Gut bacterial communities in roadkill animals: A pioneering study of two species in the Amazon region in Ecuador

Manuel Alejandro Coba-Males; Magdalena Díaz; C. Alfonso Molina; Pablo Medrano-Vizcaíno; David Brito-Zapata; Sarah Martin-Solano; Sofía Ocaña-Mayorga; Gabriel Alberto Carrillo-Bilbao; Wilmer Narváez; Jazzmín Arrivillaga-Henríquez; Manuela González-Suárez; Sandra Enríquez; Ana Poveda

doi:10.1371/journal.pone.0313263

Peer Review History

Original SubmissionMay 23, 2024
8 Sep 2024 Decision Letter - Sayed Haidar Abbas Raza, Editor PONE-D-24-20575Gut Bacterial Communities in Roadkill Animals: A Pioneering Study of Two Species in the Amazon region in EcuadorPLOS ONE Dear Dr. Poveda, 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 Oct 23 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, Sayed Haidar Abbas Raza Academic Editor PLOS ONE Journal requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Thank you for stating the following in your Competing Interests section: “On behalf of all authors, I disclose any competing interests.” Please complete your Competing Interests on the online submission form to state any Competing Interests. If you have no competing interests, please state "The authors have declared that no competing interests exist.", as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now This information should be included in your cover letter; we will change the online submission form on your behalf. 3. When completing the data availability statement of the submission form, you indicated that you will make your data available on acceptance. We strongly recommend all authors decide on a data sharing plan before acceptance, as the process can be lengthy and hold up publication timelines. Please note that, though access restrictions are acceptable now, your entire data will need to be made freely accessible if your manuscript is accepted for publication. This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If you are unable to adhere to our open data policy, please kindly revise your statement to explain your reasoning and we will seek the editor's input on an exemption. Please be assured that, once you have provided your new statement, the assessment of your exemption will not hold up the peer review process. 4. We note that Figure 1 in your submission contain [map/satellite] images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth). For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright. We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission: a. You may seek permission from the original copyright holder of Figure 1 to publish the content specifically under the CC BY 4.0 license. We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text: “I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.” Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission. In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].” b. If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only. The following resources for replacing copyrighted map figures may be helpful: USGS National Map Viewer (public domain): http://viewer.nationalmap.gov/viewer/ The Gateway to Astronaut Photography of Earth (public domain): http://eol.jsc.nasa.gov/sseop/clickmap/ Maps at the CIA (public domain): https://www.cia.gov/library/publications/the-world-factbook/index.html and https://www.cia.gov/library/publications/cia-maps-publications/index.html NASA Earth Observatory (public domain): http://earthobservatory.nasa.gov/ Landsat: http://landsat.visibleearth.nasa.gov/ USGS EROS (Earth Resources Observatory and Science (EROS) Center) (public domain): http://eros.usgs.gov/# Natural Earth (public domain): http://www.naturalearthdata.com/ [Note: HTML markup is below. Please do not edit.] 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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No Reviewer #2: Yes ****** 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 ****** 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 ****** 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: The authors of this paper have proposed an innovative method the sample the microbiome of the wildlife which are killed in the accidents, this contributes to the ethical and logistical considerations of the sample collection from live animals. The authors highlight the gut microbiome of the two species which was not studied earlier. The researchers have an edge over other works die to the research being conducted in the region with a rich biodiversity (Ecuador) which contributes to the knowledge of local wildlife. Problems Although the method is good, but the sample size is too small to eliminate any biases this limits the works ability to generalize the findings. The microbiome also needs to be compared with the live animal's sample. The only roadkill animal research makes data skewed. The hypothesis needs to be clear, not just description of the microbiome. The methods and result need to be elaborated more. Recommendation Increase sample size of the species. Include the comparative study of the related species. Clearly mention the research qu3estin and hypothesis. Describe the barcoding methods more. Mention the limitations of the study. Reviewer #2: The manuscript "Gut Bacterial Communities in Roadkill Animals: A Pioneering Study of Two Species in the Amazon region in Ecuador" reports the gut microbes of Amphisbaena bassleri and Crotophaga ani. These two are wild animal species. Amphisbaena bassleri is a lizard whereas Crotophaga ani is a bird. There are no previous reports on the gut microbiome of these species as it is very difficult to collect samples from them. The authors have used the roadkill carcass of these animals to collect the samples. This is a very good strategy as the samples are collected without harming the wildlife. The authors performed NGS on the hypervariable V3-V4 region from the 16S rRNA. NGS of the hypervariable V3-V4 region from the 16S rRNA gene is a common method for studying bacterial diversity in microbial communities. The authors identified microbial phyla in both samples including Firmicutes, Bacteroidetes, and Proteobacteria for A. bassleri, and Firmicutes and Actinobacteria for C. ani. They also discuss the identified gut microbiota from Amphisbaena bassleri in light of the known literature from other reptiles. Also got microbiota from Crotophaga ani was discussed in reference to findings from other birds. Overall the authors clearly discuss their findings along with limitations of the study. ****** 6. 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: Yes: Sabyasachi Mohanty Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] 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. https://doi.org/10.1371/journal.pone.0313263.r001
Revision 1
17 Oct 2024 Author Response PONE-D-24-20575 Gut Bacterial Communities in Roadkill Animals: A Pioneering Study of Two Species in the Amazon region in Ecuador Responses to Editor and Reviewers comments Editor: 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf Response: We have checked all of the style requirements. If we have missed anything, please let us know. 2. Thank you for stating the following in your Competing Interests section: “On behalf of all authors, I disclose any competing interests.” Please complete your Competing Interests on the online submission form to state any Competing Interests. If you have no competing interests, please state "The authors have declared that no competing interests exist.", as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now This information should be included in your cover letter; we will change the online submission form on your behalf. Response: We agree with that. 3. When completing the data availability statement of the submission form, you indicated that you will make your data available on acceptance. We strongly recommend all authors decide on a data sharing plan before acceptance, as the process can be lengthy and hold up publication timelines. Please note that, though access restrictions are acceptable now, your entire data will need to be made freely accessible if your manuscript is accepted for publication. This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If you are unable to adhere to our open data policy, please kindly revise your statement to explain your reasoning and we will seek the editor's input on an exemption. Please be assured that, once you have provided your new statement, the assessment of your exemption will not hold up the peer review process. Response: We agree. 4. We note that Figure 1 in your submission contain [map/satellite] images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth). For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright. We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission. We decided to remove Figure 1. ANSWER TO THE REVIEWERS: Reviewer #1: The authors of this paper have proposed an innovative method the sample the microbiome of the wildlife which are killed in the accidents, this contributes to the ethical and logistical considerations of the sample collection from live animals. The authors highlight the gut microbiome of the two species which was not studied earlier. The researchers have an edge over other works die to the research being conducted in the region with a rich biodiversity (Ecuador) which contributes to the knowledge of local wildlife. Problems Although the method is good, but the sample size is too small to eliminate any biases this limits the works ability to generalize the findings. The microbiome also needs to be compared with the live animal's sample. The only roadkill animal research makes data skewed. The hypothesis needs to be clear, not just description of the microbiome. The methods and result need to be elaborated more. Recommendation Increase sample size of the species. Include the comparative study of the related species. Clearly mention the research qu3estin and hypothesis. Describe the barcoding methods more. Mention the limitations of the study Answer to Reviewer #1 We thank Reviewer #1 for the comments provided, which are very helpful to improve our work. We will try to answer and argue the questions precisely. Firstly, we think it is important to explain how the sampling was achieved (previously described [1]. In this study we worked with samples obtained from a previous dedicated and time-consuming field survey that was carried out in a specific period (September 2020-March 2021) involving approximately 5-6 surveys per week, each survey requiring approximately 8 hours per day, by a team of two people (approximately 2000 hours invested). All the roadkill animals in the survey area, a specific geographical location in Ecuador, were collected, resulting in a collection of 590 specimens. Of these, we selected samples for this study that met essential requirements: 1) those belonging to animals from the same species, 2) those in which the roadkill animal (carcass) was relatively intact after the accident as required to allow dissection and obtention of uncontaminated intestine samples. From the 590 specimens collected, only nine specimens met these criteria and could be included in the study. This limitation reflects the difficulty of the fieldwork and collecting non-invasive samples from wildlife (e.g. requiring no manipulation or disturbance to live animals). These samples are described in table 1 of the manuscript. Table 1. Description of the samples from two vertebrate species analyses for this study. . Sample Species Estimated time since death Landscape Latitude Longitude SW001 Amphisbaena bassleri 0 hours Altered area -0.84761 -77.78706 SW002 Amphisbaena bassleri 0 hours Altered area -0.85528 -77.79068 SW003 Amphisbaena bassleri 2 hours Altered area -0.77437 -77.79246 SW004 Amphisbaena bassleri 6 hours Unaltered area -0.82108 -77.77457 SW005 Crotophaga ani 1 hours Unaltered area -1.10526 -77.79806 SW006 Crotophaga ani 1 hours Altered area -1.03703 -77.77536 SW007 Crotophaga ani 2 hours Altered area -1.06761 -77.63735 SW008 Crotophaga ani 6 hours Altered area -0.39962 -77.82664 SW009 Crotophaga ani 48 hours Unaltered area -1.04709 -77.78982 The reviewer recommends increasing the sample size. We agree the sample size is small and acknowledge this in the manuscript (lines 238, 267, and 322, applying tracking changes). However, we would argue that novel techniques or findings can be first reported or published based on small samples (eg first report of ingested microplastic on a dolphin based on 3 samples[2], 8 samples used in a study of stable carbon isotope diagnostics published in Plos One[3]). As explained above, we do not have additional samples we can add from our existing specimen collection. To add more samples, we could use two approaches, both of which we argue are not feasible. First, we could collect new roadkill specimens. Beyond the considerable effort this would represent, new samples, if secured, would represent a different time and conditions. It is already known that there are strong microbiome variations between individuals in the same species, even in different intestinal regions of the same individual, even more so if individuals are collected in different geographical areas, and at different times, since factors such as climate, diet or disturbed habitats can have a high impact on microbiomes [4,5]. Moreover, a new field survey does not guarantee we will secure samples (be able to find roadkill animals) for these two species in the necessary conditions. A second option to secure samples, would be to conduct field survey to locate live animals from these species that we would need to handle to collect fecal material, a highly invasive manipulation. In addition to presenting the same spatio-temporal limitations mentioned above, it is precisely a key proposal of our methods that they do not require handling or killing live wild animals that requires permits and brings substantial ethical and welfare concerns. It is worth mentioning finding live species would be particularly challenging for A. basleri, a cryptic species that is difficult to observe in nature. Cryptic species are those that are hard to detect due to their elusive behavior, camouflage, or the habitats they inhabit. They may be naturally secretive or live in environments that are challenging for humans to access, making them less visible and difficult to study [6]. This is reflected in the low number of publications of A. basleri, even less, about their microbiome. Therefore, any of the options that would allow us to increase the number of samples has its limitations and will generate variations in the results. However, we think studies based on small samples can contribute to our scientific understanding, and point to examples above. We are not claiming to have fully describe the microbiomes of these two species, but instead present a new approach to improve our understanding of wildlife without ethical or welfare concerns. Our study pioneers the study of wildlife microbiome using opportunistic sampling from roadkill in elusive and poor studied species. To our knowledge, this is also the first report of microbiota for A. bassleri or C. ani. And there are in fact, no report for related vertebrate species. As mentioned above and in the manuscript, first reports based on small samples sizes and without comparisons to live organisms are published and contribute to our understanding of nature (lines 327 – 329, applying tracking changes). This, together with the difficulty of collecting specimens in their natural habitat, make our results valuable to provide a baseline for these two species (lines 61-64, applying tracking changes). In addition to being a first report of microbiota in these two species, due to the characteristics of the samples we were also able to provide preliminary results to the question of whether the microbial community changed during the decomposition process of the animal (lines 67-70, applying tracking changes). The reviewer states that the “sample size is too small to eliminate any biases this limits the works ability to generalize the findings”. However, we do not claim (and we have rewritten parts of the text to ensure that is clear) to provide a comprehensive characterization of the microbiome in these two vertebrate species. Instead, our goal is to propose a new, non invasive method, showing it can offer insight into the microbiota of wild and difficult to sample species, and that initial decomposition (in more intact specimens as those selected in our study) does not result in a distinct microbiome. The limitations regarding the sample size in this study have been addressed in various sections of the manuscript to ensure that all interpretations are made with this consideration in mind. Furthermore, we indicate that further research using similar approaches is needed to provide more information about the microbial communities in understudied wildlife species. In summary, we agree with the comments of the Reviewer #1 regarding the limitations posed by a small sample size. However, we do not agree that should prevent the publication of this study because 1) there is no previous report from these two species (or related congeners) and the initial baseline can be valuable for further research; 2) although limited in number, samples were of suitable criteria and allowed us to describe microbiome; 3) highly biodiverse areas are at risk and mostly poorly studied, reducing this gap would be best served by promoting non invasive approaches, even if they come with some limitations, and also ensuring expectations from submitted work do not preclude work from the global south where resources and funding is more limited. We have improved the writing of the article to best clarify these points and have also expanded the description of the barcoding methods. 1. Coba-Males MA, Medrano-Vizcaíno P, Enríquez S, Brito-Zapat D, Martin-Solan S, Ocaña-Mayorga S, et al. From roads to biobanks: Roadkill animals as a valuable source of genetic data. PLoS One. 2023;18. doi:10.1371/journal.pone.0290836 2. Zhu J, Yu X, Zhang Q, Li Y, Tan S, Li D, et al. Cetaceans and microplastics: First report of microplastic ingestion by a coastal delphinid, Sousa chinensis. Science of the Total Environment. 2019;659: 649–654. doi:10.1016/j.scitotenv.2018.12.389 3. Fry B, Carter JF. Stable carbon isotope diagnostics of mammalian metabolism, a high-resolution isotomics approach using amino acid carboxyl groups. PLoS One. 2019;14. doi:10.1371/journal.pone.0224297 4. Senghor B, Sokhna C, Ruimy R, Lagier JC. Gut microbiota diversity according to dietary habits and geographical provenance. Human Microbiome Journal. Elsevier Ltd; 2018. pp. 1–9. doi:10.1016/j.humic.2018.01.001 5. Shanahan F, Ghosh TS, O’Toole PW. Human microbiome variance is underestimated. Current Opinion in Microbiology. Elsevier Ltd; 2023. doi:10.1016/j.mib.2023.102288 6. Franks DW, Noble J. Warning signals and predator-prey coevolution. Proceedings of the Royal Society B: Biological Sciences. 2004;271: 1859–1865. doi:10.1098/rspb.2004.2795 Reviewer #2: The manuscript "Gut Bacterial Communities in Roadkill Animals: A Pioneering Study of Two Species in the Amazon region in Ecuador" reports the gut microbes of Amphisbaena bassleri and Crotophaga ani. These two are wild animal species. Amphisbaena bassleri is a lizard whereas Crotophaga ani is a bird. There are no previous reports on the gut microbiome of these species as it is very difficult to collect samples from them. The authors have used the roadkill carcass of these animals to collect the samples. This is a very good strategy as the samples are collected without harming the wildlife. The authors performed NGS on the hypervariable V3-V4 region from the 16S rRNA. NGS of the hypervariable V3-V4 region from the 16S rRNA gene is a common method for studying bacterial diversity in microbial communities. The authors identified microbial phyla in both samples including Firmicutes, Bacteroidetes, and Proteobacteria for A. bassleri, and Firmicutes and Actinobacteria for C. ani. They also discuss the identified gut microbiota from Amphisbaena bassleri in light of the known literature from other reptiles. Also got microbiota from Crotophaga ani was discussed in reference to findings from other birds. Overall the authors clearly discuss their findings along with limitations of the study. Answer to Reviewer #2 We would also like to sincerely thank Reviewer #2 for their time and comments on our work https://doi.org/10.1371/journal.pone.0313263.r002
22 Oct 2024 Decision Letter - Sayed Haidar Abbas Raza, Editor Gut Bacterial Communities in Roadkill Animals: A Pioneering Study of Two Species in the Amazon region in Ecuador PONE-D-24-20575R1 Dear Dr. Poveda, 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, Sayed Haidar Abbas Raza Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0313263.r003
Formally Accepted
16 Dec 2024 Acceptance Letter - Sayed Haidar Abbas Raza, Editor PONE-D-24-20575R1 PLOS ONE Dear Dr. Poveda, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Sayed Haidar Abbas Raza Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0313263.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .