Dear Dr. Carazzone,

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: -->

Both reviewers saw merit in the manuscript, but reviewer one asks for additional methodological information (see below).

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Please submit your revised manuscript by Nov 03 2025 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.. 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.

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PLOS ONE

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[The research was supported by the announcement No. 757–2016 Doctorados Nacionales and project contract No. 44842–058-2018 from Ministerio Administrativo de Ciencia, Tecnología e Innovación (MINCIENCIAS). The financial support from the Faculty of Science at Universidad de los Andes partitioned in a forgivable loan assigned to one doctoral student (M.G.), the seed projects INV-2018-33-1259, INV-2019-67-1747 and FAPA project of C.C. The authors would like to thank the Vice Presidency of Research & Creation’s Publication Fund and the Science Faculty at Universidad de los Andes for its financial support too.]

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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?

Reviewer #1: Partly

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??>

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: The authors analyzed the hydrophilic toxins in the skin MeOH extract from the frog Colostethus imbricolus (11 specimens) collected in Colombia. Even though the frogs in this genus were previously reported to have paralytic toxins, they did not detected tetrodotoxin and its analogues using LCMSMS. I think that this is important finding. However, I could not find the detection limit and standard curves of TTX and its analogues for the analytical method they used in this manuscript. These descriptions are needed to be clearly shown.

Minor points

1. “Graphic abstract: Absence of TTX or any tetrodotoxin analogue”Should be revised to “Graphic abstract: Absence of TTX or any TTX analogues”

2. Table 2, why the molecular weights of 254.0000, 272.0000 and others are not the exactly accurate molecular weights of these analogues?

Reviewer #2: The manuscript titled “Lack of Tetrodotoxin analogues and individual metabolic profiling of the cryptic frog Colostethus imbricolus” (PONE-D-25-27319) includes a sophisticated series of analyses that allow the authors to investigate the presence of tetrodotoxin (TTX) and other alkaloid metabolites in a tropical dendrobatid frog species that has been proposed to possess TTX. The authors analyses indicate that TTX is not present in skin sample from the species but that a variety of other alkaloids are present. The authors use an interesting approach to catalog and begin to characterize these other natural products which will allow future investigation of this species as well as the natural products present in the species and genus.

The manuscript is well organized and complete. The experimental design of the project is solid, and I think the approach of using targeted and untargeted LC-MS/MS to investigate TTX and other alkaloids is an elegant solution to a tricky problem. The presence/absence of TTX in the genus Colosthethus is a long-term question that is of interest to ecologists, natural product chemists, and conservation biologists. My sense is that reporting negative results (e.g., the absence of TTX) is important and warrants publication. Because the manuscript also includes a more complete and interesting analysis of the metabolome of the species, I think it warrants publication in PLOS ONE.

The methods and results are clear, and the conclusions and discussion of the results are warranted. My training does not allow me to assess the details of their MS/MS analysis (Table 2) or the data analysis of the metabolomic profiling. However, it appears to me that the authors have provided adequate details for their approach to be replicated and/or judged by someone with more direct experience. Other elements of their analysis seem sound, and I am satisfied that their “null” results and absence of TTX in their samples is accurate and not an experimental artifact.

The discussion and conclusions in the manuscript seem warranted and appropriate for the data set. As with the MS/MS analyses by training does not allow me to assess the more granular level discussion of their data analyses but the broad conclusions and associated with the metabolic analyses appear to be accurate and supported by the data. This approach is a useful tool for building a more complete understanding of the chemical ecology of amphibians and was excited to see it included here.

A minor concern of the paper is the introduction and discussion of the origin of TTX. I am not sure that results presented in the manuscript are relevant to the ultimate origin of TTX as discussed in lines 57 to 62. There is still some disagreement about this. If the authors want to include this discussion, I encourage them to also include recent work from the Yotsu-Yamashita lab that suggests the possibility that TTX-bearing salamanders may synthesize their own TTX and at least acknowledge that for some amphibians the issue is still unresolved.

Overall, the manuscript warrants publication with some minor revisions and clean up. I have identified some specific concerns below:

Specific Concerns:

Lines 58-63. See above, this section may not belong in this paper at all but should include recent paper by Kudo from the Yotsu-Yamashita lab that maps a potential synthesis pathway of TTX from salamander skin intermediates that may not require bacteria.

Lines 70-73. I understand this sentence (I think), but the structure was confusing and could be clarified.

Line 79. This substance or these substances not as written.

Lines 91-93. Why are the authors only talking about 11-oxoTTX here? There are other analogs (e.g., 6-epiTTX) that also have terrestrial/marine distribution biases. Why discuss one but not the other?

Materials and Methods

I thought this section was well written and complete. However, one small request would be to include the molar concentration of their TTX standard in addition to the PPM concentration. It makes it easier for non-chemists to get a sense of detection limits with these approaches.

Another question that may represent ignorance on my part is the use of targeted and untargeted with reference to MS/MS analysis. As somebody slightly outside the field it took me a bit to realize what the terms were referencing (difference is MS/MS mode) it might help a broader audience to understand the results and design of the project to clarify why the two different approaches in the introduction.

Discussion

Lines 416-419. This sentence was confusing to me “in spite” of what? I was not sure what the first part of sentence was.

One thing that seemed missing from the discussion was explicit confirmation that none of the TTX analogs, potential precursors, and/or TTX metabolites (e.g., anhyrdro-TTX) are present in the metabolomes. Kudo et al. “Structures of N-Hydroxy-Type Tetrodotoxin Analogues and Bicyclic Guanidinium Compounds Found in Toxic Newts” document a range of compounds using MS/MS detection. Are these compounds included on the comparative database or annotations? Can the author’s results be used to confirm that other TTX related compounds are not present in their samples? This may not be possible, but it would be of interest if it could be confirmed.

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Reviewer #1: No

Reviewer #2: No

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Dear Dr. Carazzone,

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: -->

One of the referees did not respond to my invitation to review this version of the manuscript so I invited a third specialist to evaluate your work. While reviewer 2 was very positive towards your study, reviewer 3 raised some important concerns regarding the validation of the methods that should be addressed in the next version. Please see the comments bellow.

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

Please submit your revised manuscript by Jan 09 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.. 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 . 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,

Renato Filogonio

Academic Editor

PLOS ONE

Journal Requirements:

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.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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??>

Reviewer #2: Yes

Reviewer #3: Yes

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

Reviewer #2: Yes

Reviewer #3: 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 #2: Yes

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #2: (No Response)

Reviewer #3: This study investigates tetrodotoxin and other alkaloids in order to identify the paralysis-producing substances present in Colostethus imbricolus. Although the toxin itself was not identified, the detection of TTX and amphibian alkaloids, as well as the chemical profiling of Colostethus imbricolus and the metabolomic comparison between males and females, provide valuable information. However, the following concerns need to be addressed.

First, the statement that “TTX was not detected” simply means that its concentration may have been below the detection limit. The detection limit of TTX under the analytical conditions used in this study must be clearly stated. In addition, it is necessary to spike the frog extract with TTX and confirm that it can be detected without ion suppression. This is especially important because the SB-CN column is being applied for the first time in this analysis.

Abstract: “A notable additional outcome of this study is the first successful separation of TTX on an SB-CN column using a normal-phase gradient, enabling a novel method for TTX-targeted separation.”

To claim novelty and usefulness of the SC-CN column for TTX-targeted analysis, validation of the method—such as detection limits, quantification capability, and reproducibility—is essential. Any statements regarding the novelty or utility of the analytical conditions should be made with caution unless supported by proper validation data.

Table 1: The functional groups (R1–R4) should be clearly defined.

Table 2: 1-Hydroxy-5,11-dideoxyTTX was structurally revised in a 2020 Journal of Natural Products paper on TTX in newts, and the previously reported structure is no longer considered valid. The corrected compound name should be used.

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Reviewer #2: No

Reviewer #3: No

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Lack of tetrodotoxin analogues and individual metabolomic profiling of the cryptic frog Colostethus imbricolus

PONE-D-25-27319R2

Dear Dr. Carazzone,

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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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,

Renato Filogonio

Academic Editor

PLOS One

Additional Editor Comments (optional):

Congratulations on this accepted manuscript. For future reference, I recommend taking into account referee #3 suggestion of validating the TTX analytical method (see below), which was a similar concern as referee #1. With no further comments, I hope that this acceptance will make your celebrations all the better, and I wish the authors happy holidays!

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #3: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #3: Yes

**********

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

Reviewer #3: N/A

**********

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

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5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #3: Yes

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Reviewer #3: The reviewer considers validation of the tetrodotoxin analytical method, including determination of the detection limit, to be critically important. However, the authors have clearly stated that such verification was not performed in this study. It has also been confirmed that the description regarding the novelty of the analytical method has been revised accordingly.

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Reviewer #3: No

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