The protein and volatile components of trail mucus in the Common Garden Snail, Cornu aspersum

Kaylene R. Ballard; Anne H. Klein; Richard A. Hayes; Tianfang Wang; Scott F. Cummins

doi:10.1371/journal.pone.0251565

Peer Review History

Original SubmissionDecember 8, 2020
26 Mar 2021 Decision Letter - Joseph Clifton Dickens, Editor PONE-D-20-38531 The protein and volatile components of trail mucus in the Common Garden Snail, Cornu aspersum PLOS ONE Dear Dr. Cummins, 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. This is an interesting and important study of potential chemical signals used in communication of an economically important snail species. While it is well written and the experimental approach sound, the reviewers identify several possibilities for improvement. The authors should respond to the comments provided especially the suggestion to test the effect on heart rate of mucous from other snail species in order to provide insight on the specificity of the response. Please submit your revised manuscript by May 10 2021 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: http://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, Joseph Clifton Dickens Academic Editor PLOS ONE Additional Editor Comments: This is an interesting and important study of potential chemical signals used in communication of an economically important snail species. While it is well written and the experimental approach sound, the reviewers identify several possibilities for improvement. The authors should respond to the comments provided especially the suggestion to test the effect on heart rate of mucous from other snail species in order to provide insight on the specificity of the response. 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. 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. 3. We note that you are reporting an analysis of a microarray, next-generation sequencing, or deep sequencing data set. PLOS requires that authors comply with field-specific standards for preparation, recording, and deposition of data in repositories appropriate to their field. Please upload these data to a stable, public repository (such as ArrayExpress, Gene Expression Omnibus (GEO), DNA Data Bank of Japan (DDBJ), NCBI GenBank, NCBI Sequence Read Archive, or EMBL Nucleotide Sequence Database (ENA)). In your revised cover letter, please provide the relevant accession numbers that may be used to access these data. For a full list of recommended repositories, see http://journals.plos.org/plosone/s/data-availability#loc-omics or http://journals.plos.org/plosone/s/data-availability#loc-sequencing 4. Thank you for stating the following financial disclosure: 'Grains Research Development Corporation, Contract code is USU1903-001RSX]' 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. 5. Thank you for stating the following in the Financial Disclosure section: 'Grains Research Development Corporation, Contract code is USU1903-001RSX' We note that you received funding from a commercial source: Grains Research Development Corporation a. Please provide an amended Competing Interests Statement that explicitly states this commercial funder, along with any other relevant declarations relating to employment, consultancy, patents, products in development, marketed products, etc. Within this Competing Interests Statement, please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests). If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared. b. Please include your amended Competing Interests Statement within your cover letter. We will change the online submission form on your behalf. Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests [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: The authors provide foundational knowledge towards characterizing the physiological relevance and chemical identity of an economically important snail’s trail mucus. This research is a logical first step towards elucidating the potential role of snail mucus in communication between conspecifics. These results will inform future studies aimed at developing novel pest control strategies for snails. The manuscript is very well written. The rationale presented in the introduction is convincing, noting that I am not well versed in the chemical ecology of gastropods. I was left wondering if non-protein components, like those synthesized in mucous-producing cells, may be important for contact sensation via the mucous. I realize that this may be beyond the scope of this study, but it may be worth mentioning in the introduction or discussion. Methods: Do we know the active ingredient in On-guard Snail Gel? The methods section looks very complete. I have no concerns here. Results and Discussion: It would be great to measure heart rate responses of these snails to mucus from a closely related species and that of a more distant relative. The positive and negative controls used here work well to show there is a response but do little to address the nature of this response, which the authors acknowledge. The tangential discussions of identified mucous proteins is fantastic. Thank you for including this information. I think it would be helpful to readers to use the full name of each tissue on the bottom axis of the heatmap in Figure 3 for quicker reference. It looks like you should be able to fit them into the space available, perhaps at an angle. Likewise, you could insert the corresponding chemicals in Figure 5 at each peak. I only noticed one typographical error: Line 108 –punctuation error. It could increase visual interest to include one diagrammatic image or labeled photograph of the relevant snail anatomy. Though very basic, this could help gastropod novices become more connected to the work. This could even be a figure inset. This is a great submission. Congratulations. Reviewer #2: This paper provides useful information on the common garden snail’s mucus and has the potential to help identify signaling compounds. Given the economic importance of these animals as agricultural pests, this is a worthwhile contribution. I have some suggestions that I hope will improve this manuscript. If the authors can address these points, I feel that the manuscript is worthy of publication. When the authors measured the effect on the heart rate of contact with mucus, it would be interesting to look at one or more mucus samples from distantly related organisms, rather than just water as a negative control. Are the snails detecting common mucus molecules, or something unique to that species? What were criteria for selecting the proteins in Fig. 2? The authors choose to present 10 of the 22 proteins that were predicted to be secreted. Why were these 10 selected and not the other 12? Were they more abundant, or was the selection arbitrary? Notably, two of the contigs that had a markedly higher relative expression in foot (104, 2548) are not included in this ten; why not? In figure 3, the authors present relative expression compared to other tissues. It would also be interesting to provide the TPM values to give a sense of which were the most highly expressed proteins in the mucus. Lines 160-162 state, “A transcriptome-derived protein database was prepared by combining the foot and anterior tentacle with tissue transcriptomes of the mucous gland, central nervous system (CNS) and posterior tentacle using the CLC genomics Workbench.” It is not clear at this point where the non-foot transcriptome data comes from. Later in the paper it is identified, but it should be made clear here. Additionally, the methods only state that foot tissue was used, but later in the methods it is stated that anterior tentacle was also used. This should be cleared up. Line 172 states, “Trail mucus biomolecules were isolated using Sep-Pak plus C18 cartridges (Waters) according to the manufacturer’s instructions.” Give more information on this separation. Was this FPLC? What was the buffer system? Liquid chromatography of snail mucus is challenging due to the large glycosaminoglycans, which have a tendency to clog columns. For the second extraction method, were all visible bands analyzed by LC-MS/MS? If not, what were the criteria for inclusion? Were there notable differences in the proteomes derived from the two extraction methods? Note that C-lectins with similarity to perlucin have also been identified as centrally important in the adhesive properties of a terrestrial slug’s defensive mucus (Smith et al., 2017, “RNA-seq reveals a central role for lectin, C1q and von Willebrand Factor A domains in the defensive glue of a terrestrial slug” Biofouling). Fig 3. Provide some guidance for interpreting Z-score in the figure legend. I assume that red indicates higher relative expression. Not everyone will be familiar with Z-score. The last page and a half of the discussion could be more focused. There is some repetition that could be removed. ****** 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.] 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. https://doi.org/10.1371/journal.pone.0251565.r001
Revision 1
26 Apr 2021 Author Response Response to Reviewers for Manuscript PONE-D-20-38531 Editors Comments 1. Style requirements met 2. Reference list correct 3. Genbank accession numbers have been uploaded to NCBI and noted in manuscript 4. Added to cover letter 5. Added to cover letter Reviewer 1 The manuscript is very well written. The rationale presented in the introduction is convincing, noting that I am not well versed in the chemical ecology of gastropods. I was left wondering if non-protein components, like those synthesized in mucous-producing cells, may be important for contact sensation via the mucous. I realize that this may be beyond the scope of this study, but it may be worth mentioning in the introduction or discussion. Response: Added to discussion (Line 514). Do we know the active ingredient in On-guard Snail Gel? The methods section looks very complete. I have no concerns here. Response: In spite of examination of the label and an email to manufacturer (in UK), the active component has not been confirmed. As the product is stated as being non-toxic and natural, it is likely that it contains a combination of natural ingredients that the snails find aversive. I think it would be helpful to readers to use the full name of each tissue on the bottom axis of the heatmap in Figure 3 for quicker reference. It looks like you should be able to fit them into the space available, perhaps at an angle. Likewise, you could insert the corresponding chemicals in Figure 5 at each peak. Response: Chemical names have been included within the chromatogram to ensure easy reference while maintaining tidiness and clarity. It could increase visual interest to include one diagrammatic image or labelled photograph of the relevant snail anatomy. Though very basic, this could help gastropod novices become more connected to the work. This could even be a figure inset. Response: To help readers comprehend the anatomy, we have added an anatomical figure (as 3A), inclusive of abbreviations used in heatmaps 3B and 3C. I only noticed one typographical error: Line 108 –punctuation error. Response: Corrected. Reviewer 2 When the authors measured the effect on the heart rate of contact with mucus, it would be interesting to look at one or more mucus samples from distantly related organisms, rather than just water as a negative control. Are the snails detecting common mucus molecules, or something unique to that species? Response: Agreed, and are indeed exploring this type of analysis between related species within the scope of another research project performed by the first author, i.e profiling mucus molecules between sympatric pest land snail species. What were criteria for selecting the proteins in Fig. 2? The authors choose to present 10 of the 22 proteins that were predicted to be secreted. Why were these 10 selected and not the other 12? Were they more abundant, or was the selection arbitrary? Notably, two of the contigs that had a markedly higher relative expression in foot (104, 2548) are not included in this ten; why not? Response: The 10 that are presented were thought be the most likely to play a role in communication and are generally of more interest. Eg. Novel or uncharacterised. The remaining 12 contain sequences that match to several variants of collagen and another variant of epiphragmin. These sequences are available in the supplementary material (S2). In figure 3, the authors present relative expression compared to other tissues. It would also be interesting to provide the TPM values to give a sense of which were the most highly expressed proteins in the mucus. Response: This is the purpose of heatmap Fig 3C, which has now been more clearly explained in the figure legend. Lines 160-162 state, “A transcriptome-derived protein database was prepared by combining the foot and anterior tentacle with tissue transcriptomes of the mucous gland, central nervous system (CNS) and posterior tentacle using the CLC genomics Workbench.” It is not clear at this point where the non-foot transcriptome data comes from. Later in the paper it is identified, but it should be made clear here. Additionally, the methods only state that foot tissue was used, but later in the methods it is stated that anterior tentacle was also used. This should be cleared up. Response: Apologies for the oversight – the anterior tentacle was removed and RNA isolated at the same time and using the same methodology as the foot tissue. This has been added to the methods. (Line 152). Line 172 states, “Trail mucus biomolecules were isolated using Sep-Pak plus C18 cartridges (Waters) according to the manufacturer’s instructions.” Give more information on this separation. Was this FPLC? What was the buffer system? Liquid chromatography of snail mucus is challenging due to the large glycosaminoglycans, which have a tendency to clog columns. Response: Apologies for the oversight – The use of 100% acetonitrile for cartridge preparation and 60% acetonitrile for elution has been added to methods (Line 176). This pre-preparation, prior to LC-MS, is an approach that would remove large glycosaminoglycans. For the second extraction method, were all visible bands analyzed by LC-MS/MS? If not, what were the criteria for inclusion? Response: Yes, all visible bands were subjected to in-gel trypsin digestion and analysis and this information has now been added to methods (Line 184 – ‘All visible bands were excised….). Were there notable differences in the proteomes derived from the two extraction methods? Response: Yes, and this information has now been added to the manuscript (Line 396). Note that C-lectins with similarity to perlucin have also been identified as centrally important in the adhesive properties of a terrestrial slug’s defensive mucus (Smith et al., 2017, “RNA-seq reveals a central role for lectin, C1q and von Willebrand Factor A domains in the defensive glue of a terrestrial slug” Biofouling). Response: Thank you for drawing our attention to this very interesting paper. A reference to this paper has been added to our manuscript (Line 348). Fig 3. Provide some guidance for interpreting Z-score in the figure legend. I assume that red indicates higher relative expression. Not everyone will be familiar with Z-score. Response: Apologies for the confusion. Yes, red indicates higher expression and this information has now been added to the Figure 3 legend. The last page and a half of the discussion could be more focused. There is some repetition that could be removed. Response: Agreed. This part of the discussion has been revised and we now feel it is more focused. Attachments Attachment Submitted filename: Response to Reviewers for Manuscript PONE_2_SC.docx https://doi.org/10.1371/journal.pone.0251565.r002
29 Apr 2021 Decision Letter - Joseph Clifton Dickens, Editor The protein and volatile components of trail mucus in the Common Garden Snail, Cornu aspersum PONE-D-20-38531R1 Dear Dr. Cummins, 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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. 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 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, Joseph Clifton Dickens Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0251565.r003
Formally Accepted
3 May 2021 Acceptance Letter - Joseph Clifton Dickens, Editor PONE-D-20-38531R1 The protein and volatile components of trail mucus in the Common Garden Snail, Cornu aspersum Dear Dr. Cummins: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Joseph Clifton Dickens Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0251565.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .