-->PONE-D-25-48231-->-->Addition of a single bacterial isolate to conventional larval rearing water can impact wing size and longevity in adult male Aedes aegypti.-->-->PLOS One

Dear Dr. Short,

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.

==============================

Although the ms is well written there are important points that need to be addressed before being resubmitted and reconsidered for publication. Details in methodology need to be provided and the fact only negative results have been reported should be discussed, especially as far as SIT and IIT are concerned.-->-->The comments and remarks of Reviewer #1, with which I agree, should be carefully and comprehensively addressed in the revised ms. In addition, I have included minor rather editorial comments in the annotated pdf that should be considered as well.

==============================-->--> -->-->Please submit your revised manuscript by Jan 22 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.

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We look forward to receiving your revised manuscript.

Kind regards,

Nikos T. Papadopoulos

Academic Editor

PLOS One

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[This work was supported by the National Institutes of Health National Institute of Allergy and Infectious Diseases (grant number R21AI174093), the Ohio State University Infectious Diseases Institute, and the Ohio State University College of Food, Agricultural, and Environmental Sciences.].

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-->Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

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

Reviewer #1: Partly

Reviewer #2: Yes

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

Reviewer #1: No

Reviewer #2: 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: Yes

Reviewer #2: Yes

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-->4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.-->

Reviewer #1: Yes

Reviewer #2: Yes

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-->5. Review Comments to the Author

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

Reviewer #1: his manuscript presents an investigation into the impact of a single bacterial isolate in larval water on the wing length and longevity of Aedes aegypti adult males. Among the three tested strains (Asais sp., Kocuria sp., and Cedecea sp.), only larvae reared in water containing Cedecea sp. produced adults with significantly reduced wing length (body size) and longevity compared to control samples, while no significant differences were observed for the other strains. Although the manuscript addresses an interesting topic that might shed new light on the mass rearing of mosquitoes, including the invasive A. aegypti, in its current state, it does not yet meet the standards required for publication. Within the manuscript, major issues were spotted in terms of clarity, methodology, accuracy, and the significance of the results. The main points of concern are listed below:

- In the experimental trays, the bacterial isolates were added only one (108 CFU per tray). Without monitoring the bacterial potential variation, there is no guarantee that bacteria persisted or reached a stable colonization in the larval water.

- No sequencing or plating of larval water or adults was conducted after the treatment to assess the bacterial establishment and the dominant. Without it, we cannot conclude that the observed effects are due to the tested strain directly or to the indirect microbial interactions.

- The criteria for sex separation at the adult stage were not described, which is essential for the reproducibility of the study.

- For the wing length measurement, the sample size (number of wings measured per group) was not reported, which makes it impossible to assess the statistical power. In addition, the decision to analyze the biological replicate separately seems to reduce the robustness of the statistical analysis. This is quite evident in Figure 2, where Rep1-Cedecea group includes only two samples, which is insufficient for an accurate statistical test. On the other hand, the difference observed for Rep4-Cedecea is marginal, as it falls in the same group as Asaia. Therefore, out of the four replicates, only the results from Rep3 may be considered reliable. Thus, the overall conclusion lacks strong statistical support.

Reviewer #2: The paper is concise, and the methods appear technically sound, aligning well with other published studies conducting similar investigations. All data have been made available. The supplementary documents include abbreviations that should be defined for clarity.

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Reviewer #1: No

Reviewer #2: No

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Attachments

Attachment

Submitted filename: PONE-D-25-48231.pdf

-->PONE-D-25-48231R1-->-->Addition of a single bacterial isolate to conventional larval rearing water can impact wing size and longevity in adult male Aedes aegypti.-->-->PLOS One

Dear Dr. Short,

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 consider carefully comments of Reviewer #3 and address all of them. In addition, there are few editorial comments that need to be considered. I have attached an annotated pdf with some points indicated.

==============================

Please submit your revised manuscript by Apr 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.

We look forward to receiving your revised manuscript.

Kind regards,

Nikos T. Papadopoulos

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.

[Note: HTML markup is below. Please do not edit.]

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: (No Response)

Reviewer #2: All comments have been addressed

Reviewer #3: (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 #1: No

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: No

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: 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

Reviewer #2: Yes

Reviewer #3: 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: As I indicated in my initial review, the paper should be rejected, so I will not need to go through any main points to check if they have been properly addressed.

Reviewer #2: (No Response)

Reviewer #3: Overall, I agree with the authors that this study contains findings that are worth reporting. The experiments are well-designed. There are some aspects of the presentation and discussion of these results that I still feel could be improved.

I agree with the previous reviewer and authors choice to analyze the winglength together and include replicate effects. I actually prefer, the original figure with all data broken out which allows the reader to see that replicate effect clearly. I would suggest swapping Fig 2 with Fig S1, but defer to the authors on how best to display their data.

For the survival analysis, if I'm understanding correctly, the replicate effect was removed because its relative effect on hazard varied over time? I can see why they have removed it, but would like to see the variation between replicates. In Figure 3, this could be accomplished by including 95%CI shading or error bars or alternatively plotting each replicate separately as they have done for winglength.

The differences in sample sizes for winglength and survival among the treatments warrants some discussion. I am guessing that the Cedecea and Kocuria treatments had either higher larval mortality or something interesting happen with their sex ratio such that there were considerable (significantly?) less males to work with for winglength and survival measurements. It is a little puzzling as to why these data are not included here. While I agree that the focus of the manuscript is male adult traits, the evidence that these treatments had a significant impact on larvae bolsters the authors argument that the data demonstrate that the treatments have significantly impacted the environment in which larvae have developed and suggests that the differences in adult traits are likely related to "carry over" effects of the treatments in the larval stage. This ties in with my suggestion below about making it clear what the possible explanations are for these data without being too speculative.

There is considerable redundancy between the introduction, discussion, and conclusion section. Lines 171-188 are mostly focused on the design of the experiment, instead of what you found with this design. I would like to see some of the points that are repeated in the discussion that are more focused on the rationale for undertaking the study which were already laid out in the introduction streamlined, in favor of more explicit discussion of what these results mean for our understanding of the larval microbiome and male traits and more clearly outlining what previous work suggests about the underlying mechanism for the effect they have observed here. For example, in the discussion on Lines 223-228 I was left wondering whether a transient exposure to microbes in the larvae stage has been previously shown to cause any impact on mosquitoes and if so was this due to immune activation, toxicity, or some other mechanism? Of course, as discussed in the previous review, this should not be overly speculative, but a concise presentation of alternative explanations and future avenues that are opened up by the work would significantly improve the discussion.

Line 135: Remove extra "."

Line 137: insert ". " before To analyze survival data.

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-->7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review?   For information about this choice, including consent withdrawal, please see our Privacy Policy.-->

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

**********

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NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications.

Addition of a single bacterial isolate to conventional larval rearing water can impact wing size and longevity in adult male Aedes aegypti.

PONE-D-25-48231R2

Dear Dr. Short,

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.

Please pay some attention during proof reading to assure that "sp." and "s  pp." are not in italics and that "F" and "p" referring to statistics both in text and figures are given in italics.

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Kind regards,

Nikos T. Papadopoulos

Academic Editor

PLOS One

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