally be replaced with a full-stop. Copy editing of this manuscript is essential to ensure readability. I will not go through this one-by-one as it is too onerous. Authors and journals should be responsible for the correct use of the language.

Reviewer #3: In my opinion, the authors have done a good job in revising several parts of the manuscript which now are more accurate and sufficiently detailed.

However, a further revision of some limited but important points should be performed before the manuscript can be accepted for publication.

Line 203-204: The results obtained point at a probable genetic sex determination system with female heterogamety. However, a chromosome complement with either ZW in females or ZZ in male was not identified. In fact, on the basis of the method used, the author cannot exclude the occurrence of a multiple sex chromosome system (see e.g Gazoni et al 2018. Chromosoma 127(2):269-278).

Line 207: Change “karyotyping” with “karyotype”

Line 209: Change “karyotypic morphology” with “chromosome morphology”

Line 209: insert “pairs” before “1” and “5” and “4”. Remove “chromosome” in lines 211 and 212.

Line 228: change with something like “represent the heterogametic sex”.

Line 263: add something like “in them” after “explored”

Line 269-273: I think there is still some confusion here. In fact, the presence sex chromosome system can be either “revealed” (as suggested at line 270) or “hypothesized” (as stated at line 272). The results obtained with the methods employed by the author allow them to just hypothesize the presence of a genetic ZW sex chromosome system. Please, be more precise with similar statements in several sections of the manuscript in order to be more consistent with the results obtained and avoid confusion for the general reader.

Line 312-314: I think that it would be important to highlight that the hypothesized occurrence of sex chromosome system could be documented by a differential heterochromatinization between the heterogametic sex chromosomes (ZW) (Mezzasalma et al. 2007 Salamandra 55,:140-144).

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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 #2: Yes: Hardip R. Patel

Reviewer #3: No

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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.

Understanding the genetic sex-determining mechanism in Hyla eximia treefrog inferred from H-Y antigen

PONE-D-24-02112R2

Dear Dr. Ramos,

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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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,

Mozaniel Santana de Oliveira, 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 authors have submitted a revised version of the manuscript which feels improved and more accurate.

I have no further comments.

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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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