Response to ReviewersRevised Manuscript with Track ChangesManuscript

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-4304-636XX

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-1765-0002

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Reviewers' comments:

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: See attached comments.

Reviewer #2: Please see my general comments.

Reviewer #3: (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: See attached comments.

Reviewer #2: Please see my general comments.

Reviewer #3: (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: See attached comments.

Reviewer #2: Please see my general comments.

Reviewer #3: (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: Please see my general comments.

Reviewer #3: (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: See attached comments.

Reviewer #2: General Comment: This study evaluated a salivary gland escape barrier in several populations of Ae. aegypti. The presence of this genetically inherited barrier to transmission may be incorporated into disease prevention strategies. I am glad to see that the authors used virus detection rather than RT-PCR to detect ZIKV. The mere detection of viral RNA does not mean that any infectious virus was present. Similarly, I am glad to see that the authors used “days post-infectious blood meal” rather than the more commonly and incorrectly used “days post infection.” If it was days post infection, then all of the mosquitoes would have been infected. Overall, this is a very informative study and involved a lot of well-designed work. Below are several suggested changes. Most are merely style/format corrections.

Specific Comments:

1. Lines 81-82: Why establish WNV, JEV, and YFV if they are not used again?

2. Line 86: As this is the first time that Aedes aegypti is mentioned in the body of the paper, shouldn’t it be written out?

3. Line 100: Should “This immunity alone…” be “Thus, immunity alone…?”

4. Line 114: Shouldn’t “human” be “humans” as it is referring to more than a single person. More importantly, as this is referring to SGEB in general, shouldn’t this be “vertebrate hosts…?”

5. Line 115: This should be in the past tense, so it should be “we investigated the…” Similarly, on line 117, “explore” should be “explored…” and on line 120. “we aim to…” should have been “we aimed to…”

6. Line 255: Should “the transmission potential NAK population” be “the lower transmission potential NAK population?”

7. Line 260: As “fifty” is not the first word in the sentence and is greater than 10, it should be “50 males…”

8. Lines 331-335: Unless they are the first word in a sentence, numbers of measurement should not be written out and numerals should be used, e.g., 3 hours or 3 days. Note, this was correctly done on line 362 and others.

9. Line 351: As ZIKV was established on line 80, there is no reason to reestablished here.

10. Lines372-377: Yes, looking at Figure 1, I can see that the ability of the NAK strain to transmit ZIKV was significantly lower than that of either of the other two populations. You might want to modify the sentence to, “…varied significantly with transmission rates for the NAK being significantly (p < 0.0001) lower than that for the DMSC (Figure 1).”

11. Line 474-475: Why reestablish dpibm as it was established on line 362.

12. Lines 489-490: While it is easy to calculate, viral titers to a hundredth of a log are usually not accurate and claim a degree of accuracy not supported by data. Therefore, I suggest that you change these to 5.5-5.6 log and 5.9-6.1 log…

13. Lines 590-591: Yes, the Aaa are more likely to feed on humans than the Aaf, and thus, are much more likely to be involved in the transmission of anthropomorphic viruses such as dengue and chikungunya. See the recent studies by Agha et al. Entomological assessment of dengue virus transmission risk in three urban areas of Kenya. PLoS Negl Trop Dis. 2019 Aug 23;13(8):e0007686 and Agha et al. Vector competence of populations of Aedes aegypti from three distinct cities in Kenya for chikungunya virus. PLoS Negl Trop Dis. 2017 Aug 18;11(8):e0005860. Not only is transmission efficiency critical, but feeding preference is also important

14. References: These need to be formatted properly.

a. Only the first word and proper nouns in a reference title should be capitalized. See references 5, 15, 24 and many others.

b. Genus and species names should be in italics. See references 2, 3 4, and others.

c. Why is the name of the editor of some of the PLoS journal papers included? See references 5, 12 and others.

d. Shouldn’t PLOS ONE or PLoS ONE be PLoS One? See references 5, 12, and others? You need to be consistent.

e. Why is the title of reference 28 in all caps?

Reviewer #3: To the Authors:

The manuscript entitled: “Natural salivary gland barrier curtails Zika virus transmission in Thai Aedes aegypti” by Sartsanga et al. is well written and clear. The study reports the discovery of a heritable salivary gland barrier, which may be used to form the basis of an alternative arbovirus control strategy using population replacement strategies as well as a tool to explore mosquito arbovirus interactions. Nice work.

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Reviewer #2: No

Reviewer #3: No

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Attachments

Attachment

Submitted filename: Sartsanga PLoSNTD 2025.docx

Dear Dr. Jupatanakul,

We are pleased to inform you that your manuscript 'Natural salivary gland barrier curtails Zika virus transmission in Thai Aedes aegypti' 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,

Nikos Vasilakis

Section Editor

PLOS Neglected Tropical Diseases

Álvaro Acosta-Serrano

Section Editor

PLOS Neglected Tropical Diseases

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-4304-636XX

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-1765-0002

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

The revision has significantly improved the quality of the manuscript and critically addressed thoughtfully Reviewer's 1 concerns.

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