-->PONE-D-26-07884-->-->Cloacal microbiome variation in wild and captive Eastern Indigo Snakes (Drymarchon couperi) with and without Cryptosporidium serpentis infection-->-->PLOS One

Dear Dr. Brown,

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PLOS One

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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: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: Yes

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-->5. Review Comments to the Author

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Reviewer #1: Review comments to the Authors

I would like to congratulate the authors on their work and on the manuscript entitled “Cloacal microbiome variation in wild and captive Eastern Indigo Snakes (Drymarchon couperi) with and without Cryptosporidium serpentis infection”. This is an original study focusing on a relatively understudied aspect of reptile ecology, health, and conservation. The introduction provides a strong background and justifies the relevance of the research. Materials and Methods section adequately describes sampling of snakes, and explains metagenomic and statistical analysis methods in great detail. The discussion summerizes the results without major overstatements and creates a logical link between microbial composition and organismal health. However, there are some minor incosistencies, which I highlight below. I recommend that the authors to use the up-to-date names of bacterial phyla. While the study is technically sound, I suggest minor revision to address some small imprecisions before publication.

Reviewer #2: This manuscript describes a shotgun metagenomic study of the cloacal microbiome in the Eastern Indigo Snake (Drymarchon couperi), comparing wild and captive populations with a focus on Cryptosporidium serpentis infection. The study is timely, as reptile microbiome data remains sparse compared to other vertebrates, and the move from amplicon sequencing to a shotgun approach is a significant methodological step forward for this field.

While the study design is sound and the results are compelling, the manuscript would benefit from tightening up in several areas. The Introduction is a bit repetitive regarding the factors influencing reptile microbiomes (diet, geography, etc.) these sections should be condensed to keep the focus on the Eastern Indigo Snake and the study's specific objectives, which should be stated more explicitly at the end of the section.

In the Materials and Methods, there are some inconsistencies in the reported sample sizes for wild snakes across different paragraphs. This needs to be corrected for clarity. Additionally, please specify which reference genome was used for host read filtering, as this is critical for reproducibility.

The Results clearly highlight the higher microbial richness in wild individuals and identify taxa potentially linked to C. serpentis infection. The low detection rate of the parasite itself, despite confirmed infection, is a fair observation, and the discussion of intermittent shedding is a plausible explanation. However, the Discussion could be more concise, particularly in the sections covering bacterial and fungal associations.

A key limitation that should be more formally acknowledged is the lack of direct environmental sampling (soil, water, enclosure surfaces). Without this, the role of environmental exposure remains an inference. Furthermore, the high proportion of unclassified reads while common in non-model wildlife studies warrants a brief mention regarding the current limitations of taxonomic databases for reptiles.

The figures are generally informative, though the labeling and legends could be cleaner to improve readability. Overall, this is a well-executed study that provides valuable baseline data for reptile conservation and disease ecology.

Recommendation: Minor Revision.

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Reviewer #1: No

Reviewer #2: No

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-->PONE-D-26-07884R1-->-->Cloacal microbiome variation in wild and captive Eastern Indigo Snakes (Drymarchon couperi) with and without Cryptosporidium serpentis infection-->-->PLOS One

Dear Dr. Brown,

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 Jun 22 2026 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 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.

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We look forward to receiving your revised manuscript.

Kind regards,

Edvard Mizsei

Academic Editor

PLOS One

Journal Requirements:

1. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

2. 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 #1: (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 #1: Yes

**********

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

Reviewer #1: 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

**********

-->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

**********

-->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: Review comments to the Authors

I commend the authors for addressing my suggestions. However, there are still some minor issues that should be accounted for. I recommend a minor revision for now.

Comments:

I recommend that the p-value is reported with a lowercase italicized “p” throughout the text.

Lines 158-159: Since most individuals in the “wild” group were mostly rewilded rather than truly wild-hatched, the authors should provide a clearer justification for grouping them as “wild” as this may influence microbiome composition. Were all individuals collected in the private reserve previously reintroduced? Could they differ from truly wild individuals since hatching? I think a comparison between truly wild and headstarted individuals would be justified.

Line 232: Diversity is not the same thing as richness. The Shannon diversity index incorporates both species richness and evenness, but it is not a direct measure of richness alone. “richness” → “diversity”

Line 234: On Fig. 1A diversity is displayed, not richness Instead of “significantly richer,” I suggest “significantly more diverse.”

Lines 241-242: I think virus names should be italicized.

Lines 258-259: Fig.1, The phrasing of the title seems a little overcomplicated. I recommend revising the sentence to: “Diverse microbial taxa from multiple kingdoms identified in cloacal samples,” or something similar.

Lines 271-272: Be aware that these taxa are not all kingdoms, and are not at the same taxonomic level, so I recommend rewording the sentence

Line 282: In my opinion, it is better to write it as “p<0.001.”

Lines 330-331: I recommend recommend rephrasing the sentence, to avoid any overstatements. See Hernández et al., 2025.

Lines 338–339: “…that can influence overall organism health” — I think a few words about the link between microbial diversity and organismal health would strengthen the results.

Line 353: “phylum” instead of “phyla.”

Line 619: The references should be revised so that latin names are italicized.

Figure 1: Species names should be italicized.

Figure 2: There are dots in the legend, and it is not italicized. Also, the “p” should be lowercase.

References:

Hernández, M., Langa, J., Aizpurua, O., Navarro-Noya, Y. E., & Alberdi, A. (2025). Contrasting recovery of metagenome assembled genomes and derived bacterial communities and functional profiles from lizard fecal and cloacal samples. Animal Microbiome, 7(1), 15.

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Reviewer #1: No

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-->

Cloacal microbiome variation in wild and captive Eastern Indigo Snakes (Drymarchon couperi) with and without Cryptosporidium serpentis infection

PONE-D-26-07884R2

Dear Dr. Brown,

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,

Edvard Mizsei

Academic Editor

PLOS One

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