PONE-D-23-39281Impact of blood meals taken on ivermectin-treated livestock on survival and fecundity of the malaria vector Anopheles coluzzii under laboratory conditionsPLOS ONE

Dear Dr. Pooda,

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. Both reviewers did a detailed analysis with several suggestions that will help to improve your manuscript. Please give special attention to the questions on the colony's insecticide resistance status and the lack of correlation between ivermectin plasma concentrations and mortality.

Please submit your revised manuscript by Feb 17 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,

Pedro L. Oliveira

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 

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.

2. In your Methods section, please provide additional information regarding the permits you obtained for the work. Please ensure you have included the full name of the authority that approved the field site access and, if no permits were required, a brief statement explaining why.

3. We suggest you thoroughly copyedit your manuscript for language usage, spelling, and grammar. If you do not know anyone who can help you do this, you may wish to consider employing a professional scientific editing service. 

Whilst you may use any professional scientific editing service of your choice, PLOS has partnered with both American Journal Experts (AJE) and Editage to provide discounted services to PLOS authors. Both organizations have experience helping authors meet PLOS guidelines and can provide language editing, translation, manuscript formatting, and figure formatting to ensure your manuscript meets our submission guidelines. To take advantage of our partnership with AJE, visit the AJE website (http://learn.aje.com/plos/) for a 15% discount off AJE services. To take advantage of our partnership with Editage, visit the Editage website (www.editage.com) and enter referral code PLOSEDIT for a 15% discount off Editage services.  If the PLOS editorial team finds any language issues in text that either AJE or Editage has edited, the service provider will re-edit the text for free.

Upon resubmission, please provide the following:

The name of the colleague or the details of the professional service that edited your manuscript

A copy of your manuscript showing your changes by either highlighting them or using track changes (uploaded as a *supporting information* file)

A clean copy of the edited manuscript (uploaded as the new *manuscript* file)".

4. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. 

When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

5. Your ethics statement should only 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 ensure that your ethics statement is included in your manuscript, as the ethics statement entered into the online submission form will not be published alongside your manuscript. 

6. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. 

[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: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

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: Yes

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: Pooda and Colls investigated the effects of ivermectin treatments as an ectocide in three livestock species (Sheep, goats and pigs) on the survival and reproduction of the malaria vector Anopheles coluzzii. Different domestic animal species have been used since the pharmacokinetics of Ivermectin may vary between vertebrate hosts. This strategy could be used to complement current control tools such as LLINs and IRS to target exophagic, exophilic and zoophagic vectors. The injectable veterinary ivermectin formulation at the species-specific doses caused a significant decrease in mosquito survival for up to 7 days after injection. The number of gravid females Anopheles that survived after feeding on treated animals was also reduced, as well as the number of mature eggs in the ovaries. However, due to the short-term efficacy of single-dose treatments, repeated treatments and potentially increased dosages would be required to span the transmission season.

The methodology seems to be appropriate, and the results obtained support the conclusions realised by the authors. However, some issues and concerns should be addressed.

1. The authors considered the proportion of females carrying eggs (gravidity rate) and the number of mature eggs in the ovaries as proxies of mosquito fecundity. It is not the optimal way to assess the effect of a drug on reproductive fitness. The presence of a drug may delay ovaries and egg development without affecting the final reproductive output. A better and more direct way to assess reproductive fitness would be to allow the treated females to complete the reproductive cycle, lay the eggs, and count the number of eggs laid by each female and their hatching rate. The final number of F1 per female is the better way to quantify the effect of a drug on reproductive fitness.

2. Which is the insecticide resistance status of the An coluzzii colony used? Please add this information, if known.

3. ¨Lines 235-236: ¨The rate of blood-fed mosquitoes was, respectively, 71.52 (±4.88) %, 71.94 (±3.15) % and 57.46 233 (±2.55) % on sheep, goat and pig at the first blood meal (Figure 1)¨.

Were these values calculated using both control and Ivermectin-treated animals? Please clarify this issue.

4. Figure 1: Change DT for TD in the X-axis information. What’s the meaning of ten in the X-axis legend? This figure is difficult to interpret and contains some mistakes. I guess the pink columns are the insects fed on control animals, blue are mosquitoes fed on therapeutical dose-treated animals (TD), while green and violet colours are 2TD and 3DT in mosquitoes fed on pigs. Please clarify this and accommodate the order of columns in panel C according to panels A and B. Add ¨3TD¨ at figure legend.

5. Line 255-256: ¨The doubled and tripled therapeutic doses used to treat pigs induced as well a significant decrease in mosquito mortality rates¨

Should be ¨ a significant increase¨

6. Figure 2: There seems not to be differences in survival in IVM-treated and control goats on day 2 DPI. Please check it. Besides, the mortality in pigs is higher for TD than for 2TD on day 2 DPI. Please also check this issue. Add the time units in the X-axis (days?)

7. Line 282-283: From day 14 post-treatment and onwards, there was no significant difference between groups whatever the host species considered (Figure 1).

Should be ¨ Figure 2¨

8. Figures 3 and 4: Please, add asterisks to the statically significant differences between control and IVM-treated animals.

9. There is no correlation in the mean plasma concentrations of ivermectin (ng/mL) in the treated- sheep, goats and pigs (table II) with the mortality observed in Figure 2 and the number of eggs in Figure 4. Can the authors explain this? For example, in pigs, only the 3TD caused increased mortality on day 14 DPI, but the concentration is as high as 2TD, which did not increase mortality on that day. Besides, 2TD on pigs doubles the concentration in goats at 2 DPI and in sheep at 7 DPI, and both increased mosquitoes mortality. A deeper discussion about the lack of correlation in plasma IVM concentrations and the phenotypes observed associated with reduced survival and reproductive fitness is needed. The lack of correlation makes the interpretation of the results very difficult.

Reviewer #2: PONE-D- 23-39281

Impact of blood meals taken on ivermectin-treated livestock on survival and fecundity of the malaria vector Anopheles coluzzii under laboratory conditions

This review is by Carlos Chaccour from ISGlobal, Barcelona Institute for Global Health. I have a personal open peer-review policy as the current single-blinded system is riddled with vices.

This manuscript reports the results of an experiment conducted in Burkina Faso in which pigs, sheep and goats were treated with different doses of ivermectin and then An. coluzii mosquitoes were fed on them at different times after treatment. The authors also collected some PK data. The results are discussed on the context of a potential One Health approach to malaria control.

Albeit some minor mistakes, the manuscript is well written. The methods are appropriate for the objectives and the conclusion is supported by the result. I provide here comments for the author`s consideration.

Introduction

Consider mentioning early exit as another mosquito behavior contributing to residual transmission. This can be induced by the repellent properties of indoor insecticides or occur even in their absence.

There is no mention of two key concepts: zooprophylaxis vs zoopotentiation.

Methods

Please mention the colony`s insecticide resistant status. This is important given the potential cross-metabolic resistance with pyrethroids as they share the same CYP as ivermectin.

Please mention the calculated adipose vs lean weight of the animals.

How was the random allocation of mosquito cups done?

Were cups rotated in the insectary?

Figure 2 is pixelated, making reading it difficult. Additionally, the X axis seems compressed, giving the illusion that the curves are smooth rather than the usual K-M step by step drop. I recommend providing a higher quality image and perhaps even separating it in three different figures to ensure sufficient size. Consider also adding guiding marks to the reader such as the median survival in the control group.

Results

The increased gravity found at 28 DAI in goats does not seem to be statistically significant as the CI overlap in figure 3. If that is the case, I recommend, stating it in the text.

The same for the decrease in fecundity reported at day 28 in sheep, or 7 DAI in goats, although in these cases p-values are provided in the text, it is worth it to double check the figure given that CI-overlap.

Table II. The metric should be median and range given the samples come from only two animals.

Can the authors use the PK data to estimate the 7-day LC50?

What hypotheses do the authors have about the disparity between the ivermectin concentrations and the mosquito mortality seen in pigs?

There is no mention of toxicity in the livestock. Did the authors monitor for toxicity in pigs given three-fold doses?

Discussion

Please comment in the expected relative densities of each livestock species in the field. Are goats more common than cattle? What order of livestock treatment would you recommend? Cattle > Pigs > Goat > Sheep? Or other?

There is no mention about the milk or slaughter withdrawal periods and how this may affect deployment of the proposed strategy.

Please consider mentioning the potential impact of intense treatment schemes or long-lasting formulations on intestinal parasites resistance. What role could refugia play?

There is also no mention about the potential long-term theoretical risk of selectin a more anthropophilic mosquito population.

Minor

Lines 482. Passed away. Not “are passed away”. Also Rest in peace not “rested in peace”

**********

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: Marcos Sterkel

Reviewer #2: Yes: Carlos Chaccour

**********

[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-23-39281.docx

Impact of blood meals taken on ivermectin-treated livestock on survival and egg production of the malaria vector Anopheles coluzzii under laboratory conditions

PONE-D-23-39281R1

Dear Dr. Pooda,

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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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,

Pedro L. Oliveira

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

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: 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

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: Yes

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 manuscript has been significantly improved from its initial version. All of my comments and concerns have been resolved in this new version. I appreciate the authors' efforts in strengthening the discussion about the correlation between plasma levels and the observed phenotypes. After a second revision, I found no further issues to be addressed.

Reviewer #2: The authors have submitted a detailed "answer to reviewers" section. All my comments have been appropriately addressed. All changes in the manuscript are appropriately tracked

**********

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: Marcos Sterkel

Reviewer #2: Yes: Carlos Chaccour

**********