-->PONE-D-25-18938-->-->Anaerobic bacteria Cetobacterium sp. nov C33 plays a crucial role in the intestinal microbial balance and regulation of gene expression to immune and metabolic responses in Nile Tilapia-->-->PLOS ONE

Dear Dr. Villamil Díaz,

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Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Yes

Reviewer #4: Yes

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Reviewer #1: No

Reviewer #2: N/A

Reviewer #3: Yes

Reviewer #4: Yes

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Reviewer #4: Yes

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Reviewer #1: Major comments:

Even if the current study offers an effective probiotic to improve fish development, immunity, and reproduction, it has numerous limitations that should be taken into account, including the following implications

1- The probiotic effects of Cetobacterium sp. nov C33 were evaluated over a short supplementation period, which limits understanding of its long-term safety, stability, and sustained efficacy in tilapia.

2- The study was conducted exclusively on fingerlings, so the applicability of findings to juveniles or adults remains uncertain.

3- The trial was conducted under controlled laboratory or hatchery settings, which may not fully capture the complexity and environmental variability of commercial aquaculture systems.

4- Although immune-related gene expression was assessed, the study did not include a disease challenge to directly measure protection against specific pathogens or real-world disease resistance.

5- While 16S rRNA sequencing provided taxonomic shifts in microbiota composition, it did not include metagenomic or metabolomic analyses to clarify functional changes in microbial activity or interactions.

6- Findings are specific to Nile tilapia, and their generalizability to other aquaculture species is unknown without further comparative studies.

So, it is highly recommended to have a paragraph to discuss the major limitations of the present study before the section of the Conclusion.

Minor comments:

1- English proofreading is highly recommended.

2- No statistical analysis, so please analyse that data.

3- No line numbers.

4- Please prepare the manuscript according to Plos One's instructions. For example the title page does not follow the guidelines.

5- Check the grammer the 2nd paragraph in the introduction “The intestinal bacterial composition in tilapia is has been previously described”.

6- Please add brackets for SVCV to indicate that this is abbrreviatoon for spring viraemia of carp virus (3rd paragraph in the introduction)

7- Check the grammer the 2nd paragraph in the introduction “y Though the search or new potential probiotics” I believe you mean “through the search for new potential probiotics”, so please correct it.

8- The references you used to plan your experiments are missing from the entire methods section; please update and add them across the entire manuscript.

9- Keep the conclusion to a minimum to make it more representative and focused

Reviewer #2: Peer Reviewer s Comments

The Paper entitled Anaerobic bacteria Cetobacterium sp. nov C33 plays a crucial role in the intestinal microbial balance and regulation of gene expression to immune and metabolic responses in Nile Tilapia which has been submitted to publish in PLOS One presents a good investigative efforts but the hypothesis derived from the obtained data is scientifically and logically wrong very likely because there is a gap in present understanding on knowledge.

Low cell population e.g.108cells/g of Anaerobic bacteria Cetobacterium sp. nov C33 present in feed are practically too less to enable the fed innoculum to appear as dominating species in Nile Tilapia particularly in the case when it requires anaerobic condition to grow at 28C whereas the temperature of fish body is usually lower than 28C with limited availability of oxygen. However these cells by virtue of their high hydrophobicity can make clusters having potential to adhere with cells membrane of cells lining the gut effecting Oxygen, cholesterol and different metallic ions transport particularly Copper and Iron across the cell membranes effecting the host cells functions of target cells and also interconnected cells translating the impact across the organs connected through body systems modulating genes network operations by virtue of copper and iron balance in associated and dissociated forms effected differently by oxygen availability at cellular level, enabling cells to attain new phenotypes with altered homeostasis supported environment leading to the shift in microbial populations and shift in body cell functions over the period attaining altered normal. In response of stress conditions in gut the anaerobic bacteria Cetobacterium sp. nov C33 produce pigments and other secondary metabolites e.g. chelating agents enzymes etc which alters the iron availability and its accessibility in the cell environment which is is evident by up regulation and down regulation of different genes without activating an inflammatory pathway inspite of involving immune system of the host and without uncontrolled fluctuation in energy demand in body cells subsets.

Using yeast as a model, it has already been established that CTR1,(CTR1 gene encodes high affinity copper transporter) SOD1, superoxide dismutase 1 gene, GSH encoding genes and cholesterol cellular biosynthesis genes which are interdependently co-regulated at transcriptional level to sustain metallic ions homeostasis, energy homeostasis, cell s aging leading to cell evolution or cell death. Papers revealing the underlying molecular mechanisms have already been submitted.

The work presented in the paper seems to be very exciting but the hypothesis drawn is wrong very likely because of the gap in the knowledge.

I would recommend publishing this piece of work in PLOS one after revising the hypothesis presented in the paper on the ground mentioned above after making through independent critical analysis of published data and my published and unpublished but submitted work which can be obtained from Professor Simon Avery University of Nottingham United Kingdom by contacting him on e-mail Simon.Avery@nottingham.ac.uk if he kindly agrees to do so.

Helpful References

1) Bishop, A.L, Rab, F.A, Sumner, E.R, Avery, S.V (2007). Phenotypic heterogeneity can enhance rare-cell survival in 'stress-sensitive' yeast populations. Mol Microbiol. 63(2):507-20. doi: 10.1111/j.1365-2958.2006.05504.x. Epub 2006 Dec 14. PMID: 17176259

2) Rab, F.A. Solving a biological problem: Research methodologyVDM Verlag Dr. Müller (April 7, 2010) ISBN-10 ‏ : ‎ 9783639247497 ISBN-13 ‏ : ‎ 978-3639247497

3) Rab, F.A.(2014)Environmentally Modulated Evolution through Genetic Regulation Information Systems for Biotechnology ISB News Reports June/July

4) Rab, F.A.(2015) A Global Prospective on Research. Academe September October

5) Rab, F.A.(2016) Biotechnology and its Potentials and Challenges

EC Agriculture 3.4 : 705-707

6)Rab, F.A.(2017)(a)Who Should Come in Research? EC Nutrition 6.3:102-104

7) Rab, F.A. (2017)(b) Eat Fresh Live Young EC Nutrition RCO.01:03-05

8) Rab, F.A(2018)(a) Is Sugar an Accessory or a Neccessary.EC Nutrition 13.4:236-237

9) Rab, F.A.(2018)(b)Drug Disease Relationship and the Role of Food in Healthy Living.EC Nutrition 13.8 doing the bold one

10) Rab, F.A.(2018)(c)Genome-Nutrifortified Diets-Their Disease Protection and Remedy Potential. J.Prob Health 6 :204 doi: 10.4172/2329-8901.1000204

Rab FA (2018) Genome-Nutrifortified Diets-Their Disease Protection and Remedy Potential. J Prob Health 6: 204. doi:

10.4172/2329-8901.1000

Rab FA (2018) Genome-Nutrifortified Diets-Their Disease Protection and Remedy Potential. J Prob Health 6: 204. doi:

10.4172/2329-8901.1000

11) Rab, F.A.(2019)(a) Comparison between Safety Risks Associated with Domestically Processed Food and Commercially Manufactured Processed Food across the Food Supply Chain.EC Nutrition 14.5:414-416.

12) Rab, F.A(2019)(b) Is Hunger More Dangerous than having Mal-Nutrition or Consuming Unsafe Diet.EC Nutrition 14.12:01-05

13) Rab, F.A.(2020)(a) Hurdles in Progression of Knowledge and its Global Impact. Glob J Res Rev Vol.7 No.1:47. DOI: 10.36648/2393-8854.7.1.47

14) Rab, F.A. (2020)(b) Halal or Haram-New for Religious Scholars Muslim World and Food Supply Chain Stake Holders. Int J Nutr Sci & Food Tech. 6:3

15) Rab, F.A. (2021) Food items biologically tailored to meet nutritional deficiency challenge during Covid 19 Pandemic. J Prob Health. 9:233.

16) Abdur Rab, F., Hassan, A. (2022). Tourism, Health Promoting Food Domain and Technology Applications: Individual’s Genes Reservoir, Environmental Change and Food in Natural Health Context. In: Hassan, A. (eds) Handbook of Technology Application in Tourism in Asia. Springer, Singapore. https://doi.org/10.1007/978-981-16-2210-6_53

17) Rab et al.,(Co authored with Dr Sami Farooq, Dean of School of Management Sciences, Ghulam Ishaq Khan Institute of Engineering Sciences and Technology Swabi Pakistan) (2023) COVID 19- Technology driven Economically coordinated Supply Chain Chaos, its Remedy and Recommendations [Complete Paper and Science Policy Brief had been discussed in World Health Organization (WHO) s Meeting held on 4th May 2023 and in United Nation (UN) s Science Forum Meeting ( Science Technology and Innovation Forum e.g. STI Forum) held on 3-4 May 2023 in United States of America (USA) on the author (Dr Faiza Abdur Rab) s request which she made in capacity of Reviewer for all the Research Journals being published by The Royal Society of Chemistry. These papers are available on request].

18)Rab, F.A.(2023) Would Science Knowledge, Food and Agriculture Sector be Considered Basic Human Services or Commercial Services in this Post Covid19 Era? Decision will Determine the Destination. EC Nutrition 18.10: 01-08

19) Rab, F.A. (2023) Corona Virus (SARS-CoV-2 ) Rewinding Models in Science and Business and Redefining Illness Manifestations [submitted in Kuwait Journal of Science by Elsevier]

(20) Rab F.A (2023) Covid 19 Virus- Genomic and Molecular Gaming and the Role of Diet Preparations in Therapy [ under review in one of the well reputed Journals by Springer Nature]

21) Rab, F.A. (2024) Universal Evolutionary capacitor switch operation regulation gimmicks driving illness and natural cure [ under process in one of the well reputed Journals by Nature]

22) Colorado Gómez, M. A., Melo-Bolívar, J. F., Ruíz Pardo, R. Y., Rodriguez, J. A., & Villamil, L. M. (2023). Unveiling the Probiotic Potential of the Anaerobic Bacterium Cetobacterium sp. nov. C33 for Enhancing Nile Tilapia (Oreochromis niloticus) Cultures. Microorganisms, 11(12), 2922. https://doi.org/10.3390/microorganisms11122922

23) Chatfield, C. H., & Cianciotto, N. P. (2007). The secreted pyomelanin pigment of Legionella pneumophila confers ferric reductase activity. Infection and immunity, 75(8), 4062–4070. https://doi.org/10.1128/IAI.00489-07

24) Maret W.(2024) The Extracellular Metallometabolome: Metallophores, Metal Ionophores, and Other Chelating Agents as Natural Products. Natural Product Communications.;19(8). doi:10.1177/1934578X241271701

Reviewer #3: This manuscript presents a timely and relevant study addressing a critical need in the aquaculture industry. As one of the fastest-growing sectors in global food production, aquaculture—particularly the farming of Nile tilapia (Oreochromis niloticus)—faces increasing challenges due to rising consumer demand and the resulting intensification of production systems. These pressures often lead to compromised fish health and increased disease prevalence.

However, I have significant concerns regarding the overall presentation and writing style of the manuscript. It does not adhere to the formatting and structural guidelines typically expected for scientific manuscripts, such as those outlined by PLOS ONE. The writing style resembles that of a paper prepared for a different discipline, rather than aligning with standard scientific conventions. Additionally, the English language requires substantial editing for clarity, grammar, and readability to meet the standards of a peer-reviewed journal.

In the Materials and Methods section, please provide detailed information on how Cetobacterium sp. nov C33 was cultured under anaerobic conditions, including the specific broth medium used and any relevant growth parameters.

To improve transparency and enhance the manuscript’s clarity for readers in the field, I recommend rewriting sentences like: 'The gut-associated microbiota modulation was studied using culture independent techniques by 16S amplicons Illumina sequencing.' As currently phrased, it is difficult to understand. Consider rewording in a clear and concise manner, such as: 'The composition of gut-associated microbiota was analyzed using culture-independent 16S rRNA gene sequencing via Illumina technology.' This helps ensure that the methods and findings are easily understood and accessible to a broader scientific audience.

To improve transparency and ensure a clearer understanding, please clarify how microbial diversity was calculated and how the data were normalized. The statement, 'To estimate microbial diversity, abundance tables were normalized with Total Sum Scaling (TSS) in each sample,' lacks sufficient detail. It would be helpful to specify the exact steps used in the normalization process, including how TSS was applied and whether diversity metrics were calculated on raw or normalized data. Providing this information will strengthen reproducibility and comprehension for readers.

Reviewer #4: The abstract could be a little larger and elaborate. The overall writing is average not up to the mark.It needs more grammartical refining. More references should be used to validate the given informations properly.

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Reviewer #1: No

Reviewer #2: Yes:Faiza Abdur RabFaiza Abdur Rab

Reviewer #3: No

Reviewer #4: Yes:Md. Afif UllahMd. Afif Ullah

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Attachment

Submitted filename: Plos One Peer Review Comments.docx

-->PONE-D-25-18938R1-->-->Anaerobic bacteria Cetobacterium sp. nov C33 plays a crucial role in the intestinal microbial balance and regulation of gene expression to immune and metabolic responses in Nile Tilapia-->-->PLOS ONE

Dear Dr. Villamil Díaz,

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 Nov 05 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.

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'.
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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,

Amel Mohamed El Asely

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

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 #2: (No Response)

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

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. -->

Reviewer #2: Partly

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

Reviewer #2: No

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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.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: No

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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 #2: 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 #2: Reviewer s comments

The comments of the authors in response of the points I raised have failed to justify the given scientific status of the revised paper.

For instance technically speaking in a common person s language hypothesis is referred as a possibility. Merely substituting the synonyms cannot serve the purpose to make a scientific content authentic

The authors failed to present a scientifically authentic counter argument against my stance " Low cell population e.g.108 cells/g of Anaerobic bacteria Cetobacterium sp. nov C33 present in feed are practically too less to enable the fed inoculums to appear as dominating species in Nile Tilapia particularly in the case when it requires anaerobic condition to grow at 28°C whereas the temperature of fish body is usually lower than 28°C with limited availability of oxygen ".

Furthermore it seems that paper under revision has been written with inadequate literature search reflected as deficient understanding of relevant domains of knowledge. There is no single living entity which does operate without interplay of mineral ions. As far as production of pigments are concerned, genes encoding them may not be present on genome of the bacteria such as they can be present on plasmid or production of pigments can be an induced manifestation of a biological event or change in the environment.

Supporting scientific abstract statement

"microorganisms might evolve responses to sublethal concentrations of these metabolites, either to protect themselves from inhibition or to change certain behaviors in response to the local abundance of another species. Here, we report that violacein production by C. violaceum ATCC 31532 is induced in response to hygromycin A from Streptomyces sp. 2AW, and we show that this response is dependent on inhibition of translational polypeptide elongation and a previously uncharacterized two-component regulatory system. The breadth of the transcriptional response beyond violacein induction suggests a surprisingly complex metabolite-mediated microbe-microbe interaction and supports the hypothesis that antibiotics evolved as signal molecules".

Reference

Lozano, G. L., Guan, C., Cao, Y., Borlee, B. R., Broderick, N. A., Stabb, E. V., & Handelsman, J. (2020). A Chemical Counterpunch: Chromobacterium violaceum ATCC 31532 Produces Violacein in Response to Translation-Inhibiting Antibiotics. mBio, 11(3), e00948-20. https://doi.org/10.1128/mBio.00948-20.

The concerns raised on assessing the submission entitled Anaerobic bacteria Cetobacterium sp. nov C33 plays a crucial role in the intestinal microbial balance and regulation of gene expression to immune and metabolic responses in Nile Tilapia have not been addressed while revising the paper, scientific flaws still sustain in the revision, that builds a ground to recommend major revision of the revised version of the submission while addressing the points raised by me in capacity of reviewer prior publishing the paper.

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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 published, this will include your full peer review and any attached files.

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Reviewer #2: Yes:Dr Faiza Abdur RabDr Faiza Abdur Rab

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Attachment

Submitted filename: PLos One Peer Review comments for Revision one.docx

-->PONE-D-25-18938R2-->-->Anaerobic bacteria Cetobacterium sp. nov C33 plays a crucial role in the intestinal microbial balance and regulation of gene expression to immune and metabolic responses in Nile Tilapia-->-->PLOS One

Dear Dr. Villamil Díaz,-->-->

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Reviewers' comments:

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-->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 #5: All comments have been addressed

Reviewer #6: 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 #5: Partly

Reviewer #6: Yes

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

Reviewer #5: Yes

Reviewer #6: Yes

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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.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 #5: Yes

Reviewer #6: Yes

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Reviewer #5: Yes

Reviewer #6: 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 #5: In this manuscript, the authors investigated the effects of dietary the probiotic potential of Cetobacterium sp. nov C33, using 16S rRNA amplicon sequencing, on gut microbiota, and the transcriptomic analysis of the head kidney provided insights into immune system modulation. Further studies should be done for long rearing time (8-10 weeks) using different concentrations of this probiotic to evaluate the fish performance and the immune system modulation including the resistance against the possible pathogenic bacterial infection or any other stress.

L144: Diets preparation

L168: The alevins of Nile tilapia should be Nile tilapia juveniles

L169: at 5 fish per aquarium with four replicated tanks per experimental treatment;

L171-L173: Water quality parameters were monitored daily during the feeding trial, with the following average values: water temperature 28.0 °C, pH 7.12, and oxygen saturation 85 %. Write how did the authors monitored the water quality parameters, and the instruments used for measuring those parameters. Write the dissolved oxygen value.

L174 - L175: Shorten the subtitle to be “Modulation of intestine microbiota in Nile tilapia fingerling “

L247 - L248: Shorten the subtitle to be “Modulation of the immune system in Nile tilapia fingerling”

L301 - L302: Shorten the subtitle to be Shorten the subtitle to be “Modulation of intestine microbiota in Nile tilapia fingerling “

Fig. 1. Should be divided to be two Figs; Fig. 1 for diversity indices and Fig. 2 for the taxonomy of intestinal microbiota. And rewrite the results section accordingly.

Tables. Adjust the title of tables to be more specific such as “Changes in the intestinal microbiota of Nile tilapia fingerlings fed with Cetobacterium sp. nov C33 for five days.”

What is the difference in titles of Table 1 and Table 2?

L422: Shorten the subtitle to be “Modulation of the immune system in Nile tilapia fingerling”

Data in Tables 1 - 2 and Figs 3-4 should describe the changes in both treatments.

Discussion: This study evaluated the changes in intestinal microbiota. Then, the authors should focus their discussion on this topic. Other previous studies evaluated the fish growth and intestinal histomorhometry due to probiotics administration should be deleted to concentrate the Discussion section. The authors should compare their data between the control and probiotic-fed fish and support their findings with those of previous studies.

Reviewer #6: Please change Nile Tilapia in all the text to be Nile tilapia especially in the title, line 6 and 43

P value P must be capital and italic

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Reviewer #5: No

Reviewer #6: Yes:Awatef Hamed HamoudaAwatef Hamed Hamouda

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Anaerobic bacteria Cetobacterium sp. nov C33 plays a crucial role in the intestinal microbial balance and regulation of gene expression to immune and metabolic responses in Nile Tilapia

PONE-D-25-18938R3

Dear Dr. Luisa Marcela Villamil Díaz,

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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Amel Mohamed El Asely

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 #5: All comments have been addressed

Reviewer #6: 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 #5: Yes

Reviewer #6: Yes

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

Reviewer #5: Yes

Reviewer #6: 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.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 #5: Yes

Reviewer #6: 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 #5: Yes

Reviewer #6: 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 #5: The authors followed the reviewers' comments, and this manuscript is ready now to be accepted.

The authors followed the reviewers' comments, and this manuscript is ready now to be accepted.

Reviewer #6: Good luck with my best wishes,

the manuscript is now ready for publication after your explaining and corrections.

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Reviewer #5: No

Reviewer #6: No

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