-->PONE-D-25-48608-->-->Host plant nutrition drives fitness outcomes in the cactus specialist Drosophila mettleri-->-->PLOS ONE

Dear Dr. Noronha,

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 note that reviewer made a careful analysis of your work and raised several criticisms. However, It seems to me that most of them can be adjusted by a reinterpretation/rewriting (comments #1,2,3 and 6) or re-analysis (comment #5) of the data. In comment #4, they asked for additional data that you possibly have.

Please submit your revised manuscript by Jan 15 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.

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Pedro L. Oliveira

Academic Editor

PLOS ONE

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[This paper was supported by National Science Foundation (https://www.nsf.gov/bio/deb) grants DEB2030129, DEB1839598, and DEB1241253 to PMO.

The Cornell University Insect Collection (https://cuic.entomology.cornell.edu/) and the American Museum of Natural History Theodore Roosevelt Memorial Grant (https://www.amnh.org/research/richard-gilder-graduate-school/grants) provided financial support to LN.].

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4. Please amend the manuscript submission data (via Edit Submission) to include author Lidane Noronha.

5. Please amend your authorship list in your manuscript file to include author Lidane Audrey Noronha.

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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

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

Reviewer #1: No

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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

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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: In my review I considered the seven editorial criteria required for acceptance in PLoS One. My comments below mainly concern criterion 3 (Experiments, statistics, and other analyses are performed to a high technical standard and are described in sufficient detail) and criterion 4 (Conclusions are presented in an appropriate fashion and are supported by the data). Although I believe the manuscript does not meet these criteria in its current form, the issues I note below could be fixed in a revision by providing some clarifications, redoing some analyses, and noting caveats to interpretations of the results. In my opinion, the paper meets the other five criteria.

1. Drosophila mettleri specialization: throughout the paper, the authors treat D. mettleri as a strict saguaro specialist. For example, lines 9-10 from the abstract. But this is not the case as populations of D. mettleri are found in areas where saguaro cactus does not occur (e.g. Santa Catalina Island and Baja peninsula).

2. If I understood correctly, the line used in the study was actually collected from the Baja peninsula. Saguaro cactus is not present there; the main host is cardon. I think the authors need to acknowledge this as a caveat to the interpretation of their results. Furthermore, the line has been in the lab since 2012, which means it may be somewhat lab adapted. Again, this should be noted as a caveat.

3. Food formulations: The authors should provide more detail about how the food treatments were prepared. For example, I am unclear on exactly what the soil treatment consisted of. How was it collected, how was it stored, how much was applied (that goes for all treatments)?

4. Fitness assay: the most relevant measure of fitness in this assay is egg to adult survival, but the authors do not report this information. Instead, they analyze egg to pupa survival and pupa to adult survival as separate measures. I think these are reasonable measures to include for a more nuanced look, but ultimately fitness depends on the combined effects of both survival rates so this should be the main analysis. From looking at the data, this looks like it could change some conclusions or shift the interpretation, because it appears there might be a tradeoff between survival from egg to pupa vs pupa to adult. I would also suggest the authors not describe this as a “nutritional assay.” While there are no doubt nutrient differences between their treatments there are numerous other differences as well (e.g. toxicity, etc.). I don’t really think the experimental design allows you isolate the effects of nutritional differences specifically. There are also some contradictory statements—e.g. in the abstract it says microbial communities appear to improve developmental success (lines 12-13), while in the discussion it says that it is the nutritional properties of the host plant that are most critical (lines 318-319). Basically, I don’t think the design permits conclusions as to which is more important, or if it does, this needs to be better explained.

5. Statistical analysis of fitness assay: If I understand the experimental design correctly, the egg counts for each group of five pairs of flies comes from multiple separate vials, not just one. The fact that they have repeated measurements from a single group is not accounted for in their statistical analysis. This problem could be remedied with a different statistical approach. I would recommend a GLMM with binomial response that includes “fly group” as a random effect. This approach would deal with the repeated measures and the GLMM approach would likely provide much more power than the Kruskall-Wallis test.

6. I am not familiar with the excreta assay. Could the authors provide a bit more detail? For example, I am struggling to understand why the excreta is only measured for 3 hours at the end of several days of feeding. What if they excrete a bunch of dye right before you transfer them to the last vial? Or what if they don’t excrete in the 3 hours even though they ate dye? Some more clarifications/explanations on these points would be helpful. Also lines 194-196 they say “any developmental differences did not arise due to consumption differences.” But consumption was measured on adults, while by development I assume they mean egg to adult. It is not clear how these are connected.

7. Microbial assays: The authors discuss their results in terms of microbial diversity, but it is not clear to me that they measured diversity per se (i.e. the number of different types of bacteria/yeasts). It seems like they quantified biomass. More clarification or a change in the wording is needed. Also, they should briefly mention the caveat that these assays are only relevant for microbes that are easily cultured with the different media they used. There could be hidden diversity that does not culture well.

8. References:

a. The authors did not include one reference that is quite pertinent to their study and might help frame some expectations for differences between laboratory and cactus food: Hoang K, Matzkin LM, Bono JM. Transcriptional variation associated with cactus host plant adaptation in Drosophila mettleri populations. Mol Ecol. 2015 Oct;24(20):5186-99. doi: 10.1111/mec.13388. Epub 2015 Oct 12. PMID: 26384860.

b. Lines 58-60: the wording makes it sound as though these references show this effect specifically for saguaro, but these are in fact studies from different host plants.

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Reviewer #1: No

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

-->PONE-D-25-48608R1-->

Host plant nutrition drives fitness outcomes in the cactus specialist Drosophilamettleri

PLOS One

Dear Dr. Noronha,

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 03 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,

Pedro L. Oliveira

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.

Additional Editor Comments:

This time the reviewer has focus don three specific points. While comments 2 and 3 are very simple details to be fixed, comment #1 is still about one of the major points raised in the first round, and need attention.

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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)

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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: Partly

**********

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

Reviewer #1: No

**********

-->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: I appreciate that the author’s attempted to address my previous comments, but I still have a some concerns that I think need to be further addressed.

1. The authors conducted the GLMM for egg to adult survival as suggested. However, they still treat this as secondary to the egg to pupae, and pupae to adult analyses. It seems to me that it makes the most sense to start with egg-adult as the overall measure of fitness and then deconstruct it into stage specific analyses if they want to highlight how survival varies across developmental stage. Most importantly, the stage-specific analyses should also utilize GLMM instead of the Kruskall-Wallis test. A final point: the authors presented the results of the GLMM as a series of parameter estimates. I think they should consider summarizing the analysis with an anova table (e.g. the ‘car’ package can be used to produce an anova table of GLMM) and potential post hoc follow-up tests as this may be more easily digestible by readers. They may also consider analyzing the banana and cornmeal experiments separately.

2. I do not see a figure 4 although it is referenced in the text.

3. Lines 258-260: I do not understand how egg to adult survival reflects the consequences of parental feeding behavior. Perhaps it is meant to be parental oviposition behavior?

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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.

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Reviewer #1: No

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-->

Host plant nutrition drives fitness outcomes in the cactus specialist Drosophila mettleri

PONE-D-25-48608R2

Dear Dr. Noronha,

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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Kind regards,

Pedro L. Oliveira

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments: