PPATHOGENS-D-25-00409

Host environment shapes filarial parasite fitness and Wolbachia endosymbionts dynamics

PLOS Pathogens

Dear Dr. Martin,

Thank you for submitting your manuscript to PLOS Pathogens. After careful consideration, we feel that it has merit but does not fully meet PLOS Pathogens'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 within 30 days Jul 01 2025 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 plospathogens@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/ppathogens/ 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 editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. This file does not need to include responses to any formatting updates and technical items listed in the 'Journal Requirements' section below.

* 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, competing interests statement, or data availability statement, please make these updates within the submission form at the time of resubmission. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

We look forward to receiving your revised manuscript.

Kind regards,

Keke C. Fairfax, PhD

Academic Editor

PLOS Pathogens

Jeffrey Dvorin

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

Journal Requirements:

1) We do not publish any copyright or trademark symbols that usually accompany proprietary names, eg ©,  ®, or TM  (e.g. next to drug or reagent names). Therefore please remove all instances of trademark/copyright symbols throughout the text, including:

- ® on page: 35.

2) Please upload all main figures as separate Figure files in .tif or .eps format. For more information about how to convert and format your figure files please see our guidelines: 

https://journals.plos.org/plospathogens/s/figures

3) Some material included in your submission may be copyrighted. According to PLOSu2019s copyright policy, authors who use figures or other material (e.g., graphics, clipart, maps) from another author or copyright holder must demonstrate or obtain permission to publish this material under the Creative Commons Attribution 4.0 International (CC BY 4.0) License used by PLOS journals. Please closely review the details of PLOSu2019s copyright requirements here: PLOS Licenses and Copyright. If you need to request permissions from a copyright holder, you may use PLOS's Copyright Content Permission form.

Please respond directly to this email and provide any known details concerning your material's license terms and permissions required for reuse, even if you have not yet obtained copyright permissions or are unsure of your material's copyright compatibility. Once you have responded and addressed all other outstanding technical requirements, you may resubmit your manuscript within Editorial Manager. 

Potential Copyright Issues:

- Figures 4 and 6. Please confirm whether you drew the images / clip-art within the figure panels by hand. If you did not draw the images, please provide (a) a link to the source of the images or icons and their license / terms of use; or (b) written permission from the copyright holder to publish the images or icons under our CC BY 4.0 license. Alternatively, you may replace the images with open source alternatives. See these open source resources you may use to replace images / clip-art:

- https://commons.wikimedia.org

- https://openclipart.org/.

4) We note that your Data Availability Statement is currently as follows: "All data generated or analyzed during this study are included in this published article and its supplementary information files. Further inquiries can be directed to the corresponding author.". Please confirm at this time whether or not your submission contains all raw data required to replicate the results of your study. Authors must share the “minimal data set” for their submission. PLOS defines the minimal data set to consist of the data required to replicate all study findings reported in the article, as well as related metadata and methods (https://journals.plos.org/plosone/s/data-availability#loc-minimal-data-set-definition).

For example, authors should submit the following data: 

- The values behind the means, standard deviations and other measures reported;

- The values used to build graphs;

- The points extracted from images for analysis..

Authors do not need to submit their entire data set if only a portion of the data was used in the reported study.

If your submission does not contain these data, please either upload them as Supporting Information files or deposit them to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of recommended repositories, please see https://journals.plos.org/plosone/s/recommended-repositories. 

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. If data are owned by a third party, please indicate how others may request data access.

Reviewers' Comments:

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: This is a very interesting study that provides important insights into the role of the host immune system in shaping Wolbachia dynamics and filarial parasite fitness. Notably, this study provides new insights into the particular susceptibility of the reproductive tissues of filarial nematodes to type 2 immunity, whereby type 2 immunity selectively degrades the development of oocysts, with downstream impact on Wolbachia colonisation of those cells. Wolbachia-negative microfilariae exhibited apparently normal larval development including in the vector, but failed to reach sexual maturity in the definitive host. This is an important step in identifying the target of protective immunity against filarial nematodes, and in clarifying the causal relationships between type 2 immunity, Wolbachia dynamics, and filarial parasite fitness. The experiments are elegantly designed and the results are well presented, with excellent use of RNA fluorescence in situ hybridisation and histology.

Reviewer #2: The manuscript by Fercoq et al. presents a relevant contribution to the understanding of the interaction between the host immune system and the Wolbachia-filariae symbiosis, revealing mechanisms that directly impact the life cycle and reproductive capacity of the parasites. The results obtained provide important support for the development of more effective therapeutic strategies to combat filariasis, reinforcing the potential of the immune system as a modulator of the efficacy of anti-Wolbachia therapies.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: (No Response)

Reviewer #2: I have no suggestions for major revisions.

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: I have a few comments and suggestions that I believe would improve the clarity of the manuscript.

- In the introduction, it would be good to mention whether / how type 1 and Th17 immunity might affect filarial survival and Wolbachia dynamics. This would help to clarify the novelty of the study, and the importance of the type 2 immune response in this context.

- Although FISH images don't show any Wolbachia post DR treatment, Fig 2A suggests that DR treatment in KO mice does not completely remove Wolbachia (these figures show normalised values (x 1,000) relative to average worm size). Could these residual levels explain the ability of apparently Wolbachia-negative microfilariae and larvae to develop somewhat normally? For example, as per the statements lines 209-210 & 507-509, those low numbers of Wolbachia might still provide some benefit, which would wane as the larvae increase in size at L4 and adult stages while Wolbachia are unable to proliferate due to early “sterilisation” by DR. It would be good to clarify this point in the discussion, and if so, to temper the wording in lines 456-458.

- In the discussion, paragraph lines 479-500 seems very speculative, and could be shortened.

- Figure 6: shouldn't the arrow linking type 2 immunity (top left) to Wolbachia instead point to the worm? I wonder if here the authors might be able to graphically differentiate between the direct effects of antibiotics and type 2 immunity on Wolbachia and on the filaria's development, respectively, since it appears that the effects of immunity on Wolbachia are indirect (through the worm). This could be clarified in the figure legend.

Reviewer #2: Minor Revision

1.In the introduction section

a) Standardizing technical terms is essential. For example, when referring to the “host immune environment” or the “type 2 immune response”, consistency should be maintained throughout the text.

• "type 2 immune deficient mice", "BALB/c mice strains", "immune-competent mouse strains" → can be standardized to avoid unnecessary variations.

b). Before describing the worm's anatomy, a sentence like:

"To better understand how Wolbachia interact with their nematode hosts at the tissue level, we briefly describe the anatomy relevant to their localization."would greatly help the reader's understanding.

2. Discussion section

A) after describing the possible metabolic competition in the permissive environment (lines 573–581), there is no clear conclusion or testable hypothesis. A more explicit conclusion would help with understanding.

b) since the discussion addresses multiple immunological, metabolic pathways and anatomical sites, a schematic figure would help the reader understand the complexity of the interactions.

3. Conclusion section

The sentence "revealing their critical contributions to germline function and its stage-specific dependencies" is somewhat generic in my understanding. I understood that the great contribution of the work is precisely to show that the dependency is crucial from the L4 stage onwards.

4. Methods sextion

On line 633-635 include some detail on how the life cycle of L. sigmodontis was maintained in the laboratory. Since the infection was in females, include a comment on how the infection or immune response is more consistent or reproducible in females when compared to males. Also include how the negative control was performed in this experiment.

**********

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.]

Figure resubmission:

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. If there are other versions of figure files still present in your submission file inventory at resubmission, please replace them with the PACE-processed versions.

Reproducibility:

To enhance the reproducibility of your results, we recommend that authors of applicable studies deposit 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. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Dear Dr. Martin,

We are pleased to inform you that your manuscript 'Host environment shapes filarial parasite fitness and Wolbachia endosymbionts dynamics' has been provisionally accepted for publication in PLOS Pathogens.

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 Pathogens.

Best regards,

Keke C. Fairfax, PhD

Academic Editor

PLOS Pathogens

Jeffrey Dvorin

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************************************************

Reviewer Comments (if any, and for reference):