Dear Dr. Quigley,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration by two reviewers and myself, we feel that it has merit but does not 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. While I do not necessarily agree with the reviewer comment that additional experiments are necessary at this point (although that would certainly strengthen your manuscript), both reviewers make several really important points that you need to address. In addition, I would like to ask you to perform a statistical power analysis and interpret your results accordingly. Finally, you need to go through your writing thoroughly to eliminate any easily avoided mistakes (e.g., Line 118 or L. 175).

If you are prepared to undertake the required revisions, please submit your revised manuscript by Jan 02 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.

• 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'.
• 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 . 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,

Olav Rueppell

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for stating the following financial disclosure:

“Research Council of Norway, Grant # 335244”

Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

If this statement is not correct you must amend it as needed.

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

3. We note that your Data Availability Statement is currently as follows: All relevant data are within the manuscript and its Supporting Information files.

Please confirm at this time whether or not your submission contains all raw data required to replicate the results of your study. Authors must share the “minimal data set” for their submission. PLOS defines the minimal data set to consist of the data required to replicate all study findings reported in the article, as well as related metadata and methods (https://journals.plos.org/plosone/s/data-availability#loc-minimal-data-set-definition).

For example, authors should submit the following data:

- The values behind the means, standard deviations and other measures reported;

- The values used to build graphs;

- The points extracted from images for analysis.

Authors do not need to submit their entire data set if only a portion of the data was used in the reported study.

If your submission does not contain these data, please either upload them as Supporting Information files or deposit them to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of recommended repositories, please see https://journals.plos.org/plosone/s/recommended-repositories.

If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent. If data are owned by a third party, please indicate how others may request data access.

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

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

Reviewer #1: Yes

Reviewer #2: No

**********

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

Reviewer #1: No

Reviewer #2: I Don't Know

**********

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

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: No

**********

Reviewer #1: In this manuscript Quigley and Amdam analysed the permeability of the honeybee blood-brain barrier (BBB) during aging and under a parasitic stressor. They found that both paracellular and transcellular permeability are maintained during aging, however, transcellular permeability increases upon exposure to the parasitic mite, Varroa destructor. Overall, this manuscript presents two simple experiments with clear objectives. The experimental design and data presentation are simple and direct, and the conclusions are supported by the data. Nevertheless, some minor adjustments could enhance the manuscript.

In our experience, injecting insects with dyes presents a challenge due to high variability between individuals. Differences in the amount of dye injected in each animal or loss of haemolymph can cause significant variation in tracer concentration. This variability limits the quantitative analysis of the experiments, requiring a large number of animals to obtain a reliable representation of the population.

In this regard, the number of animals is quite variable, ranging from 8 to up 22 honeybees. While limitations in animal availability are understandable, as shown in Table 1, comparing groups of 22 animals to 8 could obscure small differences. The authors should address this issue in the discussion section.

Similarly, in Fig. 1 and Fig. 2, the data are presented as box plots. I strongly suggest that the authors include all points in the plots or provide supplementary tables showing all values. This is particularly relevant, as the authors used parametric statistics but displays median and interquartile ranges as whiskers. Additionally, bimodal distribution may be present in this type of data, making nonparametric statistics more appropriate for analysis.

Minor comments:

Why was a 0-1 mites range used? Would it be more informative to include a completely mite-free (no-stress) control group for comparison?

Additionally, could the authors discuss the possibility of an inflammatory response due to parasite-induced stress? This may be particularly relevant, as studies on Drosophila indicate that inflammation can increase the BBB permeability.

Reviewer #2: Quigley and Amdam report the use of BBB permeability assays in honey bees. These assays have widely been used to study BBB permeability in Drosophila previously. The use of these assays reveals that strong infection with mites increases transcellular BBB permeability in freshly hatched bees. The adaptation of the assays used in Drosophila is surely useful, but rather straight forward given the similarities between the insects. The finding that BBB permeability is changed upon heavy mite infection is interesting, but I have several concerns about the study.

Major concerns:

- It remains unclear if and how many independent experiments have been performed. Have animals from different hives been probed? The number of animals probed is very small and indicates just one independent experiment, which is clearly not enough to draw any general conclusion.

- The authors claim that increased transcellular diffusion over the BBB could have an effect on animal health. However, they do not show if this is really the case. Do bees with increased BBB transcellular permeability show a decrease in fitness? Or at least: Do bees that have been infected by multiple mites show a reduction in health span or fitness?

- The authors show that infection with more than two mites affects transcellular BBB permeability. They probe freshly emerged animals of the age of 1 day. However, it remains unclear if this phenotype persists. The phenotype might be an adaptation to the effect of mite infection during development (reduced resources) and might wear of rapidly after the animal got rid of the mites. In this case, it would probably not impact the animal’s health span or fitness. Thus, I suggest to analyze BBB permeability of animals that have been infected with mites during development at later stages of their life.

- The authors suggest that a follow up study should increase the number of animals tested. I would strongly suggest to do this in this study.

- The authors probe bees of different ages, the oldest age group being 2-3 weeks old, if I am not mistaken. Hoe old does a worker be become usually? Is 2-3 weeks “old” for a bee? I suggest to probe the entire lifespan of a worker to be able to draw conclusions on aging.

- The text (introduction and discussion) describing the features of the BBB contains several mistakes. E.g. the authors state that the BBB is enriched in active transport systems for the exchange of various substances (line 86). There are active transport systems, but many substances are also transported via facilitative transporters (e.g. glucose). Line 158 states that there are tight junctions between the cells – in insects the diffusion barrier is provided by septate junctions. In line 270 the authors claim that there is redundancy between different junctional proteins. Vast research in Drosophila suggests that the junctions are formed by big protein complexed, that comprise many different proteins, which are all needed to keep the complex stable (at least the core proteins). If one of them is missing the complex falls apart. Thus, I would not talk about redundancies when it comes to BBB protein complexes. There are more other imprecisions that should be corrected.

- Several sentences suggest causative connections between unrelated information. Please review the text for inconsistencies and wrong causative relationships. In general, the text does need some proof reading.

**********

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.

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 For information about this choice, including consent withdrawal, please see our Privacy Policy .-->

Reviewer #1: No

Reviewer #2: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.. Please note that Supporting Information files do not need this step.

Dear Dr. Quigley,

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.

• 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 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,

Olav Rueppell

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.

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.

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

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures

You may also use PLOS’s free figure tool, NAAS, to help you prepare publication quality figures: https://journals.plos.org/plosone/s/figures#loc-tools-for-figure-preparation.

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

Effect of aging and Varroa parasitism on the paracellular and transcellular permeability of the honeybee blood-brain barrier

PONE-D-24-43745R2

Dear Dr. Quigley,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager®  and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact  and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact billing support ..

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Olav Rueppell

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments: