PONE-D-24-25883An effective protocol to isolate and mechanically test silk fibers spun by Osmia lignaria Say (Hymenoptera: Megachilidae) fifth instar larvaePLOS ONE

Dear Dr. Wasserman,

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.

ACADEMIC EDITOR: The study initiated by Wasserman et al. is well elaborated and written. Some minor points as raised by the reviewer need to be addressed before it can be made available to the general audience.

I thank you once again for your patience and I am looking forward to received your newer version of the manuscript incorporating the suggested improvements.

Please submit your revised manuscript by Jan 10 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 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,

Adel Tekari, PhD

Academic Editor

PLOS ONE

Journal requirements:

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

1.  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. We note you have not yet provided a protocols.io PDF version of your protocol and/or a protocols.io DOI. When you submit your revision, please provide a PDF version of your protocol as generated by protocols.io (the file will have the protocols.io logo in the upper right corner of the first page) as a Supporting Information file. The filename should be S1_file.pdf, and you should enter “S1 File” into the Description field. Any additional protocols should be numbered S2, S3, and so on. Please also follow the instructions for Supporting Information captions [https://journals.plos.org/plosone/s/supporting-information#loc-captions]. The title in the caption should read: “Step-by-step protocol, also available on protocols.io.”

Please assign your protocol a protocols.io DOI, if you have not already done so, and include the following line in the Materials and Methods section of your manuscript: “The protocol described in this peer-reviewed article is published on protocols.io (https://dx.doi.org/10.17504/protocols.io.[...]) and is included for printing purposes as S1 File.” You should also supply the DOI in the Protocols.io DOI field of the submission form when you submit your revision.

If you have not yet uploaded your protocol to protocols.io, you are invited to use the platform’s protocol entry service [https://www.protocols.io/we-enter-protocols] for doing so, at no charge. Through this service, the team at protocols.io will enter your protocol for you and format it in a way that takes advantage of the platform’s features. When submitting your protocol to the protocol entry service please include the customer code PLOS2022 in the Note field and indicate that your protocol is associated with a PLOS ONE Lab Protocol Submission. You should also include the title and manuscript number of your PLOS ONE submission.

3. Thank you for stating the following financial disclosure:

“The Department of Biology at Utah State University and the Utah State University Office of Research funded and supported this work. Funds from the U.S. Department of Agriculture, Agricultural Research Service (Project#2080-21000-019-000-D) supported the rearing of the O. lignaria larvae.”

Please state what role the funders took in the study.  If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

If this statement is not correct you must amend it as needed.

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

4. 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.

Additional Editor Comments:

None

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

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript report a protocol which is of utility to the research community and adds value to the published literature?

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the protocol been described in sufficient detail?

To answer this question, please click the link to protocols.io in the Materials and Methods section of the manuscript (if a link has been provided) or consult the step-by-step protocol in the Supporting Information files.

The step-by-step protocol should contain sufficient detail for another researcher to be able to reproduce all experiments and analyses.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Does the protocol describe a validated method?

The manuscript must demonstrate that the protocol achieves its intended purpose: either by containing appropriate validation data, or referencing at least one original research article in which the protocol was used to generate data.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. If the manuscript contains new data, have the authors made this data 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 article 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 highlight any specific errors that need correcting in the box below.

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 protocol for isolation and mechanical testing of silk fibers spun by the blue orchard bee, an important pollinator of orchards in North America, offers a minimally invasive approach that allows larvae to proceed with cocoon formation after fiber isolation. This protocol allows larval rearing of this ecological important insects and thus, it is very important for investigating the relationship between silk biochemistry and mechanical properties without killing the bees. In addition, the presented protocol can also be used for testing of silk from other solitary bee species. Therefore, the relevance of this protocol is very high. The protocol is very detailed and easy to follow because of the very detailed description of the single steps. In addition, the discussion of possible limitations is very helpful to avoid errors in the implementation of the protocol by other scientists at an early stage.

Reviewer #2: The authors needs to answer certain queries provided which can be included in the manuscript:

1. What are the alternative materials that can replace 3-D printed well plates for rearing Osmia lignaria larvae?

2. How does the timing of fiber isolation affect the mechanical properties of the silk?

3. What are the optimal environmental conditions (temperature, humidity) for silk spinning in Osmia lignaria?

4. How can the isolation method be adapted for smaller bee species with different cocoon construction methods?

5. Does the method of cutting the silk mesh impact the tensile strength or elasticity of the isolated fibers?

Mechanical Testing

6. How do variations in fiber diameter correlate with mechanical properties like tensile strength and elasticity?

7. Can the tensile testing protocol be adapted for higher-throughput analyses of multiple fibers simultaneously?

8. What are the effects of different glues on the mechanical properties of silk during C-card mounting?

9. How does the choice of testing environment (e.g., ambient vs. controlled humidity) affect mechanical test outcomes?

Silk Characteristics and Applications

10. What molecular markers in the silk indicate the health and nutritional status of the larvae?

11. How does the chemical composition of Osmia lignaria silk compare to that of other bee or silkworm species?

12. Can the silk from Osmia lignaria be used as a biomarker for exposure to environmental stressors, such as pesticides?

13. What are the biodegradation properties of Osmia lignaria silk in various environmental conditions?

Comparative Studies

14. How do the mechanical properties of silk from solitary bees compare with those of social bees, such as honeybees?

15. What are the evolutionary implications of the observed mechanical differences between Osmia lignaria silk and other insect silks?

16. Are there phylogenetic correlations between the mechanical properties of bee silks and their nesting behaviors?

Protocol Optimization

17. What improvements can be made to reduce the variability in fiber diameter during isolation?

18. How does the larval diet during development influence the quality and mechanical properties of the silk?

19. Can the protocol be modified to allow for automated fiber isolation?

Broader Applications

20. What are the potential industrial applications of silk isolated using this protocol (e.g., biomedical materials, eco-friendly textiles)?

**********

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

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.]

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.

An effective protocol to isolate and mechanically test silk fibers spun by Osmia lignaria Say (Hymenoptera: Megachilidae) fifth instar larvae

PONE-D-24-25883R1

Dear Dr. Wasserman,

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,

Adel Tekari, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript report a protocol which is of utility to the research community and adds value to the published literature?

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the protocol been described in sufficient detail?

To answer this question, please click the link to protocols.io in the Materials and Methods section of the manuscript (if a link has been provided) or consult the step-by-step protocol in the Supporting Information files.

The step-by-step protocol should contain sufficient detail for another researcher to be able to reproduce all experiments and analyses.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Does the protocol describe a validated method?

The manuscript must demonstrate that the protocol achieves its intended purpose: either by containing appropriate validation data, or referencing at least one original research article in which the protocol was used to generate data.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. If the manuscript contains new data, have the authors made this data 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 article 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 highlight any specific errors that need correcting in the box below.

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: There is nothing to add to my original review. Thus I recommend to accept the manuscript in the revised version.

Reviewer #2: 1. What statistical methods were employed to analyze the large variation in mechanical property data presented in Table 1, and how were the error margins validated?

2. What measures were taken to ensure the reproducibility of fiber isolation and testing techniques across different trials or by different researchers?

3. How do the mechanical properties of Osmia lignaria silk fibers compare to those of other insect silk (e.g., silkworms or spiders) as discussed in the results and supported by the data in Table 1?

**********

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

Reviewer #2: No

**********