PONE-D-22-33692Virus infections in honeybee colonies naturally surviving ectoparasitic mite vectorsPLOS ONE

Dear Dr. Oddie,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, 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. Specifically, reviewer 2 was very critical and has explained several important issues. They are concerned about the rigor of your methods and terminology and also raised the possibility that this manuscript was previously reviewed and then submitted here without sufficiently addressing previous reviews. This would be a waste of everybody's time and therefore I would urge you to careful pay attention to the criticisms of that reviewer if you chose to revise and resubmit.

Please submit your revised manuscript by Mar 04 2023 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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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.

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We look forward to receiving your revised manuscript.

Kind regards,

Olav Rueppell

Academic Editor

PLOS ONE

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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: Yes

Reviewer #2: No

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

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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: The paper by Oddie et al. focuses on the analysis of viral infections (prevalence and viral loads) in Norwegian honey bee colonies that naturally survived infestation by Varroa destructor. They found lower V. destructor infestation levels in surviving colonies compared to Varroa susceptible colonies, and detected BQCV, DWV-A, LSV1, LSV2 and SBPV in all the colonies. The prevalence and viral load of Deformed Wing Virus (DWV-A) was lower in the mite surviving colonies in contrast to BQCV. Since the former virus is vectored by the mite they concluded that their findings suggests that lower mite levels may account for reduced prevalence and titers DWV-A rather than other mechanism of virus resistance in the surviving colonies

The study is interesting, well performed and described and it contributes to our knowledge on virus-resistance observed in V. destructor naturally surviving honey bee colonies. The conclusion is sound and it paves the way for further investigation of these mechanisms at the colony and individual bee levels.

The statement at lines 79-81 that indicates that the IKA viruses (IAPV, KBV and ABPV) and SBPV are not vectored by V. destructor should be changed since these viruses can be vectored by the mite as stated in Yañez et al. 2020, Bee Viruses: Routes of Infection in Hymenoptera.

Reviewer #2: Editors,

The manuscript entitled, “Virus infections in honeybee colonies naturally surviving ectoparasitic mite vectors” by Oddie et. al describes differences in virus abundance between a reported samples obtained from colonies that are reportedly mite-resistant (i.e., survivor honey bee stock) compared to samples obtained from conventionally managed colonies. This is an interesting topic, but as currently presented it was too difficult to interpret the data to ensure accuracy, and the text requires additional work prior to publication.

Points to clarify or address before publication include:

1. Figures – In general, virus abundance data be better presented as box and whisker graphs that include and data point for all samples (i.e. rather than bar charts), so that readers can get a better understanding of the values and actual variability in the data.

2. Abstract Line 21 / Introduction – While the citations in the introduction provide insight into mechanisms / behaviors associated with mite resistance and indicate that these adaptations can occur within 5 years (which seems short, if selection is acting at the colony level). It would be good for the authors to elaborate on this topic in regard to selection at the individual and/or colony level, etc. and/or revise the first line in their abstract and other place in the text. The researchers should be in other examples of hosts that have developed resistant to parasites over a similar relative time period (i.e., discuss typically ‘generation time’ for a honey bee colony, which is likely 1-2 years+ , and the number of years of mite exposure in Norway, etc.) in order to better describe how the mite-resistance described in this manuscript is/may be the result of natural selection.

3. Abstract Line 22 - The Abstract should be changed since if the colonies were selected from mite resistance, that may result in lower viral load since mites are amplifying vectors of several honey bee viruses and likely passive vectors of many others – but that does not mean that the honey bees have developed resistance to the viruses. While the Abstract gets to this point it begins by stating “It is possible that resistance to the viruses spread by this parasite may contribute to colony survival,. . .” which is misleading/ confusing.

4. Abstract – “The damaging variant of DWV, (DWV-A) .. “ is incorrect, both DWV-A and DWV-B can be detrimental to honey bees, as well as recombinant viruses.

5. The authors need to include a list of primers used in this study, including the primers used to distinguish DWV-A vs. DWV-B. Were the viruses sequence? Were DWV-recombinants assessed? Without this information, some of the DWV abundance in this study may not be accounted for, and therefore the conclusions would only be based on some of the DWV. Regional differences in RNA viruses, which have required new primers for detection and quantification may be needed (e.g., see Daughenbaugh et al Viruses 2021 doi: 10.3390/v13020291, Moore et al. J. Gen. Virol. 2010 doi: 10.1099/vir.0.025965-0, and others).

6. The Virus nomenclature needs to be revised throughout the manuscript, Lines 77-80+, to match ICTV guidelines. Virus nomenclature suggestion based on ICTV: , “black queen cell virus” and “deformed wing virus”, etc. be written in lower case, when referred to in-general (i.e., not a specific strain or species) should be written in lower case and not-italicized.

See: https://talk.ictvonline.org/information/w/faq/386/how-to-write-virus-species-and-other-taxa-names

Considering “deformed wing virus-A” is a virus name, “A virus name should never be italicized, even when it includes the name of a host species or genus, and should be written in lower case. The first letters of words in a virus name, including the first word, should only begin with a capital when these words are proper nouns (including host genus names but not virus genus names) or start a sentence.” Alternatively, for specific virus species, the authors could use “Deformed wing virus-A” (i.e., first letter capitalized, and italicized word), but this is rarely used.

7. Figures, were all colonies assessed on the same date? The supplemental information is good, but would be improved by inclusion of sample date (not just season) or at least including the exact sample dates in the methods. Since the virus abundance in any colony changes over time.

8. The use of the word “titres” is inaccurate and should be revised throughout the manuscript.

Virus titer or viral titre = titre typically refers to the concentration of infectious viral particles

The authors present “viral abundance” or “viral load” data in the form of RNA or cDNA copies per bee (which would be better presented as copies/ xxx ng RNA), therefore the text should be revised.

9. Virus abundance data is a major component of this study – yet it is unclear how this was determined in this study, and the reporting on figures, etc. requires major modification.

Specifically –

(a) Figure 3 – Log copies per colony is not the correct axis label.

It should be log(10) copies per 1000 ng total RNA (if they scaled results to represent total RT reaction).

The reaction volumes for RT and qPCR reactions should be included in the methods section and it should be clearly stated if Line 139 “3 ul cDNA” is really “3 ul of the 1:10 cDNA” – since, as written it is impossible to compare the virus copy number reported in this paper to other studies.

The researchers do not know the copy number per colony, since the assessment is done per x ul of a cDNA reaction – it represents some amount of RNA sample obtained from a subset of bees sampled from one colony.

(b) Lines 146 – 152, it is unclear how virus standards were made since simple dilutions of an unknown stock would not work to calculate “virus copy numbers” .

What did the authors use as a virus standard?

The standard data should be included and graphed to evaluate copy number and the qPCR efficiency.

PCR efficiency typically expressed as a percentage

(i.e., 10^(1/slope “3.32) – 1 = 1 or 100%

I am not sure how the authors calculated efficiency.

(c) The qPCR standard curves used to calculate virus abundance per qPCR well, and the math required to make that number representative of a given amount or RNA need to be clearly presented in this manuscript.

10. Virus abundance in individual colonies varies over time within a colony and the variation between individual colonies with the same apiary and/or region can be high.

The sample size in this study, which was n=10 mite-tolerant colonies, and n=11 conventionally managed colonies for virus abundance data was low. This sample sizes should clearly stated in the figures / figure legends The methods section reports higher numbers of both colonies in other sections making it difficult to see that the virus data came from a subset of the total number of colonies described.

The authors should revise the text describing putative virus resistance vs. tolerance since with a total of 21 colonies (i.e., 10 or 11 colonies per group) it would be difficult to try to assess the potential of virus resistance vs. tolerance.

For pupae and mite assessment it seems, 29 samples were obtained from mite resistant colonies located in three apiaries and 18 conventionally managed mite-susceptible colonies from five apiaries.

Since sample sizes vary for each figure, they must be included in the figures and figure legends, they should be included in every figure or figure legend.

11. Discussion – Although the virus levels reported in honey bees in this study were lower in the “mite-resistant stock” were lower than the “conventionally managed susceptible stock” the number of mites were also lower – so the conclusion stated at the beginning of the discussion may not be accurate (i.e., Line 252), lower mite numbers may be associated with lower virus numbers.

12. As written the discussion is difficult to read, and included big overarching conclusions based on the data obtained from a relatively few number of colonies – that may or may not be exhibiting “mite-resistance” phenotype given that the levels of mite in the brood are similar in the different stocks (although they are missing the autumn time point).

There may be other areas to comment on in the discussion, but since the virus data was difficult to review/interpret it is not possible to fully evaluate the discussion.

Minor points to clarify or address before publication include:

1. Fig. 2 – y-axis does not need decimal places (e.g., 10%, instead of 10.00%)

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Reviewer #1: Yes: Nor Chejanovsky

Reviewer #2: No

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PONE-D-22-33692R1Virus infections in honeybee colonies naturally surviving ectoparasitic mite vectorsPLOS ONE

Dear Dr. Oddie,

Thank you for submitting your manuscript to PLOS ONE. As you can see below, I sent the manuscript out to one of the previous reviewers who was very critical before but is now almost satisfied. Therefore, we invite you to submit a revised version of the manuscript that takes their remaining concerns into account. One issue that was not directly stated in the review but was brought to my attention was your assertion in line 45. While I (and the reviewer) agree that the potential exists, I would suggest to caution readers that this potential may not be realized. Encouraging "Darwinian beekeeping" may be your goal, but I think it is only fair to put in more cautious language for beekeepers that may well read an open-access article and gamble their livelihood on that potential. The survivor populations exist, but they are not necessarily the rule (which makes them particularly interesting).Also, please check for spelling and other minor mistakes throughout. Some mistakes are not caught by automatic spell-checkers (e.g., line 65: "be" instead of "me").

Please submit your revised manuscript by May 26 2023 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 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 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:

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

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

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

Reviewer #2: Partly

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

Reviewer #2: 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

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

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

Reviewer #2: (No Response)

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6. Review Comments to the Author

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

Reviewer #2: 

The manuscript entitled, “Virus infections in honeybee colonies naturally surviving ectoparasitic mite vectors” by Oddie et. al was significantly improved by the review process.

Points to clarify or address before publication include:

1. While I understand that the abstract “European honeybee populations,

Apis mellifera, “ is indicating “Apis mellifera” populations in Europe, since some papers still define Apis mellifera as the European honey bee (as opposed to the Western honey bee) – the authors should revise the abstract to make sure this is clear – since PONE has a general readership.

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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 #2: No

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[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 figures@plos.org. Please note that Supporting Information files do not need this step.

PONE-D-22-33692R2

Virus infections in honeybee colonies naturally surviving ectoparasitic mite vectors

PLOS ONE

Dear Dr. Oddie,

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 Jun 23 2023 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 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 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:

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:

Thank you for re-submitting your manuscript to PLOS ONE. As indicated in our previous correspondence, I felt obligated to it out for additional review and I was fortunately able to quickly secure two additional reviewers. As you can see, both have additional comments and concerns. While I do not think that the demand of reviewer #3 for additional experiments is warranted, I do think that both reviewers raise many important points. Most importantly, they confirm my concern about the language that could mislead beekeepers and cause them to stop treating for Varroa. I quote an excerpt from their confidential comments to the editor in this case: "The most important point, however, is that the wording concerning the evolution of mite tolerance must be adjusted (first sentence of the abstract and l. 45-47 in the Introduction)." Reviewer #4 also indicates a number of other necessary changes (for example Table 1 now seems to be missing from the manuscript?). If you are prepared to perform the necessary revisions, please follow the directions below. If you are not willing to do so, please just indicate this in an email to save the work of another submission because I will be forced to reject the manuscript in that case.

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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 #3: (No Response)

Reviewer #4: (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 #3: Partly

Reviewer #4: Yes

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

Reviewer #3: Yes

Reviewer #4: Yes

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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 #3: Yes

Reviewer #4: Yes

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

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

Reviewer #3: Yes

Reviewer #4: 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 #3: The paper by Oddie et al. analyzes viral infections (prevalence and viral loads) in Norwegian honey bee colonies that apparently survived infestation by Varroa destructor naturally. However, as a reviewer, I find the provided information insufficient to verify the authors' statement. The term "naturally survival" is not explicitly defined, and there is no information on the management system or procedures involved in the study. The lack of fundamental information, such as the size and strength of the colonies at the beginning of the experiment, feeding schedules, and honey collection, makes it difficult to evaluate the article's argument thoroughly.

The researchers found lower V. destructor infestation levels in surviving colonies compared to Varroa-susceptible colonies, which confirms the results from Dr. Oddie's previous work. The researchers detected BQCV, DWV-A, LSV1, LSV2, and SBPV in all the colonies. The prevalence and viral load of Deformed Wing Virus (DWV-A) was lower in the mite-surviving colonies, in contrast to BQCV. The authors concluded that their findings suggest that reduced mite levels may account for the lower prevalence and titers of DWV-A, rather than other mechanisms of virus resistance in the surviving colonies.

The study is interesting, but caution should be exercised when interpreting the results. In honey bee research conducted in the field, it can be challenging to obtain reliable control groups. Thus, it is uncertain what diseases or conditions were initially present in the colonies at the beginning of the experiment, which could interfere with the intended measurements.

Although the authors measured some pathogens, it is clear that unmeasured pathogens interfere with the replication of another pathogen, making it difficult to draw firm conclusions.

My suggestion is to include the missing information management information and perhaps perform an additional experiment by injecting bees with purified viruses and measuring replication efficiency in these two honey bee populations.

This experiment could provide the authors with a more robust indication of resistance selection mechanisms than those proposed in this manuscript.

Without more information, any conclusion regarding the mechanisms by which these "naturally" surviving honey bees survive varroa mite infestations is pure conjecture.

Reviewer #4: In this study, the authors hypothesized that in honey bee populations that survive V. destructor infestations without treatment, that is, that are "mite survivors," the bees may also have increased tolerance or resistance to viral infections vectored by the mite. The results presented here from analyzing honey bee colonies originating from the "mite-surviving" population and from a "normal", hence, mite susceptible population over three successive seasons in 2013/2014 are interesting, the paper is nicely written, however, some amendments and corrections are still necessary:

l. 16-17: The wording needs to be amended. This reads as if Western honey bees in general are known to survive V. destructor infestation. But this is not the case. It is true that there are reports and research on a few populations that survive mite infestation without treatment, e.g. in France, Sweden, Norway. But - to the best of my knowledge - these "adapted" bees lost their "adaptation" once transferred to other environments/regions of Europe.

l. 19-20: This is a very simplistic view on virus infections and host tolerance. Host induced reduction of viral titers may be one possibility but sustaining higher viral titers may be another one and selection of less virulent virus variants may be yet another one.

Please reword the sentence and make clear that you only test one of several different possibilities here.

l. 25-26: What about DWV-B? That is the relevant, clearly mite associated DWV-variant. Why are you concentrating on the less virulent variant DWV-A? Please explain and justify your choice of variant.

l. 37: Please replace the term "recombinants" by the term "variants", because recombinants are only a subclass of the different variants within the DWV quasispecies.

l. 38: The wording is wrong: It is NOT the virus that developed new transmission routes but a new transmission route for DWV has opened up through the mite.

l. 45-47: The wording is misleading: Actually, small adapted populations have been reported from several European regions and have been analyzed for many years by now to identify the mechanisms behind these so far rather rare evolutionary events. Although these populations are worth to be studied, the sentence, as it reads right now, implies that every honey bee population would become "adapted" if left untreated. However, that such a development (evolution) would automatically occur within 5 years, if the colonies would be left untreated, has never been shown. Moreover, bee colonies from these allegedly mite-tolerant populations were no longer able to survive mite infestation without treatment when brought to other regions in Europe. The question still remains whether it is the bee or the mite or both that adapt to "lack of treatment".

Therefore, I request that the authors rephrase this sentence in a way that it better reflects the current knowledge and does not leave the impression that stopping mite treatment for five years will solve the mite problem and result in mite surviving honey bees all over Europe.

l. 54-55: Especially the Primorsky bees and the Gotland bees were tested for survival without Varroa treatment in Germany and they were no longer able to survive mite infestation without treatment. This has been published and should not be ignored because it is a very interesting aspect regarding the evolution of mite tolerance in honey bee populations.

l. 78: SBV and CBPV are known to be vectored by V. destructor, please read the literature.

That the vector capacity of the mite has not yet been shown for the other viruses mentioned does not mean that the mite is not able to vector them. There are just too few comprehensive studies on viruses in mites. Please correct and rephrase.

l. 172: It would be better to use the same terminology in the text and in the table, i.e. either worker infestation rate or phoretic mite infestation levels. Please correct.

l. 174: Will the supporting information (raw data in VirusVarroa.zip) also be available to the reader? If so, please refer here to this information but remove the names of the beekeepers in the file.

l. 182: (i) Please indicate whether mean +/- SD or +/- SEM is shown. (ii) The number of the colonies differs considerably between spring, summer, autumn. Why? Did colonies die? Did all "surviving" colonies really survive the winter season and how many of the "susceptible" colonies did not survive the winter season? (iii) Acc to MatMeth, the samples were collected in Sept 2013, April 2014 and June 2014. I assume that spring is April 2014, summer is June 2014 and autumn is Sept 2013. The proper order of the seasons in the figure then were autumn, spring, summer. Please rearrange and add the year. It will then become clear that information on winter mortality must exist because the colonies were observed over winter. Please provide this information. It is absolutely necessary for interpreting the data. Are the “surviving” colonies really surviving, are the susceptible colonies really more susceptible (more of them collapsing over winter) than the “surviving” colonies? Plesase add the requested information and consider it in the interpretation of your data.

l. 194: I assume that all colonies that were sampled in April and June 2014 survived the winter 2013/2014. Hence, strictly speaking, all colonies were "surviving" colonies". If this is not the case, please specify and differentiate between truely surviving colonies and replaced colonies in both groups.

I think it is better to keep writing mite-surviving and mite-susceptible colonies because otherwise the impression is given that the other colonies have not survived - but they had, because they were sampled!? Or didn't they? Then please specify.

Please amend the terminology throughout the manuscript.

l. 202, test stat (X2) 15.30: In Table 1, the value 15.03 is given. Please correct. Please explain "(from qpcr negative results)" given in the Table.

l. 203, test stat (X2) 6.75: In Table 1, the value 4.61 is given. Please correct.

l. 203, p value 0.009: In Table 1, the value 0,03 is given. Please correct.

l. 204: Acc to MatMeth, for virus prevalence pooled samples were collected in April and June 2014. Please provide this information somewhere in the legend of the Fig and explain/justify why you combined the results from two sampling time points.

l. 209, figure 4: It must be Fig. 3. Please correct.

l. 217: Please stick to one terminology! For instance, either write “mite-surviving colonies and mite-susceptible colonies” or “colonies from the mite-surviving population and the mite-susceptible population”, but don't switch between susceptible, mite-susceptible, treated etc. colonies.

This is especially relevant if you use a term here (treated) that cannot be found in the Figure or elsewhere.

l. 223: I cannot find results for individual adult workers in Table 1, only for (i) individual worker brood, (ii) pooled adult worker, and (iii) individual viral loads pupae: Likewise searching for X2 33.751 did not yield a result. Please correct.

l. 225: Please be consistent: If the legend of the axis says "wandering mites" don't use "phoretic mites" in the figure legend.

l. 238: For pupae, I can only find data for individual viral loads but not for prevalence in Table 1. Please correct.

l. 240: In Table 1 the term "viral load" is used, here it is "virus abundance". Please be consistent and rather take viral load throughout.

l. 246: Fig. 5 is about prevalence and abundance, not only prevalence. “Abundance” is better named viral load. Please correct.

l. 246: Fig. 5 is supposed to show surviving pupae and associated mites (see l. 241), not adult workers and phoretic mites. Please correct.

l. 247: Fig 5 is a box plot showing more than just means. Please describe correctly what is shown and specify the box and error bars (SD? SEM?).

l. 249: Here the use of "surviving" colonies is especially problematic. Since these data are from autumn, the use of the term surviving implies that the data of surviving colonies from both groups (mite survivjng and mite susceptible) are shown here, but I assume that surviving actually means “mite surviving”. Please correct.

l. 256: Please provide data on what happened over winter. Did the described differences had any influence on the mortality rate? Did the highly infested and virus infected colonies all die or did they survive? This information is lacking but highly relevant. Please add this information.

l. 259: Please also mention and properly discuss the possibility that the experimental design (pools of entire bees were analyzed, only DWV-A was analyzed instead of DWV-B or even better both variants) was not optimal for answering the question.

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PONE-D-22-33692R3Virus infections in honeybee colonies naturally surviving ectoparasitic mite vectorsPLOS ONE

Dear Dr. Oddie,

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.

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Journal Requirements:

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:

I can see that this ms has been reviewed by 4 people at various times and 3 suggest minor revisions. The final and latest one suggested major revision which I am satisfied you have addressed fully in your changes to the final versions and in the response letter. As the new editor for the ms I have carefully read the most recent version and suggest the following minor changes before final acceptance as I am not requesting any more reviews so these are the final changes.

Line 39 remove full stop after ‘many cases’

Line 81 reword (BQCV and LSV),

Line 104 Change 100 to ‘One hundred’

Line 272, maybe add 'as suggested by Grindrod and Martin (2021)'?.

Line 327 remove full stop before refs

Check refs for Latin names and put in italics.

Both these previous studies found lower DWV viral loads in resistant populations so worth adding into your discussion as it strengthens your findings:

de Souza FS, Allsopp M, Martin SJ (2020) Deformed wing virus prevalence and load in honeybees in South Africa. Arch Virol (166): 237–241.

Mendoza Y et al., (2020) Unravelling Honey Bee–Varroa destructor Interaction: Multiple Factors Involved in Differential Resistance between Two Uruguayan Populations. Vet Sci (7):116.

Also, Grindrod and Martin (2021) states in the discussion that “a reduced mite burden also reduces the number of viral vectors” and you come to the same conclusion so maybe worth citing this ref in that context to support your ideas.

Grindrod I, Martin SJ (2021) Parallel Evolution of Varroa Resistance in Honey Bees; a common mechanism across continents? Proc. R. Soc. B 288: 20211375.

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Virus infections in honeybee colonies naturally surviving ectoparasitic mite vectors

PONE-D-22-33692R4

Dear Dr. Oddie,

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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Stephen J. Martin

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

A nice study

Reviewers' comments: