PONE-D-23-03395Knockdown resistance allele L1014F introduced by CRISPR/Cas9 is not associated with altered vector competence for O’nyong nyong virus in Anopheles gambiae”PLOS ONE

Dear Dr. Reimer,

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.

The major weakness of this paper is that the original mosquito strain seems not to be sensitive to the  ONNV that largely question the usefulness of the data, however, thanks to the carefull review that was done by the reviewer, the results seems to deserve to be published we moderation of the conclusion. This is an impact on infection susceptibility rather than on competence efficiency as highlited.

Please submit your revised manuscript by Apr 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,

Pierre Roques, Ph.D.

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

[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?

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: Partly

**********

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

Reviewer #1: Yes

**********

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

**********

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

**********

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 article presented here is well introduced and the objective is clear. The proposed method is original and well justified (use of CRISPR/Cas9 to introduce a SNP into a line, allowing to have two lines with close genetic backgrounds varying only by this SNP). I cannot judge the method of creating the strain by CRISPER/Cas9. I am not a specialist in this field. The creation of the strain having been the subject of another published article, I consider that it has already been evaluated and validated by other reviewers.

Several points should be clarified in the method section, and I propose several modifications to the analysis and presentation of the results, notably not to compare your 7 dpi (reflecting the replication of the virus in the mosquito) with the "0 dpi" (control of the titre of the blood meal provided to the mosquitoes).

From a general point of view, I think that the title and the conclusions should be slightly moderated by the authors. Indeed, the Kis strain (without kdr) infects well (45% of bodies positive at 7 dpi) but does not disseminate and no saliva is positive. This strain is therefore not competent for the ONNV strain used. The same results were observed for the Kis-kdr strain. But can we really speak of an absence of impact of kdr on vector competence, given that the Kis strain is already not competent?

I would therefore say that “kdr has no impact on infection” rather than “on competence”.

Also, be careful to not over-interpret IT results. I agree that it is an interesting addition method, but IT does not measure vector competence (i.e., no measurement of EIP).

specific comments:

Line 32: put kdr in italics.

Line 42: I would delete "modest". It is indeed a SNP, but kdr has a major impact on the survival of Anopheles under insecticide pressure.

Line 58 - 60: Although the cited paper does not determine the role of these kdr-associated SNPs, perhaps specify that they could be associated with a reduced cost on kdr fitness? At present, it is not clear what information this sentence provides.

Line 130-134: the numbers are a bit limited for this type of test but seem sufficient from figure 1.

Line 152: at 0 dpi? specify that this is to check the titre of the inoculum.

Line 171: please describe a little better what is counted here and how the viral titre is calculated or cite a reference describing the method.

Line 190: cite R rather than R studio (type > citation()" in the console).

Line 193: Throughout the paragraph, you can round percentages without decimal (the numbers are small, so such precision is unnecessary). Do not write XX.0%.

Line 214-215: Please round the percentages. The numbers are also very small (n=20).

Line 218: Was the Fisher test done on a proportion calculated on n=20 per class, or only on the infected (n=8-9)? This is not specified in the text.

Line 221-222: I do not understand this comparison. Line 209-212 states that the 0 dpi test was done on whole mosquitoes to check the viral titre of the blood meal. This is a experimental control. It is not possible to compare a whole mosquito assay that has just completed a blood meal (mosquito + blood + virus) with the mosquito body at 7 dpi (reflecting mosquito infection and viral replication). This sentence should be deleted, and Figure 2 should be amended by deleting the 0 dpi and the whole mosquito category.

Lines 229-228: to be amended (see previous comment).

Line 240-246: same comment on percentages to be rounded.

Line 246-247: I think the sentence is incomplete.

Line 248-256: If the statistical tests are not significant, it means that the H0 hypothesis (no difference) cannot be rejected in view of your results. From there, why talk about an increase in viral titre? This section should be moderated in my opinion.

Figure 3: As for figure 2, representing the 0 dpi does not make sense. It is not a variable measured in the experiment, but an initial condition of the manipulation. In my opinion, 0 dpi should be removed from the figure.

Line 297 - 298: ITs are not a measure of vector competence (i.e., EIP), but only of the susceptibility of mosquitoes to infection.

Line 309 - 310: Yes, I agree with you on this point.

**********

6. 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

**********

[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.

Knockdown resistance allele L1014F introduced by CRISPR/Cas9 is not associated with altered vector competence of Anopheles gambiae for o’nyong nyong virus

PONE-D-23-03395R1

Dear Dr. Reimer,

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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

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,

Pierre Roques, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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

**********

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: Yes

**********

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

**********

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: 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 #2: The revised version of the manuscript has addressed all concerned raised by previous reviewers. The revised version has improved the manuscript.

**********

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

**********