-->PONE-D-25-53545-->-->Genetic structure of Rattus rattus  populations in an endemic plague focus in Madagascar: implications for rodent surveillance and management-->-->PLOS ONE

Dear Dr. Brouat,

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Reviewer #1: Yes

**********

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

Reviewer #1: Yes

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-->3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #1: No

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Reviewer #1: Yes

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-->5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)-->

Reviewer #1: The manuscript aimed to report the genetic structure of the black rat in plague focus district of Madagascar.

Comment (to the journal?): Line numbers should be added.

‘microsatellite’ in the keywords should be in plural with regards to the plurality of biomarkers, or ‘microsatellite markers’

Abstract:

As you have focused the paper on data from two trapping sessions, one in March-May 2019 and another in March-May 2020, is ‘temporal structure’ still relevant? Does significant change of genetic structure occur in such duration?

Change ‘Our results show’ to ‘…. showed’

‘… combining flea control within houses’ may be ‘… flea control inside houses’?

Introduction:

‘… in close contact with people’ => ‘… with humans’?

‘central highlands’ to ‘Central Highlands’

‘above 800 m high’ to ‘at elevation above 800m’

‘to the relief in the central highlands’ => ‘…. Central Highlands’

‘inside village houses’ => ‘inside houses’

Materials and methods :

‘central Highlands (mean altitude of 1,190 m)’ => ‘Central Highlands…’

‘altitude’ => ‘elevation’

‘namely: Ambohitromby’ => ‘namely Ambohitromby’

‘Hills slopes’ => ‘Hill slopes’

Trapping sessions: as you are focused on two trapping sessions, it may not be necessary to mention ‘Six small TS…’

Specify meaning of ‘night-traps’ (calculation) to ease understanding of 3,888 going from 18 houses during three nights.

Is there any particular reason why trapping efforts were different between habitats? 18 houses (36 traps?) against 84 traps outside villages.

‘Species, sex, external measurements (head-body length, tail length, ear length and hind foot length, in mm) and weight (in g) were recorded on each individual.’ You can arrange sentence to say that body mensuration (or external measurement) allowed identifying rodent at species level.

‘Kidney of each individual was sampled then stored in 95° ethanol for subsequent molecular investigation purpose. Furthermore, blood was collected by cardiac puncture and centrifuged to obtain sera samples for serological analyses.’ I think blood sampling goes before kidney sampling?

‘For each individual, total DNA was extracted from kidney using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) as recommended by the supplier’. Manufacturer instead of supplier.

A table showing microsatellite marker features would be helpful. How 18 markers were pooled during multiplex reactions? What is the expected amplicon size per locus? Here there are 18 markers whereas in the abstract, there are 17. Also, can authors explain the use of markers developed for brown rat to test black rat? Isn’t it required to use species-specific microsat markers?

‘Detection was conducted using an ABI 3130xl Genetic Analyzers (Applied Biosystems, Foster City, CA, USA) and genotypes were red, analysed…’. Detection of ….? Read instead of red?

‘Each group of samples from a given habitat × locality × session was considered as a distinct subpopulation in the subsequent analyses.’ You can change to ‘Individuals belonging to a given habitat (inside house or outside village), locality (the same site?), and sampling session (2019 or 2020) were considered as a distinct subpopulation in the subsequent analyses’

‘Nei’s unbiased genetic diversity (HE, [28]) and FIS’. Change to ‘………….. and the inbreeding coefficient FIS’.

Can authors spell out LD methods?

For IBD analysis, please specify what is considered as subpopulation? Still pop from a habitat?

‘Rats captured inside houses during March-May 2020 in Antakavana and Ambolotarakely were excluded from the dataset due to low sample sizes (n = 6 and n = 7, respectively) (Fig 1).’ You can also cite Table 1 in addition to Fig1.

Table 1:

HS meaning?

Results:

‘STRUCTURE was used to investigate whether rat populations where spatially structured in Ankazobe district.’ Were instead of where.

Discussion:

‘During our 2 year-long survey’. If I’m not wrong, samplings were performed once per year during two years.

As the genetic diversity varied according to habitat (higher outside village), how is the connection/gene exchanges between populations from inside houses with those from outside? In the bibliography, rat would move from field to inside houses following harvest.

‘the observation of different ectoparasites on rats’ change to ‘… different fleas…’

**********

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Reviewer #1: No

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-->PONE-D-25-53545R1-->-->Genetic structure of Rattus rattus  populations in an endemic plague focus in Madagascar: implications for rodent surveillance and management-->-->PLOS One

Dear Dr. Brouat,

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 Mar 16 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:-->

• A 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 https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Tzen-Yuh Chiang

Academic Editor

PLOS One

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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 #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

**********

-->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

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: 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

Reviewer #2: Yes

Reviewer #3: No

**********

-->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

Reviewer #2: Yes

Reviewer #3: 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 #1: My previous comments are addressed by authors. The revised version was highly improved.

I just have one comment/question:

Lines 125-126 : eight or seven markers ?

Reviewer #2: This is my first review of this manuscript. I understand it has undergone previous review, and I acknowledge the work done to reach this stage. The manuscript is clear and the results are relevant for pest control.

However, my main question relates to the clarity and justification of the research objective concerning the temporal genetic structure between the two sampling years, which are only one year apart. The introduction does not sufficiently establish why investigating genetic changes from one year to another (2019 vs 2020) is a worthwhile question for this system.

See specifics below:

Ln68: Could you please elaborate on these previous results, particularly their spatial extent? While the importance of the spatial scale is clear, the relevance of the temporal scale is less obvious. What is the generation time of the rat population? Is the consideration of time primarily due to seasonal variations in rat abundance? Or, is it because rats are routinely exterminated within houses each year, leading us to expect that rat colonization events are an annual occurrence? Please make clear justification of the objectives regarding the temporal question. Relying on the reader to piece it together from the introduction and methods reduces the text's clarity and impact.

Ln97-99: In terms of distance, what average distance was from inside the house’s vs outside of the village? The distance between villages is provided, but which were the min. and max. distances between the two sampling habitats? This is relevant to interpreting the results.

Table 1. Session S1 and S2 relate to the two sampling years? Why not instead to put the years S2019, S2020

L223: among sampling sessions

Ln265-268: I don’t have clear why genetic structure was analyzed separately for inside and outside villages?

Ln317: this information should be in the introduction, I have still no clarity of why comparing two sampling years.

Ln359-362: Expand further the management recommendation provided; I don’t see much difference between what is being done and what is being recommended. Again, the initial question regarding year to year differences is not clear, and not linked to a specific biology of the species or related to a particular pest control management.

Reviewer #3: Body of work presents technically sound original research.

Would be nice to see comparisons made to somewhat similar work published in the Canadian Journal of Zoology, 2007. This work feels like a scale up of what was done there?

Transparency is important and I feel the authors should share how the analyses were done, instills confidence that the work was done as written and imparts knowledge to others.

Microsatellite genotyping section - please indicate temporal distinction of the samples genotyped. Also, based on the rates of evolution for some molecular marker's, I am not sure one can expect to see temporal difference with microsats over a year's time, even if you managed to catch the same individual?

Data analyses section - please indicate what tools what were used at the very start or first few lines of the paragraph. Also, may just mention 'R v 4.4.2' instead of 'R software version....'.

Not sure non-parametric tests will give anything remotely definitive on plague leaving a trace on genetic diversity? unless the objective is to just give an association? somewhat superficial relationship? Certain you are aware non-parametric tests don't provide a cause-effect relationship.

Line 183: did not see mention of STRUCTURE and DAPC analyses being used to test temporal variation? unless I missed whether this was stated?

In my experience STRUCTURE and DAPC rarely give you similar answers, always a '1 K' margin of error...of course this is dependent on inherent differences in the marker of choice and algorithmic biases...please review all mentions of DAPC as confirmatory analyses and suggest that changes to them being secondary analyses.

Great work.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

**********

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-->

-->PONE-D-25-53545R2-->-->Genetic structure of Rattus rattus  populations in an endemic plague focus in Madagascar: implications for rodent surveillance and management-->-->PLOS One

Dear Dr. Brouat,

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 May 16 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:-->

• A 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.

As the corresponding author, your ORCID iD is verified in the submission system and will appear in the published article. PLOS supports the use of ORCID, and we encourage all coauthors to register for an ORCID iD and use it as well. Please encourage your coauthors to verify their ORCID iD within the submission system before final acceptance, as unverified ORCID iDs will not appear in the published article. Only  the individual author can complete the verification step; PLOS staff cannot  verify ORCID iDs on behalf of authors.

We look forward to receiving your revised manuscript.

Kind regards,

Tzen-Yuh Chiang

Academic Editor

PLOS One

Journal Requirements:

1. 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.

2. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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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: All comments have been addressed

Reviewer #4: (No Response)

Reviewer #5: (No Response)

Reviewer #6: (No Response)

**********

-->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: Yes

Reviewer #4: Yes

Reviewer #5: Partly

Reviewer #6: Yes

**********

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

Reviewer #2: Yes

Reviewer #4: Yes

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.-->

Reviewer #2: Yes

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: No

**********

-->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

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: 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 #2: I have read the revised version of this work and I found that the authors have addressed all my comments satisfactorily. I have no further comments.

Reviewer #4: The manuscript is clear and well structured, and the objectives, methods, and results are presented in a coherent and understandable manner. The authors have adequately addressed the issues raised during the review process, and the revised version meets the standards for publication. The study contributes relevant data to the understanding of rodent population dynamics in relation to disease surveillance.

I have no additional comments or concerns regarding research ethics or publication ethics. I support the publication of this manuscript.

Reviewer #5: This is my first revision of Parany et al. The study aims at quantifying genetic structure of rat populations occurring inside houses and outside houses across the Ankazobe district in the Central Highlands of Madagascar. This study is relevant in the context of plague outbreak, since rats are one of the major reservoir of plague spread. The authors used 17 microsatellites loci of >400 individuals sampled over two seasons (March-May 2019 and March-May 2020) to investigate genetic diversity, effective population size and genetic structure inside and outside houses, and across sampling seasons. They found higher genetic diversity and effective population size in the outside houses populations compared to the inside houses. There are also evidence of stronger genetic structure in the inside house populations. Overall, they conclude that the higher genetic diversity in the outside populations is likely due to the fact that there is higher dispersal among outside populations than inside populations. Higher dispersal in the outside populations suggest that rodent sureillance plans should not carry out on single subpopulations, but considering the whole network of rat connectivity.

The manuscipt is well written and clear. I think that they also present well the context. That being said I am not an expert on disease spread, plague and rat-plague co-dispersal so I cannot judge if the state of the art of the topic has been accurately presented.

I think that the authors have a quite unique dataset, although currently limited to microsatellites. I have some questions and suggestions that may help increase the impact of this work. I suggest the publication of this work upon minor revision.

General Comments

I might have missed it but it would have been interesting to see a quantitative comparison of plague seroprevalence between the inside vs outside houses rat populations. If the authors have enough data to quantfy such differences, it would also be interesting to assess whether differences in seroprevalence between inside and otside houses rats correlates with measures of genetic diversity (He and Fst) in the respective populations (inside and otside houses). Also, did the author computed seroprevalence per sampling seasons? Are there difference between those?

Also, I noticed that the authors reported genetic IBD statistics for the inside houses populations and overall sampling, but not among outside houses rat populations. Did I miss it or there is a specific reason for not doing that?

I think that in this context spatial analyses of genetic structure are quite relevant to understand how rat dispersal impact plague spread. I would suggest the authors to add few other spatial analyses which are related to genetic IBD but different, such as spatial autocorrelation or EEMS analysis (which measure rate of relative dispersal across a landscape using either microsatellite or SNP data) [https://github.com/dipetkov/eems].

Detailed Comments (numbering refers to the newest version of the manuscript I received)

Line 296: I think this sentence should be rephrased. The genetic IBD appears to be significant but negative in slope. I see what the authors meants, but I think it should be rephrased.

Line 297-299: what about genetic IBD for outside houses populations?

Line 282: I think it could be nice to better point to the south and northern populations in Fig. 3. I feel it is very difficult to see that at the moment.

Line 358-360: replace “larger sizes” with “larger effective population size”. Higher genetic diversity or Ne do not directly translate into higher census size, but it might be related to patterns of dispersal, higher in the outside houses than inside houses populations (as also the authors points out in the discussion).

Line 362-363: Could you measure relative abundance based on rat capture rate over the two sampling seasons and subpopulations?

Reviewer #6: Reviewer's report: Genetic structure of Rattus rattus populations in an endemic plague focus in

Madagascar: implications for rodent surveillance and management

This is my first review of the Parany et al. manuscript and I can see the authors have been thorough in responding to comments of all previous reviewers. This exploratory study provides an interesting comparison of genetic structure among rat subpopulations sampled from two environments: inside houses versus surrounding fields. The authors make key inferences regarding the rapid recolonization of rats, which is significant for plague control efforts in Madagascar.

Overall, the population genetic analyses are robust at both district-wide and local scales. However, some of the inferences are overstated, such as interpreting the genetic differentiation of Ankazobe (ANK) and Ambohitromby (AMB) as being a north-south pattern. ABK (a southern location) does not support a north-south hypothesis, based on the data presented. An alternative explanation that seems more plausible is given in the Discussion; namely, proximity to major urban centers which could potentially lead to gene flow into ANK and AMB. I recommend removing text related to a north-south pattern in the Discussion, and instead focus on the finding that ANK and AMB are members of a genetic group that is distinct from the other four locations.

Major Revisions:

1. Introduction: I saw the main hypothesis described in the Methods on lines 203-206. I think it would make the Introduction stronger if you also mentioned this hypothesis (briefly) in the last paragraph of the Introduction (perhaps after line 91). I especially liked the explanation you provided to Reviewer #2, along the lines of : “Because rats living indoors may function as relatively discreet demographic units (Rahelinirina et al. 2010), we tested the hypothesis that genetic structuring might be more detectable among house-dwelling subpopulations than among outdoor individuals.”

2. Methods: It would benefit your audience to briefly describe 2019-2020 rat control operations in Ankazobe district. Mention if rat control efforts had taken place at your specific study sites before trapping occurred.

3. Methods: Related to point#2, it would also be helpful to mention whether rat removal during your trapping efforts would be expected to significantly reduce rat numbers in residences and surrounding habitat. I am curious to know if rat removal could explain the lower Ne estimates inside houses.

4. Methods: Please add a few more details on the trapping design. Outside the village, you placed 84 traps spaced by 10 m. Were these placed in a single long transect 830m long, or multiple shorter transects? Or in a grid design? If a grid, please give the size of the grid area.

5. Results Line 267: One of the most important results is that genetic structure is higher among rat subpopulations sampled inside houses than among subpopulations sampled outside villages. Please add the two Fst values to this sentence (one for inside houses, one for outside villages) and the range of pairwise Fst values. In fact, it would help readers to see all pairwise Fst values from inside the houses in a table format (probably as a diagonal matrix). A separate diagonal matrix could be used for samples from outside villages. If the authors are willing to do so, these diagonal matrices could be added as supplementary material. This would give a clear understanding of variation among Fst values and show whether the pattern is driven by a few outliers, or is consistent across all pairwise Fst comparisons.

6. Results: Related to point#5 above, is the genetic structure of rats in houses due to relatives remaining in this habitat instead of dispersing? You could answer this by using FSTAT to run a “Comparison among groups of samples” procedure, and choose the Relatedness option to compare samples from inside houses vs. outside villages. This test would provide additional insight into the aspects of rat biology that shape genetic structure at a fine scale.

7. Discussion Line 385: What is meant by “environmental-based management towards reduction of human-rodent contact”? Please give 1 or 2 specific examples. (for instance, do you mean house improvements to prevent holes that allow rat access, or storing food in a shed separate from the living space?)

Minor Revisions: The manuscript is generally well-written, but some revisions are needed for clarity. Here are my suggestions for re-wording specific sentences.

8. Line 22: Change to “In this context, our study investigates…”

9. Line 25: Change to “…and in habitat outside of villages”

10. Line 28: Change to “…relatively similar among villages”

11. Line 36: “Because of the re-colonization problem, an integrated approach…”

12. Line 47: “within or near these natural foci”

13. Line 78: “…which may be associated with the heterogeneous”

14. Line 90: …effective population size (Ne),

15. Line 90: “and examined the short-term stability”

16. Line 132: Remove “purpose”.

17. Line 149: Change to “and manually corrected in GeneMapper…”

18. Line 191: “genetic groups”

19. Line 214: Change human to “humane”

20. Line 237: I suggest replacing homogeneous with “similar” (but this is up to the authors)

21. Line 253-254: Remove the text in this sentence, it repeats the same information as the first sentence. Keep the statistical reporting by adding it to the first sentence.

22. Line 268: Replace “…in one session or in the other” with “…within individual sessions”.

23. Line 272: after Fst = 0.095, add the range of pairwise values.

24. Line 288: use lower case for north-south.

25. Line 326: change “in four areas” to “in four other highland areas”.

26. Line 376-377: Remove the word “although” from the start of the sentence and place it after the first comma “, although with marked seasonal fluctuations, and our results reveal…”.

27. Line 379-381: I recommend merging these two sentences in the following way: At the end of the first sentence add “…fluctuating units, leading to population stability at the district scale. I would then remove the second sentence (Line 380-381) because the term “limited natural turnover” seems confusing in this context.

28. Line 386-387: Consider removing “on the one hand….on the other hand”.

29. Table 1: The order of subpopulations in the table is not the same as in Figure 2 (or Fig. 3). I suggest re-arranging the Table to match the order given in Figure 2 (ABK, AMB, ANK, ANT, KIA, TLA). This will make it much easier for your readers to follow the reporting of results.

30. Fig 1: Increase the size of the inset showing Madgascar, so that readers will be able to find Ankazobe.

31. Fig. 1 caption: Add a period after “district.” Also, end with “…two sessions (2019 and 2020).”

32. Fig 2 caption: Change NE to “Ne”. Remove “s” from subpopulations

33. Fig. 3 caption: add “s” to house. Spell out full name of Rattus rattus for all figure and table captions.

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Reviewer #2: No

Reviewer #4: No

Reviewer #5: Yes: Gabriele Sgarlata

Reviewer #6: No

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Genetic structure of Rattus rattus  populations in an endemic plague focus in Madagascar: implications for rodent surveillance and management

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Reviewer #5: All comments have been addressed

Reviewer #6: All comments have been addressed

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Reviewer #6: Yes

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Reviewer #5: Yes

Reviewer #6: Yes

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Reviewer #2: I found that the authors have made a substantial effort to adequately respond to all reviewers comments including myself. I have no further comments and I support the acceptance of the manuscript.

Reviewer #5: The authors have carefully replied to my comments. Therefore, I support the publication of the manuscript.

Reviewer #6: The authors have made extensive edits that have significantly improved the manuscript. I appreciate their mindful consideration of comments from all reviewers. I have no remaining concerns and only a few final edits to suggest.

Minor edits:

1. Line 114: In Fig. 1 legend, please capitalize “March”

2. Line 137: Change to “one kidney from each….”

3. Line 168: Please consider revising this sentence to “Genetic diversity of each subpopulation was estimated with FSTAT v2.9.4 using three estimators: 1) allelic richness..., 2) Nei’s ..., and 3) the inbreeding coefficient...”. This will make it easier for your readers to understand.

4. Line 170: Nei should not be italicized

5. Line 248: Remove “relatedness” because it is redundant. You already mentioned this term in the first word of the sentence

6. Line 252: Table 1 title, remove “s” from subpopulations

7. Line 356: Remove “of”

8. Line 366: Change “observed within” to “of the”

9. Line 375: Change “with frequent people interactions” to “frequent human movements”

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Reviewer #2: No

Reviewer #5: Yes: Gabriele Sgarlata

Reviewer #6: No

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