Aquificae overcomes competition by archaeal thermophiles, and crowding by bacterial mesophiles, to dominate the boiling vent-water of a Trans-Himalayan sulfur-borax spring

Nibendu Mondal; Subhajit Dutta; Sumit Chatterjee; Jagannath Sarkar; Mahamadul Mondal; Chayan Roy; Ranadhir Chakraborty; Wriddhiman Ghosh

doi:10.1371/journal.pone.0310595

Peer Review History

Original SubmissionNovember 5, 2023
27 Dec 2023 Decision Letter - Durgesh Kumar Jaiswal, Editor PONE-D-23-36526Antibiosis helps Aquificae surmount competition by archaeal thermophiles, and crowding by bacterial mesophiles, to dominate the boiling vent-water of a Trans-Himalayan sulfur-borax springPLOS ONE Dear Dr. Ghosh, 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 Feb 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. 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The funders played no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Please include your amended statements within your cover letter; we will change the online submission form on your behalf. Additional Editor Comments: Dear Authors, After carefully evaluated the manuscript, we find that the study is very interesting however, before considering the manuscript for publication is required the major revision. So we have requested the authors to follow the reviewers comments and improvised the manuscript according it. Thanks [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: No Reviewer #3: No Reviewer #4: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A Reviewer #2: N/A Reviewer #3: I Don't Know Reviewer #4: N/A ****** 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 Reviewer #3: Yes Reviewer #4: 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: No Reviewer #2: No Reviewer #3: Yes Reviewer #4: 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: I wish to express my gratitude to the author for their diligent efforts in conducting the study titled "Antibiosis helps Aquificae surmount competition by archaeal thermophiles, and crowding by bacterial mesophiles, to dominate the boiling vent-water of a Trans-Himalayan sulfur-borax spring". It contributes to enhance our understanding on functional profile of microbiome from geothermal region. However, there is a significant need for improvement in the method section, particularly considering the nature of the sequencing platform. All claims in the study rely solely on results from gene calling, assembly, taxonomic assignment, and functional annotation. Substantial enhancements in the methodological approach would strengthen the study's validity and reliability. I have outlined certain aspects that require attention from the authors. I encountered difficulty in understanding the English used, particularly in the abstract and introduction. Sentences are excessively long, featuring unnecessary words and a lack of coherence between phrases separated by commas. Please aim for clarity and conciseness. The author utilized the eggNOG database for functional analysis, last updated in 2019. However, it's worth noting that the COG database (https://www.ncbi.nlm.nih.gov/research/cog) is the most recent, updated in March 2022. The COG database provides dedicated resources for both bacteria and archaea. I strongly recommend that the author considers using the COG database for more up-to-date and comprehensive functional profiling. The author might want to consider including Quality Check (QC) results of the FASTQ files in the supplementary section, both before and after read filtration. This additional information can offer insights into the data quality and the impact of the filtration process. Consistency should maintain in providing parameter information for the tools used in the analysis. For example parameter information for Prodigal v2.6.3 is missing. If default parameters were used, please mention this in the documentation. The study references the searching of reads against the rrnDB database, a crucial step for taxonomic delineation. However, there is limited information on read mapping and extraction. It would be beneficial if the author could provide a detailed workflow, including steps, parameters, and intermediate files used for the mapping and extraction of reads. Additionally, if any other tools were employed in this process, their inclusion as an workflow would be appreciated. Given that the Shotgun sequence is not the most suitable option for taxonomic profiling with publicly available 16S databases, I suggest that the authors utilize a dedicated pipeline for shotgun metagenome data, such as MEGAN Community Edition. The study employed the UBCG Pipeline v3.0 for core gene phylogeny, which has limitations in identifying less core gene due to its reliance on a universal core genome database.Consequently, it overlooked several actual core genes in the user dataset. I recommend utilizing the best-bidirectional hits (BBH) method for core gene identification, as it is sure to offer a more comprehensive core gene set from the given genomic dataset. MAGs with accession numbers JARJND000000000 and JARJNC000000000 exhibit a completeness of less than 50% in Table-1, indicating significant incompleteness and poor quality. It is advisable to exclude these MAGs from the analysis for more robust and reliable results. Reviewer #2: This manuscript presents data from a unique ecosystem that is understudied and worthy of better characterisation. For that reason, I am recommending a major revision before this manuscript can be published. I had a hard time reading this manuscript due both to the way it is written and the choice of data analysis and visualisation. The manuscript is excessively long, which compromises readability. Throughout the paper the tendency for long sentences, that usually focus on different ideas, is maintained. In many sentences the choice of words is unusual, to say the least. As an example of what I am saying the sentence in the Introduction: "Idiosyncratic to the above community structure axiomatic for hydrothermal ecosystems" - what is an axiomatic ecosystem, and what idiosyncrasy are you referring to? Please consider getting someone to help revising the English. As it is, it reads as if a machine has tried to come up with dictionary synonyms for words that are more commonly used, even in scientific writing. There is a lack of detail in the Material and Methods section regarding data analysis and visualisation. You detail how you get MAGs and OTUs and then no more information is provided. After you get the MAGs and the 16S reads how do you analyse them? For example, compare the legend in Fig 3 and Fig 6, the latter explains the analysis that was done. Also asking as this is not the most common way of presenting these data. It is also very uninformative, as none of your Figures help to visualise the conclusions you draw from your data. Figure 5 is particularly unhelpful, and if I understood correctly the only thing that is related to your work is the legend? Your data analysis and visualisation should show and support the main points you make in your conclusions. As an example, in your title you use the word Antibiosis, but nowhere in the paper do you show that is possible. The fact that you find a potential gene does not mean that it is being expressed. You have no data from RNA, therefore any activity must be considered as potential. Either you do that, or you rephrase the manuscript to reflect the data analysis that you are presenting. Please note that these comments are meant to be read as examples of issues with the whole manuscript, which should be thoroughly revised to improve readability, reduce length and provide data visualisation and analysis that is in line with the most common methods. Reviewer #3: The paper by Mondal et al. is devoted to the microbiome of the hot spring (namely, geyser), called Lotus Pond, located in the Puga Valley on the Changthang plateau. The authors applied the metagenomics analysis, including MAGs binning and their functional analysis, 16S rRNA amplicon sequencing and enumeration of microbial cells, filtered out from the hot spring water, with different fluorescent dyes. The main hypotheses of this work are: - that genomes of Aquificota possess many genes of antibiotic biosynthesis and even virulence functions, as well as antibiotic resistance genes, that helps them dominating in the hot spring, overlapping the other thermophiles, especially Archaea; - and bacterial mesophiles are not random components of this hot spring community and their genomes are replete with genes crucial for hydrothermal and other stress adaptation; However, the data, obtained by in silico analysis of MAGs, does not support these conclusions. Please, find all comments and observations in the attached document Reviewer #4: Interesting and very well written manuscript. I have only one reservation - you analyzed just the metagenome, so, the conclusions of the article and partially even the title of the article are not based on experimental data. The presence of a large number of "resistance" genes is not an evidence that these genes are actually expressed. For many bacterial taxa, it is well documented that these genes or gene clusters are not actually expressed. Without additional transcriptomic data, in my opinion, the title of the article is inappropriate ****** 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: Sushanta Deb Reviewer #2: No Reviewer #3: No Reviewer #4: Yes: Peter Pristas ******** [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. Attachments Attachment Submitted filename: The paper by Mondal et al.docx https://doi.org/10.1371/journal.pone.0310595.r001
Revision 1
11 Mar 2024 Author Response Response to the comments of Reviewer 1 I wish to express my gratitude to the author for their diligent efforts in conducting the study titled "Antibiosis helps Aquificae surmount competition by archaeal thermophiles, and crowding by bacterial mesophiles, to dominate the boiling vent-water of a Trans-Himalayan sulfur-borax spring". It contributes to enhance our understanding on functional profile of microbiome from geothermal region. However, there is a significant need for improvement in the method section, particularly considering the nature of the sequencing platform. All claims in the study rely solely on results from gene calling, assembly, taxonomic assignment, and functional annotation. Substantial enhancements in the methodological approach would strengthen the study's validity and reliability. RESPONSE: We thank the Reviewer for appreciating our detailed exploration of the microbiome architecture of the geobiologically unique Trans-Himalayan sulfur-borax spring system. We agree with the suggestions pertaining to the strengthening of the methods section, so have now dealt with these concerns primarily by bringing to the main text all those procedural details that were previously narrated in the Supplementary Methods. Thus, in the revised Article File, we have now elaborated the previous Methods section called “Metagenomics” under three new sections titled as - Metagenome extraction and sequencing - Assembly and annotation of the metagenomic sequence - Direct annotation of metagenomic reads corresponding to rRNA gene sequences besides significantly substantiating the Methods sections - Construction and taxonomic characterization of metagenome-assembled genomes (MAGs) - PCR-amplified 16S rRNA gene sequence analysis. I have outlined certain aspects that require attention from the authors. I encountered difficulty in understanding the English used, particularly in the abstract and introduction. Sentences are excessively long, featuring unnecessary words and a lack of coherence between phrases separated by commas. Please aim for clarity and conciseness. RESPONSE: We agree with the Reviewer’s concerns regarding the apparently difficult English used in certain sections of the manuscript. We have now tried our best to make the sentences simple and short, yet comprehending some of them may still require additional attention. The geochemical distinctiveness of the hot spring explored and the uniqueness of the microbiome thriving therein are both quite complex. Accordingly, the intricate issues pertaining to the geomicrobiological peculiarities of Trans-Himalayan sulfur-borax springs, compared with other well studied hydrothermal ecosystems, need to be put in their proper perspectives without over-simplification. The author utilized the eggNOG database for functional analysis, last updated in 2019. However, it's worth noting that the COG database (https://www.ncbi.nlm.nih.gov/research/cog) is the most recent, updated in March 2022. The COG database provides dedicated resources for both bacteria and archaea. I strongly recommend that the author considers using the COG database for more up-to-date and comprehensive functional profiling. RESPONSE: We agree, so have now removed all COG-related information from the eggNOG-derived annotation of CDSs shown in Table S2 (previously numbered as Table S1), and provided fresh data obtained by searching the Prodigal-derived putatively translated gene catalog against the COG Little Endian version of the Conserved Domain Database of NCBI located at https://ftp.ncbi.nih.gov/pub/mmdb/cdd/little_endian/Cog_LE.tar.gz, using COGclassifier v1.0.5 (please see lines 241-245, 498-517 of the Track Changes file). The author might want to consider including Quality Check (QC) results of the FASTQ files in the supplementary section, both before and after read filtration. This additional information can offer insights into the data quality and the impact of the filtration process. RESPONSE: We agree, so have now included Quality Check (QC) results for the raw read set as well as the one obtained after quality filtering (please see the new Table S1 of the Revised Manuscript). Consistency should maintain in providing parameter information for the tools used in the analysis. For example parameter information for Prodigal v2.6.3 is missing. If default parameters were used, please mention this in the documentation. RESPONSE: We agree and have now included the parameter information for all the bioinformatic software programs that have been used in this study, including Prodigal v2.6.3 (please see lines 197-321 of the Track Changes file). The study references the searching of reads against the rrnDB database, a crucial step for taxonomic delineation. However, there is limited information on read mapping and extraction. It would be beneficial if the author could provide a detailed workflow, including steps, parameters, and intermediate files used for the mapping and extraction of reads. Additionally, if any other tools were employed in this process, their inclusion as an workflow would be appreciated. RESPONSE: We agree and have now added the warranted details of read mapping, extraction, and classification, to the new Methods section titled “Direct annotation of metagenomic reads corresponding to rRNA gene sequences” (please see lines 254-261 of the Track Changes file). Given that the Shotgun sequence is not the most suitable option for taxonomic profiling with publicly available 16S databases, I suggest that the authors utilize a dedicated pipeline for shotgun metagenome data, such as MEGAN Community Edition. RESPONSE: We used the MEGAN6 Community Edition to taxonomically classify the metagenomic reads that were detected by searching the raw dataset against the SILVA_SSURef_138.1_NR99 database with the help of Minimap2-2.26 (Li, 2018). Curiously, this led to the detection of no read ascribed to Crenarchaeota / Thermoproteota, Thermotogae / Thermotogota, Cyanobacteria, Chloroflexi / Chloroflexota, Actinobacteria / Actinomycetota, Synergistetes / Synergistota, even though 396, 196, 131, 56, 21, and 2 reads were detected for these taxa respectively, in our existing RDP-based analyses. Betaproteobacteria and Alphaproteobacteria, for which 1345 and 595 reads were identified in the RDP-based annotation, also went undetected in the MEGAN-based analysis. In fact, no phylum other than Aquificota, Proteobacteria (only the Gamma class), Firmicutes, Deinococcota, Bacteroidota were detected in the MEGAN6-based classification. Given that the metagenome under consideration is that of a boiling vent-water community, in situ presence of phyla such as Thermoproteota (this included genera such as Aeropyrum, Pyrobaculum and Stetteria) and Thermotogota, as detected in the existing RDP-based classification, is obviously true; accordingly, a bioinformatic tool which fails to identify these native taxa cannot constitute a technique of choice in the present case. In the revised manuscript, therefore, we retain the contextually more acceptable RDP-based classification of raw metagenomic reads corresponding to 16S rRNA genes. The study employed the UBCG Pipeline v3.0 for core gene phylogeny, which has limitations in identifying less core gene due to its reliance on a universal core genome database. Consequently, it overlooked several actual core genes in the user dataset. I recommend utilizing the best-bidirectional hits (BBH) method for core gene identification, as it is sure to offer a more comprehensive core gene set from the given genomic dataset. RESPONSE: We agree that the UBCG Pipeline identifies relatively less number of core genes due to its reliance on the universal core gene database. However, since the MAG dataset involved in the current analysis includes entities diverged at the level of the phylum, we have no other choice but to use the universal core gene database (hence UBCG) to accommodate all the MAGs in one phylogenetic tree. Furthermore, when we used Roary - The pan genome pipeline v3.23.0, as well as GET_HOMOLOGUES v3.6.2, to identify core genes using the best-bidirectional hits (BBH) method, we were unable to identify any gene with a conservation level of >90% sequence similarity across the MAGs. MAGs with accession numbers JARJND000000000 and JARJNC000000000 exhibit a completeness of less than 50% in Table-1, indicating significant incompleteness and poor quality. It is advisable to exclude these MAGs from the analysis for more robust and reliable results. RESPONSE: It’s true that the completeness levels of the MAGs having accession numbers JARJND000000000 and JARJNC000000000 are 30% and 34% respectively. However, it is noteworthy that the two MAGs have contamination levels of only 0.3% and 1.1% respectively, so they cannot be regarded as being poor in quality. In the current analysis, 19, 14, and 30 MAGs were initially generated using Metabat2, MaxBin2, and CONCOCT, respectively. Comprehensive refinement and optimization of the 63 population genome bins taken together, using DASTool, finally qualified 15 MAGs as the best representatives of the metagenome, notwithstanding the low completeness level of a few shortlisted MAG. This result needs to be presented upfront in its entirety without any modification/subtraction of convenience. This is important for the sake of the comprehensiveness as well as the integrity of the data. Moreover, in the context of the microbiome, it is most important to note that the two MAGs JARJND000000000 and JARJNC000000000 belonged to the family Thermodesulfobacteriaceae and the genus Paracoccus respectively. Thermodesulfobacteria is the most predominant taxon of true thermophiles within the Lotus Pond ecosystem after Aquificae, while Paracoccus strains isolated during previous expeditions of Lotus Pond have been studied in-depth for their thermal endurance (Mondal et al. 2022: Microbiology Spectrum 10, e01606-22). All the above issues strongly support the essentiality of a robust documentation of the two MAGs in the present manuscript, even if their completeness is currently low (notably, generation of more metagenomic sequence data from this sample in the future can well increase the completeness of the two MAGs). Response to the comments of Reviewer 2 This manuscript presents data from a unique ecosystem that is understudied and worthy of better characterisation. For that reason, I am recommending a major revision before this manuscript can be published. I had a hard time reading this manuscript due both to the way it is written and the choice of data analysis and visualisation. RESPONSE: We agree, and believe that the Reviewer appreciated our detailed exploration, as well as the underlying phenomenology, of the microbiome architecture of the geobiologically unique Trans-Himalayan hot spring system. As suggested, we have now overhauled the manuscript by focusing on the main findings and making the overall text (especially the language) more lucid. The manuscript is excessively long, which compromises readability. Throughout the paper the tendency for long sentences, that usually focus on different ideas, is maintained. In many sentences the choice of words is unusual, to say the least. As an example of what I am saying the sentence in the Introduction: "Idiosyncratic to the above community structure axiomatic for hydrothermal ecosystems" - what is an axiomatic ecosystem, and what idiosyncrasy are you referring to? RESPONSE: We have now tried our best to abridge the text, remove the asides, and make the sentences simple and short. Yet following some of the sentences may still require additional attention. The geochemical distinctiveness of the hot spring explored and the uniqueness of the microbiome thriving therein are both quite complex. Accordingly, the issues pertaining to their peculiarities (idiosyncrasies), compared with other well studied hydrothermal systems and their microbiota, need to be put in their proper perspectives without over-simplification. As for the sentence in question, we have now assorted the information into two separate sentences, and added precise terminologies (such as “low-biodiversity community structure” instead of “above community structure”, and “extra-ordinarily diversified vent-water microflora” instead of “extra-ordinarily high habitability”) to bring more clarity to the message conveyed (please see lines 93-98 of the Track Changes file). In relation to the Reviewer’s queries “what is an axiomatic ecosystem", and “what idiosyncrasy had been referred to”, a careful reading of the previous sentence “Idiosyncratic to the above community structure axiomatic for hydrothermal ecosystems, a geochemically unusual category of Trans-Himalayan hot springs …... have been reported to feature extra-ordinarily high habitability” points out that - no ecosystem is being called axiomatic here but the typical community structure described in the previous paragraph is being termed as axiomatic for hydrothermal ecosystems in general (please note the preposition “for” after the word “axiomatic”); - and, the extra-ordinarily high habitability of Trans-Himalayan hot springs is being referred to as idiosyncratic to the aforesaid community structure that in turn is axiomatic for hydrothermal ecosystems (in this case the preposition “to” after the word “idiosyncratic” is to be noted). Please consider getting someone to help revising the English. As it is, it reads as if a machine has tried to come up with dictionary synonyms for words that are more commonly used, even in scientific writing. RESPONSE: We have now considerably shortened the text by deleting the issues of tangential importance (please see lines 65-80, 646-653, 686-692, 746-798, 838-876, 886-913, 1182-1237 of the Track Changes file). The new text focuses on the main findings and their implications. We have also made the sentences simpler by splitting them wherever possible. That said, some sentences may still found complicated and warrant additional attention as the issues dealt there are complex and cannot be oversimplified. There is a lack of detail in the Material and Methods section regarding data analysis and visualisation. You detail how you get MAGs and OTUs and then no more information is provided. After you get the MAGs and the 16S reads how do you analyse them? For example, compare the legend in Fig 3 and Fig 6, the latter explains the analysis that was done. RESPONSE: In the previous manuscript, all the details of the bioinformatic procedure were given in the Supplementary Methods. We have now brought all those critical information to the main text to add to the reader’s satisfaction as well as the study's reliability (please see lines 167-321 of the Track Changes file). Thus, in the revised Article File, we have now elaborated the previous Methods section called “Metagenomics” under three new sections titled as - Metagenome extraction and sequencing - Assembly and annotation of the metagenomic sequence - Direct annotation of metagenomic reads corresponding to rRNA gene sequences, besides significantly substantiating the Methods sections - Construction and taxonomic characterization of metagenome-assembled genomes (MAGs) - PCR-amplified 16S rRNA gene sequence analysis (for the upgraded methods pertaining to MAGs and OTUs please see lines 263-321 of the Track Changes file). Also asking as this is not the most common way of presenting these data. It is also very uninformative, as none of your Figures help to visualise the conclusions you draw from your data. Figure 5 is particularly unhelpful, and if I understood correctly the only thing that is related to your work is the legend? RESPONSE: In the revised manuscript, Figure 1 depicts the topography of the study site. Figure 2 shows representative picture of the fluorescence microscopic fields based on which microbial cell density was calculated in the vent-water sample of Lotus Pond. Figure 3 illustrate the overall microbiome composition by providing an overview of the taxonomic distribution of (A) the CDSs identified in the assembled metagenome of Lotus Pond, and (B) the metagenomic reads which corresponded to 16S rRNA genes. Figure 4 presents the phylogen Attachments Attachment Submitted filename: Response to Reviewers.doc https://doi.org/10.1371/journal.pone.0310595.r002
3 Sep 2024 Decision Letter - Marcos Pileggi, Editor Aquificae overcomes competition by archaeal thermophiles, and crowding by bacterial mesophiles, to dominate the boiling vent-water of a Trans-Himalayan sulfur-borax spring PONE-D-23-36526R1 Dear Dr. Ghosh, 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, Marcos Pileggi, 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 #1: All comments have been addressed ******** 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: (No Response) ****** 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 #1: Yes: Sushanta Deb ******** https://doi.org/10.1371/journal.pone.0310595.r003
Formally Accepted
16 Oct 2024 Acceptance Letter - Marcos Pileggi, Editor PONE-D-23-36526R1 PLOS ONE Dear Dr. Ghosh, 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. Marcos Pileggi Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0310595.r004

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