Dear Dr. Yang,

Thank you very much for submitting your revised manuscript "Sex-specific distribution and classification of Wolbachia infections and mitochondrial DNA haplogroups in Aedes albopictus from the Indo-Pacific" for consideration at PLOS Neglected Tropical Diseases. Your manuscript was reviewed by the editorial board and by the independent reviewers. Based on the reviews, we are likely to accept this paper for publication, providing that you modify the manuscript according to the additional recommendations made the reviewers.

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[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).

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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,

Pattamaporn Kittayapong, Ph.D.

Associate Editor

PLOS Neglected Tropical Diseases

Louis Lambrechts, Ph.D.

Deputy Editor

PLOS Neglected Tropical Diseases

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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: 1. Mitochondrial COI as a DNA barcode and The trimmed 623 bp sequence was analysed with Geneious 9.18 software (Kearse et al., 2012) to investigate SNP variation among samples. These methods may not be sufficient to identify species.

2. ML sex classification with ddRAD-seq ，How reliable is this method?

3. Wolbachia detection via qPCR assay，What are the advantages and confirmations of this method compared with general PCR.

Reviewer #2: (No Response)

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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: Figure 6. wAlbA density and wAlbB density in individual females (A-F) and males (G, H) across populations.Why do some have linear correlation and some don't? What is the significance of this difference? Why?

Test set confusion matrix for sex prediction of mosquitoes based on SNP markers identified from a Torres Strait dataset but tested against sexed samples from other populations. How accurate is the prediction accuracy of SNP markers?

Reviewer #2: (No Response)

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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: modification

Reviewer #2: (No Response)

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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)

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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: Sex-specific distribution and classification of Wolbachia infections and mitochondrial DNA haplogroups in Aedes albopictus from the Indo-Pacific， it is Scientifically significant.

Reviewer #2: Yang et al analysed population genomic data and tested for cryptic species in 160 Ae. albopictus sampled from 16 locations across Indo-Pacific region and found that Ae. albopictus can partitioned into three groups, but with no evidence of cryptic species. They also show wAlbA and wAlbB infections showed markedly different patterns in the sexes, with most population carrying superinfection and males having more single wAlbB infection than females. These results provide important background information for developing Wolbachia to control dengue transmitted by this mosquito vector. The manuscript is well written. Below are some minor comments.

Figure 4, What is the host gene used to normalize the Wolbachia copy? This information should be included in method. There is no PNG (the highest one) density in Fig.4A. Statistic information should be provided in the figure.

Why not validate the sex through assay of Nix gene by PCR? Is this more straightforward and easier than ddRAD-seq?

The age of sample can be a big factor affecting the variation of Wolbachia infection between sexes as male can lose wAlbA with aging. This probably is more important reason than others in term of higher rate of wAlbB in males than females in the tested samples.

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Reviewer #1: No

Reviewer #2: No

Figure Files:

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References

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.

Dear Dr. Yang,

We are pleased to inform you that your manuscript 'Sex-specific distribution and classification of Wolbachia infections and mitochondrial DNA haplogroups in Aedes albopictus from the Indo-Pacific' 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,

Pattamaporn Kittayapong, Ph.D.

Associate Editor

PLOS Neglected Tropical Diseases

Louis Lambrechts, Ph.D.

Deputy Editor

PLOS Neglected Tropical Diseases

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