Dear Dr. Ross,

Thank you very much for submitting your manuscript "Persistent deleterious effects of an unstable deleterious Wolbachia infection" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.  

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript. 

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Sassan Asgari

Guest Editor

PLOS Neglected Tropical Diseases

Robert Reiner

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: The methods seem appropriate to reach the conclusions. I have only a couple of comments concenring the methodology.

First, the strain described in this paper is not the Drosophila wMelPop, but a very particular wMelPop variant: wMelPop-PGYP (Woolfit et al. (2013) Genome Biol. Evol.). This variant differs from the Drosophila wMelPop is several ways, the most striking being the deletion of the Octomom genomic region, responsible for wMelPop virulence in Drosophila (Chrostek and Teixeira (2015) PLoS Biol.). This region, present in one copy in wMel and wMelCS, is amplified in wMelPop, while wMelPop-PGYP has zero copies of this region (Woolfit et al. (2013) Genome Biol. Evol.).

Lines 280-281 – wMelPop-CLA, the variant passaged through mosquito cell lines, which was introduced into the Aedes aegypti mosquitoes (from mosquito cell lines), already replicates much less than wMelPop in Drosophila melanogaster (McMeniman et al. (2008). Appl. Environ. Microbiol.). It is also a null for Octomom and seems like a more fair comparison for the wMelPop-PGYP assayed here.

Line 300 – I would replace “fecundity” with “egg production”.

Line 112-113 – What was the age of females blood-fed for colony maintenance? wMelPop variants causes a range of pathologies late in life, but young insects seem to be normal.

Figure 4 - The loss of wMelPop from populations crossed to uninfected males seems very fast. Could 2% of transmission failure and ~20% fewer eggs explain an almost complete Wolbachia loss over 4 generations? More detailed method description would help to understand this result – eg. what was the age of the females when they were crossed to uninfected males? Were the eggs stored before hatching to provide advantage to the uninfected individuals? Does the model described in lines 461-470 predict the loss of infection under these circumstances?

Reviewer #2: Line 128 Improper italics on Alb. Also line 130. Check throughout manuscript.

Line 132-138 Belongs in the results not methods. Reader will be confused/skeptical until they get the full explanation.

The methods are generally appropriate although the design is not ideal for comparing fitness and other measures across time under different circumstances, etc. The authors note this on line 275. Regardless, the data are worth examining with a grain of salt.

The authors are experts at all of the insect measures taken here (hatch rates, CI, fitness, etc) and they have used the appropriate statistical analysis.

Reviewer #3: The objectives are clearly articulated, with clear testable hypotheses. There are no concerns about statistics or ethical requirements.

The paper would have been strengthened by the addition of a wMelPop line from a different laboratory, although this may not have been possible. The generality of the conclusions is questionable given that it is only one line/ one colony effectively. Although the experiments are well designed to maximise sample size within that one colony, it is still one colony. The colony was also collected from the field, which may have imposed very strong selection on the original stock. It would have been interesting to compare with a colony that has not left the lab.

Specific comments:

Line 109: by North Queensland material, do the authors mean Wolbachia-uninfected material? Presumably but please specify as North Queensland material can also mean Wolbachia infected.

Methods – please also provide detail on rearing of larvae

Line 120 – what does this mean? Over how many years? Which experiments were done in which years?

Line 128 – please give primers for the wAlb

Line 151 – it would be easier for the reader if the authors denoted wMelPop with + and wMelPop negative populations with a minus

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: The results are clearly presented, I have just a single suggestion in this department:

Fig. 2C – “incompatible females” on the axis is misleading. How about “Females with 0 viable progeny” or “infertile females”? No CI-defining pathologies were scored here, and the incompatibility of these females is a hypothesis.

Reviewer #2: On line 271 are these females or a mix of M and F? Hard to know how this compares to the next few lines. Put the data on the same footing.

Line 288. Same as above. What does ‘severely mean’? Do you have a number to compare to the next sentence?

Reviewer #3: The analysis is consistent, with results generally well presented and clear.

Line 269 – this is fine but it is confounded by the fact that wMelPop mosquitoes were collected from the field, which itself may have resulted in perhaps lower densities or selection in the field for mosquitoes which are better able to tolerate wMelPop infection; it would have been useful if the authors had also been able to access the original laboratory colony?; at the very least the field origin should be discussed

Line 319 – what is meant by population effect here? What exactly is the population?

Line 323 – could the authors clarify if experimenter is person that the mosquitoes are feeding on?

Figure 2: I cannot see the data for uninfected females (2019) in A and B panels - why is this missing?

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: Overall, the conclusions are supported by the data, and the challenges of comparing them with the data from other authors obtained ten years ago are commented on. Minor comments are listed below.

Line 426 – limited, rather than “no” attenuation. Lifespan shortening phenotype, differences in feeding behavior, and number of eggs per female seem to have changed since 2009, improving the outcomes for infected colonies.

Lines 436-438 – Duplication in Octomom is easy to reverse, but it is not the cause of virulence of wMelPop-PGYP1.

Lines 493-494 – or heads or legs?

The last paragraph contains a mix of vector control ideas, including CI-based suppression and population replacement and subsequent crashing. To make this speculation more complete, assessment of the current susceptibility of wMelPop-PGYP to viruses could be proposed. Also, it is difficult to miss the fact that if wMelPop variants are already difficult to maintain in laboratory mosquito colonies, they are likely to be difficult to deploy in the field.

Reviewer #2: Line 501 What do you mean? “deleterious effects and monitoring issues”

Line 505 Explain why "isolated areas"? Do you mean because there won't be invasion from the outside by Wolbachia free? I think to discuss this properly you need rehash the failed releases a bit - what did the failures look like. Possibly better in the discussion.

Reviewer #3: Yes, the conclusions are generally supported by the data presented.

The public health relevance is somewhat tangential.

Comments:

Discussion at lines 472-475: mating-based transmission would surely lead to significant rates of false positive identification only if there was a large proportion of wMelPop uninfected females already in the population, therefore the underlying reason for loss must lie elsewhere.

Discussion at line 482: this appears to be a phenomenon restricted to wMelPop and wMel in this particular study, not wAlbB; has this been observed in Drosophila lines infected with wMelPop?; it is an important observation so would it be feasible for the authors to perform additional experiments with Drosophila flies infected with wMel and wMelPop?

Further point on the Wolbachia mating transmission experiments: females that have wMePop transferred via seminal fluid would be weakly positive in qPCR of whole bodies, as the abdomen only qPCRs are close to Cq of 29 (line 394). Generally values close to 30 in qPCR should be suspect (for any pathogen) and require repetition. Therefore, it should be possible to screen out weakly positive females from a laboratory line.

Line 493: unless adults are held singly post hatching from the field it seems somewhat difficult to implement this recommendation of only screening unmated adults

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: I recommend minor revisions and suggestions for modifications are listed in the sections above. The most important change required for publication is clarification which Wolbachia strain was under investigation here.

Reviewer #2: (No Response)

Reviewer #3: No editorial suggestions, as the writing is very clear and the figures are fine.

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: The manuscript “Persistent deleterious effects of an unstable deleterious Wolbachia infection” is a thorough analysis of the phenotypes of a pathogenic Wolbachia strain transferred from Drosophila melanogaster to mosquito cell lines to Aedes aegyptii over ten years ago. Important life history traits have been measured and compared to historical data. This approach detected only a small attenuation of deleterious Wolbachia phenotypes over time.

Reviewer #2: This is an interesting study and that few if any other authors could do, since almost no labs have this strain. The authors also have access to some historical data for comparison. Their findings may help revive the use of wMelPop that has largely been dismissed as unworkable for the field. There are large caveats, however, associated with comparing fitness measures years apart.

Wolbachia transmission by males transmission is a novel contribution to the field, that other researchers may wish to look out for.

I am not entirely convinced of the author's premise that rearing in the laboratory should have selected for less virulent Wolbachia. Life shortening does not kick in until late in life. Most labs, knowing the line is sensitive, rear it carefully. That means taking only the first or second gonotrophic cycle (well before aging or fitness effects kick in) and and not leaving eggs dried down for too long. I think the authors need to give some room for this explanation as to why the virulence has not changed.

Additionally, to make this more than an intellectual exercise, the authors need to do a better job of explaining how, despite failure to spread in Vietnam and Australia, that the wMelPop strain could still be used in the field. Expand/explain. Also in the discussion. And what about temperature (26 in the lab vs much hotter in the field). This strain may be particularly affected.

The writing is largely clear. Just some areas that need to be expanded for the uninitiated or better fleshed out with respect to their arguments.

Reviewer #3: In general the paper is well written and the studies are adequate. I have two principal concerns as discussed above:

1) we are seeing patterns largely restricted to one colony;

2) one of the most interesting interesting findings - of male to female transmission of wMel and wMelPop during mating - isn't explored further through additional experiments; is this specific to Aedes aegypti or does it occur in Drosophila? what happens to Wolbachia in the spermathecae? there is no dissection of spermathecae to look at viability or what happens to the bacteria over time post mating.

I should note that wMelPop deleterious effects are also observed after many, many generations in Drosophila. This isn't really discussed anywhere.

The title suggests wMelPop is unstable - yet it seems very stable in general over the decade being investigated, except for one particular instance when the infection was lost. Therefore it is unclear what the authors mean by this.

--------------------

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

Figure 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. 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 us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, PLOS recommends that you deposit laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see http://journals.plos.org/plosntds/s/submission-guidelines#loc-materials-and-methods

Dear Dr. Ross,

We are pleased to inform you that your manuscript 'Persistent deleterious effects of a deleterious Wolbachia infection' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Sassan Asgari

Guest Editor

PLOS Neglected Tropical Diseases

Robert Reiner

Deputy Editor

PLOS Neglected Tropical Diseases

***********************************************************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: The methods are adequately described.

**********

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: The Results are adequately described.

**********

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: Yes

**********

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: Yes

**********

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: This paper describes a very nice piece of research and the authors have addressed all of my comments.

Reviewer #2: The authors have addressed the concerns.

Reviewer #3: The authors have addressed many of the critical points of the paper.

**********

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: Yes: Ewa Chrostek

Reviewer #2: No

Reviewer #3: No