Claustrophilic oviposition: oviposition performance depends on container size in a novel forced oviposition method for Culex quinquefasciatus and Aedes aegypti

PLOS Neglected Tropical Diseases

Dear Dr. Burgess,

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Kind regards,

Adly M.M. Abd-Alla, Prof asso.

Section Editor

PLOS Neglected Tropical Diseases

Adly Abd-Alla

Section Editor

PLOS Neglected Tropical Diseases

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-4304-636XX

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-1765-0002

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1) Please ensure that the CRediT author contributions listed for every co-author are completed accurately and in full.

At this stage, the following Authors/Authors require contributions: Kendra A. Dagg, Alden S. Estep, Cason E. Bartz, and Edwin Burgess. Please ensure that the full contributions of each author are acknowledged in the "Add/Edit/Remove Authors" section of our submission form.

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Reviewers' Comments:

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods:

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: 1. The study’s aim of “comparing egg numbers” is not associated with a clearly defined threshold; it fails to distinguish expected outcomes for oviposition rate, egg number, and hatch rate. Additionally, potential confounding factors such as mosquito age or degree of blood engorgement were not controlled for in assessing oviposition performance.

2. The definition and observation criteria for "claustrophilic" behavior lack rigorous quantification.

3. The tube sizes used in Experiment 2 (1.5, 5, 15, and 50 mL) are not proportionally scaled or logically progressive, making it difficult to establish a consistent gradient for interpretation. Furthermore, moisture conditions differed between species—Cx. tubes retained a surface water layer, whereas Ae. tubes had excess water removed—which could significantly affect oviposition behavior. The criteria for determining oviposition were not clearly described, including whether this was assessed visually and whether all germination papers were collected and systematically examined.

Reviewer #2: Yes the methods are well described and appropriate

Reviewer #3: The nature of the wild mosquitos need more description (time collected, location, identification method...). Was the tap water used dechlorinated, fluoridated, harvested rain water or borehole water?

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Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: (No Response)

Reviewer #2: Yes to the above

Reviewer #3: Well presented and of sufficient quality.

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Conclusions:

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: The conclusion that the mosquitoes “exhibited claustrophilic behavior” is not well-supported, as it is based solely on oviposition rates without behavioral recordings or exploratory data. The study also lacks discussion of potential effects of the method on mosquito longevity or offspring quality, and does not address possible impacts on sex ratio or population fitness in downstream research.

Reviewer #2: yes to the above

Reviewer #3: (No Response)

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Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: (No Response)

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Summary and General Comments:

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: This study proposed and evaluated a modified forced oviposition method applicable to Culex quinquefasciatus and Aedes aegypti, finding that both species exhibited higher oviposition success rates in small-volume containers and displayed "claustrophilic" oviposition behavior. The method uses inexpensive materials and a single-mosquito setup, making it suitable for oviposition induction in both laboratory and wild mosquito populations, and holds promise for facilitating colony establishment and genetic experiments in vector research.

The background lacks a systematic review of existing forced oviposition methods, their limitations, and comparative analyses. The "claustrophilic" hypothesis was introduced late in the manuscript instead of being explicitly stated and biologically contextualized in the introduction. There is no literature-based or physiological support for the mechanism of “small space–induced oviposition,” making the hypothesis largely speculative.

Reviewer #2: This is a very well written detailed paper that can be useful for programs that has important implications for enahcing vector-borne disease research. Minor edits are:

Line 108 and 109 in Materials and Methods: Culex and Aedes already mentioned so can abbreviate the genus

Line 290 aegypti is capitalized

Could explain in line 307 what claustrophilic behavior is

Reviewer #3: The study presents a practical forced oviposition method adapted for Culex and Aedes species adapted from Anopheles methods highlighting its usefulness in generating sufficient F1 eggs for colony establishment despite their differing natural oviposition behaviors.

It is well written and easy for the reader to follow through and replicate the experiments.

The technique’s simplicity—requiring minimal female mosquitoes and low-cost lab materials—makes it highly accessible for vector control programs and various research settings.

A more elaborate description of the wild strains used will enhance repeatability and reveal any confounding factors on the outcome of the experiments including habitat type and environmental conditions pre-testing that might affect behavioral traits.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Reproducibility:

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Attachments

Attachment

Submitted filename: PNTD-D-25-00520_reviewer.pdf

Dear Dr. Burgess,

We are pleased to inform you that your manuscript 'Claustrophilic oviposition: oviposition performance depends on container size in a novel forced oviposition method for Culex quinquefasciatus and Aedes aegypti' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Adly M.M. Abd-Alla, Prof asso.

Section Editor

PLOS Neglected Tropical Diseases

Adly Abd-Alla

Section Editor

PLOS Neglected Tropical Diseases

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-4304-636XX

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-1765-0002

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Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: (No Response)

Reviewer #2: The methods are clear and the minor edits have made improvements to the manuscript.

Reviewer #3: (No Response)

**********

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: (No Response)

Reviewer #2: The results are clear.

Reviewer #3: (No Response)

**********

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: (No Response)

Reviewer #2: Improvements to the conclusions have been made and increased the clarity of the findings.

Reviewer #3: (No Response)

**********

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: (No Response)

**********

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: The review concerns have been sufficiently addressed and this is a much improved version of the MS.

**********

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

Reviewer #3: No

Attachments

Attachment

Submitted filename: 00 Response to Reviewers.docx