-->PONE-D-25-46577-->-->The venom gland transcriptome of Tityus paraguayensis reveals a diverse array of bioactive molecules from the Brazilian Cerrado-->-->PLOS ONE

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PLOS ONE

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MNL This work was conducted with support from the Federal University of Mato Grosso do

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FHS FAPESP     23/17182-6

ACS FAPESP     22/15264-2

MNL This work was conducted with support from the Federal University of Mato Grosso do

Sul—UFMS/MEC—Brazil. This study was also financed in part by the Coordenac.o de Aperfeicoamento

de Pessoal de Nivel Superior—Brasil (CAPES)—Finance Code 001.”

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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: Partly

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

Reviewer #1: No

Reviewer #2: N/A

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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: No

Reviewer #2: No

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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: No

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: Overall, the manuscript “The venom gland transcriptome of Tityus paraguayensis reveals a diverse array of bioactive molecules from the Brazilian Cerrado” presents important and significant results; however, the way these findings are currently presented requires improvement. I recommend reassessing the actual objective the authors wish to convey with this study, in order to allow for a more focused presentation of both the Results and the Discussion.

Point-by-point comments:

Data availability must be public. The authors appear to have inserted placeholder names (?) in the data availability statement. The underlying data should be deposited in a publicly accessible online repository.

The use of two specimens is mentioned, but it is not clear whether RNA extracts were pooled or analyzed independently for each individual. This information is important, as individual-level analyses are generally preferred for transcriptomics to capture biological variability.

Regarding RNA quality, the Methods section indicates Bioanalyzer assessment, and the supplementary Results text reports a clear 18S rRNA peak with absence of 28S, which prevented RIN calculation. Although this partially explains the missing RIN, the criteria used to deem the samples suitable for sequencing should be explicitly stated (and reporting such QC solely in Results/supplementary material is not ideal).

There is no indication of overall sequencing depth (e.g., total reads per library/sample), which is essential to evaluate dataset robustness.

The handling of redundant contigs and any filtering of very short transcripts are not described—both directly affect transcriptome quality. Likewise, the pipeline should specify explicit ORF selection criteria (e.g., minimum length thresholds, presence of conserved domains, and/or homology evidence).

There is no mention of scorpion euthanasia procedures or the final disposition of individuals after experimentation (e.g., placement in an institutional collection). Such information is typically required to document ethical compliance.

The section titled “RNA extraction, quality control, preprocessing, and de novo assembly” reads as Methods, not Results, and should be relocated or reframed accordingly.

Page 11, lines 216–217; Page 13, lines 256–259; Page 16, line 330. What was the basis for these comparisons (this must be described in the Methods)? What were the reference sequences? Although identification codes appear in the figures, the Results must precisely describe these elements in the text.

The alignment procedures should be detailed in the Methods, including the software used and how percentage identity/similarity among peptides was computed.

The correct term is molecular mass, not “molecular weight”; values should be reported in Daltons (Da) in scientific articles.

Each Results subsection presents a representative peptide for the respective class; what was the selection criterion for these peptides? Was a different criterion applied in each subsection?

Figures 5, 7, 9, and 11 do not convey meaningful structural information. They could be consolidated into a single figure or removed entirely.

The first two paragraphs of the Discussion are unnecessary. It is common in transcriptomic studies to conflate methodological QC with actual results; this work aims to establish functional relevance and does not need to dwell on such information.

The remainder of the Discussion is almost as descriptive as the Results and follows the same order of topics, but without a coherent thematic synthesis. Transcriptome data are important, but the manuscript’s objectives should be reassessed so that the Discussion provides substantive interpretation.

The authors themselves have a study on the venom of this scorpion yet chose not to cite it, which is unusual. Including it would strengthen the functional assessment of proteins (https://doi.org/10.1016/j.toxicon.2025.108332).

In the Conclusion, the phrase “523 potential toxins” may imply all are validated toxins. A more accurate formulation would be “523 toxin-like transcripts” or “523 putative toxin-related sequences,” clarifying that these are transcriptome-based predictions without functional validation.

Reviewer #2: The manuscript entitled “The venom gland transcriptome of Tityus paraguayensis reveals a diverse array of bioactive molecules from the Brazilian Cerrado” focuses on the transcriptomic analysis of the venom gland of Tityus paraguayensis. I have carefully reviewed the manuscript, and several points require revision and further attention, which are discussed below:

-The authors stated in the manuscript that "Scorpions are venomous arthropods in the class Arachnida and the order Scorpions, which includes 22 families, …", whereas according to credited databases like “https://wwwntnuno/ub/scorpion-files/” and “https://pmc.ncbi.nlm.nih.gov/articles/PMC11598449/”, there are 24 families of scorpions. Please correct it according to mentioned references and include the references in the text.

-The authors stated in the manuscript that " … most of which have been found in subtropical regions", whereas according to article “https://www.sciencedirect.com/science/article/abs/pii/S0041010124006810”, and some other references, scorpions are widespread in tropical and subtropical regions. Please correct it and include the reference.

-Reference is needed for this statement: “Because of the diverse compounds in scorpion venoms, they are considered promising sources for discovering molecules with potential pharmacological applications“. The authors may consider using references such as “https://www.sciencedirect.com/science/article/abs/pii/S0041010125003782”, “https://link.springer.com/article/10.1186/s12864-023-09851-y”, and “https://pmc.ncbi.nlm.nih.gov/articles/PMC6706747/” to support it.

-The authors homogenized the telsons of two scorpions. What was the rationale for doing so, considering that using a single scorpion could have provided unique, individual-specific results? A comparative approach (e.g., male vs. female, juvenile vs. adult) would have allowed for more precise analysis. By homogenizing samples from different individuals, important intra-species variations may have been overlooked.

-The raw transcriptome data from T. paraguayensis venom glands (BioProject PRJNA1268009, BioSample SAMN48739814, and SRA SRR33699524) that the authors claimed to have submitted to NCBI is currently not available in the database.

-In the methodology section, for a proper and accurate extraction, the venom gland should have been separated from the telson and used for RNA extraction. In this way, the unusually high percentage of peptides obtained would not have originated from glandular tissue.

-The Discussion section needs to be rewritten, especially the first paragraph, which should typically provide a concise summary of the study’s significance, the existing knowledge gap, and finally the research conducted and its main findings. However, such structure is not observed in the current version of the Discussion.

-The manuscript requires English editing by a native speaker to improve clarity and readability.

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Reviewer #1: No

Reviewer #2: No

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<p>The venom gland transcriptome of Tityus paraguayensis reveals a diverse array of bioactive molecules from the Brazilian Cerrado

PONE-D-25-46577R1

Dear Dr. Lucena,

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.

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Kind regards,

Karen de Morais-Zani

Academic Editor

PLOS One

Additional Editor Comments (optional):

Only one remaining comment from Reviewer 4, which can be addressed during the proofreading stage: "Only one remaining comment from Reviewer 4One minor point for refinement concerns Lines 670–671, which state that confirming the actual presence and amounts of toxins would require mass spectrometry. It would be more accurate to note that this would require a quantitative proteomic approach or targeted protein purification, with LC–MS/MS serving as an analytical tool to support such quantification, rather than mass spectrometry alone".

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

Reviewer #4: 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

Reviewer #4: Yes

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

Reviewer #3: N/A

Reviewer #4: 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 #3: (No Response)

Reviewer #4: 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 #3: Yes

Reviewer #4: Yes

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-->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: (No Response)

Reviewer #4: PONE-D-25-46577R1 — The authors have addressed the reviewer queries satisfactorily. The conclusions are supported by the data, the technical descriptions have been appropriately updated, and the study’s limitations are now clearly acknowledged.

One minor point for refinement concerns Lines 670–671, which state that confirming the actual presence and amounts of toxins would require mass spectrometry. It would be more accurate to note that this would require a quantitative proteomic approach or targeted protein purification, with LC–MS/MS serving as an analytical tool to support such quantification, rather than mass spectrometry alone.

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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: Yes: Mohamed A. Abdel-Rahman

Reviewer #4: No

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