PONE-D-23-20467First report of V1016I, F1534C and V410L kdr mutations associated with pyrethroid resistance in Aedes aegypti populations from Niamey, NigerPLOS ONE

Dear Dr. Badolo,

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Additional Editor Comments:

Manuscript should be carefully reviewed and many parts significantly overhauled. All reviewers note questions on methodology- these must be clarified/answered as they are critical for interpretability/repeatability.

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Reviewers' comments:

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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

Reviewer #2: Partly

Reviewer #3: Yes

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

Reviewer #1: I Don't Know

Reviewer #2: I Don't Know

Reviewer #3: Yes

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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

Reviewer #3: 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: No

Reviewer #3: 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: 1. Introduction: 'Data on Aedes aegypti bionomics and insecticide resistance status are still lacking' - There have been various insecticide resistance reports in Aedes aegypti in West Africa. 

2. Methods: Bioassay tests were performed according to World Health Organizations (WHO) protocol using Anopheles diagnostic doses [27] - Justify why Anopheles diagnostic doses were used when Aedes diagnostic doses are available for resistance testing. 

3. Methods: Temperature and humidity during the tests were not stated.

4. Methods: How many samples were used for Allele-specific - PCR for the detection of F1534C, V1016I and V410L mutation

5. Methods: What were the modifications done on the protocol of Granada et al.  2018?

6. Methods, Results, Discussion: 'Elevated activity of oxidase and esteraseenzymes were detected and may also be involved in metabolic resistance to pyrethroids' was mentioned in the abstract but the methodologies and results were not mentioned and discussed. The elevation of activity should not be based on a synergist test. 

7. Results: 'A total of 10 tri-locus combinations genotypes were found out of 27 possible (Figure 4).' - Rewrite to clarify.

8. What was the history of insecticide use in the study areas?

Reviewer #2: 1. Please add numbered lines throughout the manuscript; pointing out the reviewer's comments is difficult.

2. Methodology

CDC bottle bioassays interpretations

It would be necessary to update the interpretation of the resistance status based on the % mortality in the bottle bioassays. Since the authors refer to the WHO 2016 manual for selecting DD, in the same manual, the interpretation of the results in bottle and cylinder of the WHO is established in the same way.

The methodology for detecting kdr mutations is confusing. Please clarify. AS-PCR should be detailed. Please describe the primers used. Are you comparing two methods?

Which mosquitoes were submitted to each method? You said that 50 non-exposed mosquitoes were genotyped by qPCR as described in the methodology but in AS-PCR, what type of mosquitoes were used, and how many?

What do you mean by exposed mosquitoes, alive or dead?

What do you mean by non-exposed mosquitoes, field mosquitoes?

Please explain it the detailed in the methodology.

3. Results

It is confusing the way of expressing the kdr genotypes. Please use two alleles for each locus.

The tri-locus haplotypes CIL, CVV, and CIV were significantly associated with permethrin resistance ( p < 10-6 , p = 0.014 and p = 0.016, respectively) (S3 table).

Those results are in Table 2.

Discussions are limited to investigations done on the continent. There is a vast literature on the association of kdr mutations and pyrethroid resistance worldwide that should be included.

Reviewer #3: 1. The authors state bioassays were conducted as per Ref #29 - Guideline for Evaluating Insecticide Resistance in Vectors Using the CDC Bottle Bioassay. While the stated diagnostic was used for the CDC bottle bioassays, the authors state that mosquitoes were exposed to the insecticides for 1 hour while the diagnostic timepoint for the CDC bottle bioassays at the diagnostic concentration used is stated as 30 minutes in the reference. The authors do not explain why this modification was made in the submitted manuscript.

2. Throughout the manuscript - the authors alternate between "Ae aegypti" and "Aedes aegypti" - please consider improving consistency

3. Knockdown should be defined.

4. Clarification is required on whether knockdown was recorded for a total of 60 minutes throughout the exposure period or if knockdown was recorded after the 60 min exposure.

5- Allele specific PCR - please specify what modifications were made to the published protocol.

6- Statistical analysis - "Mortalities" should be "mortalities"

7. Major consistency and formatting errors in the references section.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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PONE-D-23-20467R1First report of V1016I, F1534C and V410L kdr mutations associated with pyrethroid resistance in Aedes aegypti populations from Niamey, NigerPLOS ONE

Dear Dr. Badolo,

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 22 2024 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,

Nicholas C. Manoukis

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 addressing the comments, I believe the paper is much improved. The last remaining issue is deposit of the study data - which some aggregate information from resistance assays is provided it is not in primary form (by replicate) and I was not able to find allele typing data in the SI (ex. data of alleles for each locus from each of the 50 specimens screened via TaqMan assay- I think only aggregate is provided in Table 1). I believe it would be appropriate to deposit all the raw data from the study in an online database and cite it in the paper.

Table 2 seems to have some typesetting issues in the pdf.

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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 #1: 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 #1: Yes

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

Reviewer #1: 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 #1: 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 #1: 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 #1: The revision for PONE-D-23-20467R1, entitled "First report of V1016I, F1534C and V410L kdr mutations associated with pyrethroid resistance in Aedes aegypti populations from Niamey, Niger"is satisfactory. Thank you for addressing my comments

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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 #1: No

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First report of V1016I, F1534C and V410L kdr mutations associated with pyrethroid resistance in Aedes aegypti populations from Niamey, Niger

PONE-D-23-20467R2

Dear Dr. Badolo,

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.

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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,

Nicholas C. Manoukis

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Thank you for addressing final comments, and congratulations on completing a very nice study!

Reviewers' comments: