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PONE-D-24-03915Susceptibility status of Aedes aegypti (Diptera: Culicidae) to public health insecticides in Southern Afar region, EthiopiaPLOS ONE

Dear Dr. Seid,

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 Apr 14 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.

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PLOS ONE

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Additional Editor Comments:

All reviewers in felt that the manuscript lacked sufficient detail to fully evaluate. In particular they requested more detail on the methodology, including the sampling methods and statistical analyses performed. They have also listed additional specific changes that should be addressed prior to resubmission.

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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: Yes

Reviewer #2: Yes

Reviewer #3: No

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

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: No

**********

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: No

Reviewer #3: No

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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

**********

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 authors conducted a study to evaluate the susceptibility status of Aedes aegypti (Diptera: Culicidae) to public health insecticides in the Southern Afar region, Ethiopia. The findings revealed that populations of Ae. aegypti in the study area showed phenotypic resistance to carbamates such as propoxur and bendiocarb. However, they were susceptible to certain pyrethroids, including permethrin, alphacypermethrin, and deltamethrin, as well as the organophosphate insecticide primiphos-methyl. The study provides compelling evidence of an emergence of insecticide resistance within Aedes aegypti populations in the country. While the study provides valuable insights into the emergence of insecticide resistance within Aedes aegypti populations, the writing of the paper requires revision to enhance its overall quality.

Major comments

1. What criteria underlie the selection of insecticides in this study, specifically the inclusion of three pyrethroids and two carbamates? Additionally, are these insecticides currently in use for public health purposes?

2. According to the WHO guideline cited in the methodology section, the recommended discriminating doses for susceptibility tests of Aedes mosquitoes using pyrethroids are 0.25% Permethrin, 0.03% alpha-cypermethrin, and 0.03% deltamethrin. However, it appears that different discriminating doses of pyrethroids were used for the study. Why it is different?

3. Why only phenotypic resistance studied? Was there a specific reason for not investigating the resistance mechanisms at play in the mosquito populations?

4. How did you identified the Aedes mosquitoes used for the susceptibility tests were all Ae. Aegypti?

5. How and why was the association between pyrethroid resistance in the study area and agricultural practices established?

Minor comments

1. Are Ae aegypti and Ae bromeliae the only two species these the only two Aedes species existing in the country? For example Ae. vittaus and Ae. africanus also exist in the country.

2. Why the Knockdown effect of permethrin and alpha-cypermethrin not elaborated like that of deltamethrin?

3. The vertical axis maximum value should not exceed 100 (Fig 2). This works for all graphs

4. The susceptibility test of alphacypermethri in Awash Arba town repeated or not?

5. Why is integrated vector control strategies recommended without supporting data from the study findings?

6. How are pyrethroids currently utilized in the control of vectors?

Suggestions

Suggestions and comments are provided in the main document

Reviewer #2: In recent years arboviral diseases have emerged as a major public health problem in Africa, calling attention for implementing effective disease control approaches, especially the insecticide-based interventions. Effective chemical interventions require an up-to-date knowledge of insecticide resistance in vector species, therefore, this study is very relevant for Ethiopia.

I suggest improvement in the manuscript and offer the following comments and suggestions:

Overall: correct numerous typos and improve English language; ensure the references cited in the text match with those in the References list.

Abstract:

- Background: In the last sentence of the “Background”, mention names of the insecticides instead of stating “public health insecticides”.

- Methodology: Modify the sentence “Mortality was recorded after 24 hours” to “The mortality in mosquitoes was recorded 24 hours after 60 minutes of exposure”. Delete the abbreviation “KDT” for knockdown rate since later you have used KDT for the knockdown time.

- Results: In this sentence KDT95 implies Knockdown time, so modify the sentence to “Aedes aegypti from Awash Arba showed high knock down time (KDT95 26.5 minutes) to deltamethrin ….”.

- Results: Rename “alphacypermethrin” to “alpha-cypermethrin”, “delthamethrin” to “deltamethrin” and “pirimiphos methyl” to “pirimiphos-methyl” throughout the manuscript (in text, figures, and tables).

- Conclusion: delete “control” at the end of the second sentence. Mention the insecticide discriminating concentration for each insecticide impregnated paper used. Also state that further studies are required to confirm resistance to alpha-cypermethrin.

Introduction

- Correct typos: change “Ae. albopictus[11, 12].” to “Ae. albopictus [11, 12].”, “Ae .aegypti” to “Ae. aegypti”, “Fenitrothion” to “fenitrothion”.

Material and Methods

Study sites:

- Correct many typos (e.g. “820masl” to “820 masl” and so on.

- Exactly what insecticide spraying is practiced in the study areas for the control of malaria and arboviral diseases to give an indication of insecticidal interventions used.

- Figure 1 is not mentioned in the text under the study sites.

Insecticide susceptibility:

- Reference # 16 attributed to the WHO procedure is incorrect. Please recheck correctness of all references used in the text.

- Correct numerous typos such as “delta-methrin” to “deltamethrin”.

- A correct number of mosquitoes (100) were exposed in the treatments but not in the control (40 instead of 50). This is a slight deviation from the WHO tube test procedure.

- “fed on 10% sugar solution” is an incorrect statement; instead, state “were offered cotton wool pads soaked in 10% sugar solution”.

Data analysis

- Correct this statement “suspected for resistance were with a mortality 90-98%” to “suspected of resistance were with a mortality of 90–97%”.

- Reference # 34 is incorrect; the correct reference should be: WHO (2022). Manual for monitoring insecticide resistance in mosquito vectors and selecting appropriate interventions. https://iris.who.int/handle/10665/356964.

- Correct the abbreviations “50% (KD50) and 95% (KD95)” to “50% (KDT50) and 95% (KDT95).

Results

- In Table 1 header, change “KDT50±SE” to “KDT50 ± SE” and “KDT95±SE” to “KDT95 ± SE”

- Correct numerous typos in the text and figures.

Discussion

- Is there any publication supporting the following statement “Ae. aegypti is generally considered as exophilic, exophagic …....”.

- While KDT95 for deltamethrin in one town was higher, the tube test does confirm full susceptibility of Ae. aegypti.

- Some of the WHO insecticide discriminating concentrations for Aedes aegypti adults have changed in 2022 following the publication of the above said WHO manual based on a WHO multi-centre study in which the authors’ institution was a collaborator, as follows:

o deltamethrin 0.05% to 0.03%

o permethrin 0.75% to 0.40%

o bendiocarb 0.1% to 0.2%

o pirimiphos-methyl 0.25% to 60 mg/m2

o alpha-cypermethrin 0.05% : no change

o propoxur 0.1% : no change

Since Ae. aegypti was found resistant to bendiocarb 0.1% concentration while its concentration has been increased to 0.2%, discuss that further tests should be conducted with 0.2% impregnated papers to confirm resistance.

References

- I did not check correctness of each reference cited, but I noticed at least two incorrect references, therefore authors should recheck all references cited.

Reviewer #3: The authors have a good reason for conducting this research, the manuscript is well organized and written clearly enough to be accessible to non-specialists. However, lacks a detailed description of the method used to collect larvae and pupae. It is understood that they collected in three places in the South of the Afar region of Ethiopia, where vector-transmitted infections occur, and that they did so from July to October 2022. However, they do not specify how many times each of the locations were visited during that 3 to 4 months for the collections to obtain a variety of samples, nor how many breeding sites they found from where they collected the specimens. The latter is important because it is not known if the offspring they collected come from the same progeny or from few of them, hence the importance of collecting in different breeding sites and at different collection times in the same place. The material collected on the same day at various breeding sites of a locality must be combined so that there is mixing of the genes and thus the results of resistance to insecticides may correspond to several parental mosquitoes originated from the collected site. It is important to describe the number of samples collected on the dates in each of the locations. If possible, describe the number of larvae and pupae collected or an approximate number.

By other hand, it is not specified how many experiments were carried out for the tests with each insecticide. It is understood that only 100 mosquitoes were used and 40 for two control tubes, so that makes only a test. However, at least two other replicates of collections separated in time must be carried out for the same collection location. In this way you can work with an average mortality for more precise data on the locality that is being collected, and if diferences in mortalities are found in the three collections could be a good motive for discussion in this manuscript to enrich the discussion.

Discussion, 3rd paragraph, at the end: reffering to the phrase "the findings observed in the present study were lower than results reported in Sudan, Tanzania and Nigeria" it would be good to add if those from Sudan, Tanzania and Nigeria were reported as resistant or only tolerants to deltamethrin, and maybe is good to mention their KDT50 and 95 values, so we can have an idea for comparison.

A sentence could be added in this section about the resistance mechanisms that could be present in the mosquitoes detected resistant to propoxur and bendiocarb, but not to organophosphate. It is known that the two groups of insecticides share the same site of action, and that if resistance to both groups has been diagnosed, such a situation could explain a resistance based on a point mutation in the site of action, however this is not the case.

Minor corrections:

Introduction, 3rd paragraph. "Fenitrothion" cjhange to "fenitrothion"

Material and Methods: 1st paragraph. "Were" change to "Werer"

Insecticide susceptibility paragraph: "delta-methrin" change to "deltamethrin" and "bendio carb" change to bendiocarb"

Results, 1st paragraph, Table 1. "Ae. Aegypti" change to "Ae. aegypti"

Discussion, 4th paragraph. "propxur" change to "propoxur"

Conclusion. "perimethrin" change to "permethrin"

Check the double spaces between words throughout the writing and correct them to one space.

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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: Yes: Eba A.Simma

Reviewer #2: Yes: Dr Rajpal Singh Yadav

Reviewer #3: No

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Attachments

Attachment

Submitted filename: PONE-D-24-03915.pdf

Attachment

Submitted filename: PONE-D-24-03915_MS.pdf

PONE-D-24-03915R1Susceptibility status of Aedes aegypti (Diptera: Culicidae) to public health insecticides in Southern Afar region, EthiopiaPLOS ONE

Dear Dr. Seid,

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. In particular, Reviewer 1 still felt like there was insufficient detail in describing the justification to use non-standard doses of insecticide for the susceptibility tests, and Reviewer 2 felt like there were still too many grammatical errors in the draft. Please ensure these comments are addressed in any subsequent draft versions.

Please submit your revised manuscript by Jul 06 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,

James Colborn

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.

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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: (No Response)

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 #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

**********

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

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 #1: No

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 #1: The authors have responded to numerous comments, but there is still a gap in explaining why the recommended discriminating doses for susceptibility tests of Aedes mosquitoes, as outlined in the WHO guideline cited in the methodology section, were not followed. This discrepancy must be addressed transparently in the methodology, providing genuine explanations for the deviation. The authors may suggest that the failure to adhere strictly to the WHO-recommended discriminating doses might be due to logistical constraints, such as the unavailability of insecticide-impregnated papers at the time of the trial. This practical impediment necessitated a pragmatic approach where alternative doses were explored. Therefore, the methodology section should clearly state the reasons for the deviation from the recommended doses.

Reviewer #2: I still noticed many grammatical errors and some typos, and have mentioned them in red fonts at some places in the PDF file to draw attention of the authors to carefully check the text.

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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: Yes: Eba Alemayehu Simma

Reviewer #2: Yes: Dr Rajpal Singh Yadav

**********

[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.

Attachments

Attachment

Submitted filename: PONE-D-24-03915_R1_reviewer_2.pdf

PONE-D-24-03915R2Susceptibility status of Aedes aegypti (Diptera: Culicidae) to public health insecticides in Southern Afar region, EthiopiaPLOS ONE

Dear Dr. Seid,

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. Although the reviewers felt that their major comments had been addressed, there were still many grammatical errors that need to be addressed prior to publication. The reviewers recommended having a native english speaker review for content. Please ensure the grammar and language are of publication quality in the final submitted version.

Please submit your revised manuscript by Jul 27 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,

James Colborn

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.

[Note: HTML markup is below. Please do not edit.]

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

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 #1: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

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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

Reviewer #2: 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

Reviewer #2: No

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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 authors have addressed my concerns, and I appreciate their thorough response. They have justified their use of the discrimination doses of insecticides according to WHO recommendations for testing insecticide susceptibility in Aedes mosquitoes. This indicates that they employed the recommended doses for testing Anopheles mosquitoes as outlined in the standard operating procedure for testing insecticide susceptibility in adult mosquitoes using WHO tube tests (WHO, 2022), rather than following the guidelines for monitoring and managing insecticide resistance in Aedes mosquito populations: Interim Guidance for Entomologists (WHO, 2016). Therefore, I suggest updating the reference accordingly.

Reviewer #2: I have inserted numerous corrections in the PDF file. Authors should carefully look at the suggested corrections to further improvise the manuscript.

Additionally, two specific comments are as follows:

1. At the end of many lines, words are broken e.g. on page 1 lines 13, 14, 21, 24. Please correct these errors in the text throughout.

2. Page 10: line 6: Aedes aegypti is generally endophilic and endophilic whereas the authors have stated that they are generally exophilic and exophagic. Can they support this statement with any published information?

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Reviewer #1: Yes: Eba Alemayehu Simma

Reviewer #2: Yes: Dr Rajpal Singh Yadav

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Attachments

Attachment

Submitted filename: Comment.docx

Attachment

Submitted filename: a. PONE-D-24-03915_R2_reviewer_comments.pdf

Susceptibility status of Aedes aegypti (Diptera: Culicidae) to public health insecticides in Southern Afar Region, Ethiopia

PONE-D-24-03915R3

Dear Dr. Seid,

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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James Colborn

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 #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

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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 authors have addressed the comments in the revised version of the manuscript. It is in now in a good form and ready for acceptance for publication.

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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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