PONE-D-19-29030

Resting behaviour of malaria vectors in a highland and a lowland site of western Kenya: Implication on malaria vector control measures

PLOS ONE

Dear Dr Afrane,

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 paper has now been seen by two reviewers. Both require some rewriting. Reviewer 1 suggests some extensive rewriting of the discussion but also some important rewording and structuring of the results. I agree with reviewer 1. Also please do not report values like '57.3' - only two digits are meaningful, so instead such a value should simply read 57. This in addition to the comments by the reviewers will make the paper an easier read.

We would appreciate receiving your revised manuscript by Dec 28 2019 11:59PM. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Friedrich Frischknecht

Academic Editor

PLOS ONE

Journal Requirements:

1.  When submitting your revision, we need you to address these additional requirements.

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

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for stating in your Funding Statement:

" This study was supported by grants from the National Institute of Health (R01 A1123074, U19 AI129326, R01 AI050243, D43 TW001505)

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.".

i) Please provide an amended statement that declares *all* the funding or sources of support (whether external or internal to your organization) received during this study, as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now.  Please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement.

ii) Please include your amended Funding Statement within your cover letter. We will change the online submission form on your behalf.

3. We note that [Figure 1] in your submission contains [map/satellite] images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth). For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:

1. You may seek permission from the original copyright holder of Figure [1] to publish the content specifically under the CC BY 4.0 license. 

We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:

“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”

Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission.

In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].”

1. If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.

The following resources for replacing copyrighted map figures may be helpful:

USGS National Map Viewer (public domain): http://viewer.nationalmap.gov/viewer/

The Gateway to Astronaut Photography of Earth (public domain): http://eol.jsc.nasa.gov/sseop/clickmap/

Maps at the CIA (public domain): https://www.cia.gov/library/publications/the-world-factbook/index.html and https://www.cia.gov/library/publications/cia-maps-publications/index.html

NASA Earth Observatory (public domain): http://earthobservatory.nasa.gov/

Landsat: http://landsat.visibleearth.nasa.gov/

USGS EROS (Earth Resources Observatory and Science (EROS) Center) (public domain): http://eros.usgs.gov/#

Natural Earth (public domain): http://www.naturalearthdata.com/

4.

We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For more information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.

In your revised cover letter, please address the following prompts:

a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent.

b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories.

We will update your Data Availability statement on your behalf to reflect the information you provide.

5. Please ensure that you refer to Figure 1 in your text as, if accepted, production will need this reference to link the reader to the figure.

6. Your ethics statement must appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please also ensure that your ethics statement is included in your manuscript, as the ethics section of your online submission will not be published alongside your manuscript.

[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

Reviewer #2: Yes

**********

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

Reviewer #1: No

Reviewer #2: 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: No

Reviewer #2: 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

Reviewer #2: 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 study by Afrane and colleagues investigates resting behavior, host preference and Pf infection of malaria vectors in two settings of western Kenya. The paper is in parts fairly well written; in other parts very repetitive and unstructured. The methodologies are routine in field entomology, the findings are robust and valuable. They support a large body of evidence of high levels of resistance in the area.

The paper would benefit from some serious re-writing but the paper The intro is gives detailed background information without being overly long. It is also well written and would not need much work. The Results, on the other hand, can be greatly shortened. A lot of details are provided in the long first paragraph. All percentages that are reported should come with some indicator of precision. Either a confidence interval around the proportion or mentioning numerator and denominator (n/N). The discussion section is not very focused and would benefit from re-writing. It could, without problems, be shortened with 25-33% to make the paper more attractive, focusing on time trends in resistance or feeding behavior in Western Kenya and the implications for control. The structure of the discussion can also be greatly improved: dealing with each of the important elements in a single clearly structured paragraph (instead of mixing all messages throughout the discussion) would really improve this part of an, otherwise valuable, paper.

SPECIFIC COMMENTS

The abstract lists many findings without a very clear selection or any statistical tests. It could be improved by presenting only the key results: sporozoite rate, tube assay (?), resistance genetic findings, and present them in a bit more detail and with stats.

Abstract. Trapping methods may be listed in the abstract.

Abstract: confidence intervals or details of the underlying number of mosquitoes (n/N) should be mentioned in the abstract. Also a statement ‘None of the outdoor collected mosquitoes in Kisian tested positive for sporozoite infections’ can only be interpreted if the number of examined mosquitoes is given.

Abstract: .’Vgsc-1014S was observed at a slightly higher frequency in An. gambiae s.s hereafter(An. gambiae) resting indoor than outdoor (89.7 vs 84.6% and 71.5 vs 61.1%) in Bungoma and Kisian respectively’ is unclear. Vgsc is not defined in the abstract. And the estimates are possibly not statistically significant. If so, the finding should receive less prominence in the results section.

Abstract: ‘high densities of insecticide-resistant… [mosquitoes]’ in the conclusion can only be presented if also mosquito burden is presented in the abstract. What was the average number of mosquitoes per trapping night? It is really high? Or did the authors (simply) want to say that among mosquitoes there was a large proportion of resistant mosquitoes?

Intro, line 112. ‘Insecticide resistance could make mosquitoes respond numerically…’ is unclear to me. What is intended? This sentence could be simplified, probably by splitting the sentence in two.

Methods, line 152. Clay pots are notoriously difficult to standardize. A useful reference is Van den Bijllaardt, Acta Tropica 111 (2009) 197–199 where very detailed procedures are explained. For the current method, more details should be given on where traps were placed and how sampling was done. This applies to all trapping methods, not just clay pots.

Methods, line 195/196. The authors may want to comment on reports from SE Asia (Coosemans and team) that there is cross-reactivity in ELISA results in mosquitoes that fed on cattle blood. This might have contributed to the fairly high sporozoite rates observed in this study.

Methods: the dry versus wet season sampling is useful but needs some indication to where in the season samples were taken. How many weeks after start of the rains was ‘wet season’? How dry was the dry season?

Results, line 218-220. ‘Overall, the proportion of Anopheles species resting indoors was significantly higher by 82.4% than outdoor location 17.6% across the study sites (z = -8.47, p < 0.0001) ‘. This is very imprecise. I am not sure what is compared here. Is it the proportion of anophelines among caught mosquitoes? Or is it the absolute number of anophelines caught indoors vs outdoors? If so: is this appropriate? The sampling surface was probably very different. Only sporozoite rates and blood meal sources can be compared indoors vs outdoors since trapping methods differed profoundly.

Similarly, line 232 is unclear ‘There were more An. gambiae and An. funestus resting indoors than outdoors (80.7 vs 19.3% and 97.8 vs 2.2% respectively; X2 =122.96, df = 2, p < 234 0.0001). ‘ How can this be a percentage? Is it the percentage of all gambiae that was caught indoors? I find these presentations very difficult and dangerous since the sampling intensity was so different. I would advise to simply present for indoor and outdoor sampled mosquitoes separately: numbers per trapping night, species composition, sporozoite rates, resistance phenotype, genotype, blood source. Compare the % sporozoite positive between indoor vs outdoor but do not compare absolute numbers! Reconsidering what the authors really want to present would also allow this very long and difficult first section of the results section to be shortened considerably. That would really improve the paper. At the moment, it is the weaker part of the paper. Make use of the display figures (tables and figures) and do not add too much detail on proportions etc in the main text.

Throughout the results, give n/N or confidence intervals. Percentages without any indication of precision (either numbers of confidence intervals) are not very informative.

For blood sources, the authors make statements on higher vs lower between species and indoor vs outdoor. Present statistics and indicate precision of estimates. If a finding is not statistically significant, do not claim differences.

I am puzzled by Figure 2. There seems to be much less variation in mosquito exposure than is typically reported. Are these only successful trapping efforts or are zeroes (no mosquitoes caught) also included in the figure? These findings typically follow a Poisson or Negative Binomial distribution. The current presentation suggests a normal distribution with very few negative trapping events. That would be unusual. It is important to explain this and present the % of traps with at least one mosquito and the distribution of mosquitoes in positive traps.

Figure 3: adjust title. This figure doesn’t present abundance. Only composition.

Discussion

Line 331 ‘…leading to increased vector-human contact and ongoing malaria transmission in the region despite the high coverage and usage of LLINs’ is not an appropriate sentence. This was in no way proven by the data. It is pure speculation and should be recognizable as such (and probably placed somewhere later in the discussion and not in the crucial first paragraph).

As indicated above, the discussion is slightly unstructured. Findings and interpretations are mixed. It would be recommended to really work in clearly structured paragraphs that could explain in detail and with good references:

Paragraph 1. Conclusion. Pretty much as it is now.

Paragraph 2. Species composition in the areas. How does this relate to other findings?

Paragraph 3. How does resistance differ between species?

Paragraph 4. The resistance phenotype and genotype indoors and outdoors. Did this change compared to earlier studies in the area? Is this an indicator that biting increases indoors as a consequence of increased resistance (which again could be because of increased pressure due to widescale use of interventions)

Paragraph 5. Sporozoite rate per species among outdoor mosquitoes and for wet versus dry season. How does this compare to other studies? What does it mean for what vector is most important for transmission in the region? Include the blood meal preference in this section. Its relevance lies in this (explaining human biting and thus vector importance)

Paragraph 6. Final conclusion.

Reviewer #2: This manuscript describes the composition, bionomics and epidemiology of Anopheles malaria vectors in Western Kenya. The work is technically well done. The results are valuable and interesting, because malaria transmission continues and has even rebounded in parts of Africa despite decades of vector control.

The manuscript is well written and contains thoughtful analysis. It can be published after only several very minor points that should be corrected, as follows.

line 199 (and other locations): "anopheline" is an adjective, not a Latin name, and should not be italicized or capitalized.

line 252: unclear wording where it says "High resistance levels" of 36.6% or 65.5%, but the numbers refer to the 24 hour mortality rate, not actually resistance. Please reword this.

line 319, 320 (and other locations): be consistent about Latin names. Names should be spelled out at first use, and thereafter genus is abbreviated.

line 352 (and other locations): be consistent about "kdr", which is not capitalized, but here is shown as Kdr

**********

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

Reviewer #2: 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 to be viewed.]

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 us at figures@plos.org. Please note that Supporting Information files do not need this step.

Resting behaviour of malaria vectors in  highland and  lowland sites of western Kenya: Implication on malaria vector control measures

PONE-D-19-29030R1

Dear Dr. Afrane,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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.

With kind regards,

Friedrich Frischknecht

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Thanks for returning a well done revision, in my view you addressed all the important issues raised by the reviewers as well as their minor comments. After careful reading your revised manuscript, I thus decided not to send it to the reviewers again. There are still a few minor improvements possible in terms of wording (e.g. first sentence of abstract needs an 'and'; first sentence of discussion needs attention) and the reference list needs attention as the cited literature is not all in the journal style. I trust you can make this changes without a need for further external review. Congratulation to a nice study.

Reviewers' comments: