Gene silencing for invasive paper wasp management: Synthesized dsRNA can modify gene expression but did not affect mortality

Mariana Bulgarella; James W. Baty; Rose McGruddy; Philip J. Lester

doi:10.1371/journal.pone.0279983

Peer Review History

Original SubmissionSeptember 29, 2022
25 Nov 2022 Decision Letter - Volker Nehring, Editor PONE-D-22-26973Gene silencing for invasive paper wasp management: synthesized dsRNA can modify gene expression but did not affect mortalityPLOS ONE Dear Dr. Bulgarella, 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. Two reviewers have praised the manuscript and had only minor concerns: The authors should take care to add all the necessary details for the dsRNA production and qPCR methods. I also agree with the reviewers that the figures could be improved. I do think it is useful to have the photographs in the main manuscript, but the current solution isn’t very nice. I wonder if the photos would work better as smaller inserts inside the bar graphs, or as separate figures? Also, there seems to be some inconsistency – no photograph with figure 2, but the corresponding picture seems to be in the supplement? Please submit your revised manuscript by Jan 09 2023 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. 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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, Volker Nehring 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. 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. Thank you for stating the following in the Acknowledgments Section of your manuscript: "We thank Richard Toft for logistical assistance during wasp collection and Neil MacMillan for help collecting wasps. This project was funded with a Smart Ideas Grant from the New Zealand Ministry of Business, Innovation and Employment number 61270-ENDSI-RSCHTUSTVIC." We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: "This study was funded by a Smart Ideas grant number PROP-61270-ENDSI-RSCHTRUSTVIC from the New Zealand Ministry of Business, Innovation and Employment, awarded to PJL. https://www.mbie.govt.nz/science-and-technology/science-and-innovation/funding-information-and-opportunities/investment-funds/endeavour-fund/application-and-assessment-information/ The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 4. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. 5. 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. [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: No 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: Dear authors, This paper examines the effects of RNAi on the wasp, Polistes dominula. The experiments are reasonable and well-written. I have made some comments, please see below. It is strange that the results are shown here as Fig1; I think it would be better to separate the top and bottom of Fig.1 and just explain the methodology here using the bottom of Fig.1 (as a supplementary figure). Was RNA extraction done with the whole body of the wasps? As you cited, some genes are upregulated by RNAi (Fig.1; L.333-335). Could this overlap between the region where dsRNA was created and the region where it was quantified? Related to the question above, the sequence of the primers used for dsRNA synthesis and qPCR should be provided, even if it is only supplementary information. Minor comments L. 32-35 These two sentences say almost the same thing. L. 74 Delete “conjugation”. L. 353 Delete “feeding wasps”. L.488 DVSFN7 -> DVSNF7 L. 499 same -> similar Reviewer #2: The authors did several different experiments attempting to identify feasible dsRNA candidates for pest control in the Polistes dominula invasion. They used both injected and oral delivery, with and without protective substances for the dsRNA, all in adult P. dominula. They found that both the injected and oral dsRNA (at least when protected with lipofectamine) were able to significantly reduce expression of some genes, but that none of them increased mortality. I think this is a thorough and well-designed set of experiments. I appreciate the variety of approaches that were taken to investigate this question. I’m glad to see negative results like this coming through. This is the sort of thing that can easily be written off as a failed experiment, so it’s good to see the negative results being reported. I think that this article adds a very important contribution to the work being done with dsRNA in hymenoptera and to the work on the Polistes dominula invasion. Major issues: 1. Please include the method used to produce dsRNA. Minor issues: 1. Figure 1. The gene names trickling onto the picture makes it less clear. This would just benefit from some visual clean up. 2. Line 114: I think this should be “colonies were collected” 3. Given that previous studies have focused on targeting larval development with dsRNA and whole colonies were given dsRNA I find it disappointing that no data on larval development were reported and no measurements of gene expression were done in the larvae. I don’t think another experiment is required, but some discussion of this would be beneficial. ****** 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: Yudai Nishide Reviewer #2: Yes: Susan Weiner ******** [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.0279983.r001
Revision 1
8 Dec 2022 Author Response Replies to comments from Reviewer 1 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: No We understand that Reviewer 1 would like to see more details about the dsRNA production and the qPCR methods. We have now expanded on both these sections as suggested. 5. Review Comments to the Author Reviewer #1: Dear authors, This paper examines the effects of RNAi on the wasp, Polistes dominula. The experiments are reasonable and well-written. I have made some comments, please see below. Thank you very much for your time and useful comments. Much appreciated. It is strange that the results are shown here as Fig1; I think it would be better to separate the top and bottom of Fig.1 and just explain the methodology here using the bottom of Fig.1 (as a supplementary figure). Thank you for this suggestion. We have now separated the photographs from the graphs as the way in which we presented them together seemed distracting to the Reviewers and the Editor. There are now more figures in the manuscript but hopefully they make interpretation of the results easier. Was RNA extraction done with the whole body of the wasps? Yes, it was. We have clarified this fact now in lines 297-299. As you cited, some genes are upregulated by RNAi (Fig.1; L.333-335). Could this overlap between the region where dsRNA was created and the region where it was quantified? This is only the case in three of the genes targets evaluated, calmodulin (CaM), DVSNF7 and RPB7 for which the primers necessarily overlapped the dsRNA region as stated in Table S2. We explained that this is the case for CaM in a paragraph in lines 392-398 that reads: One benefit of this experiment was to confirm that wasps were feeding on the dsRNA. For the calmodulin (CaM) gene target, the qPCR primers unavoidably overlapped the dsRNA region. The RT-qPCR clearly demonstrated CaM dsRNA consumption as shown in the apparent massive increase in CaM gene ‘expression’ due to the qPCR primers binding to the reverse-transcribed dsRNA, rather than indicating an increase in gene expression (S1 Fig). This result also indicated that the lack of gene knockdown in other experiments is not due to the wasps not feeding on the dsRNA. We also provided a separate figure for calmodulin ‘expression’ in the Supplemental material (S1 Fig). Related to the question above, the sequence of the primers used for dsRNA synthesis and qPCR should be provided, even if it is only supplementary information. Reviewer 1 might have missed that all primer pairs used in our study are presented (and were in the original submission) in Supplementary Table 2. Minor comments L. 32-35 These two sentences say almost the same thing. You are right. We have combined the two sentences in one. L. 74 Delete “conjugation”. Done. L. 353 Delete “feeding wasps”. Done. L.488 DVSFN7 -> DVSNF7 Fixed, thank you! L. 499 same -> similar Changed. Replies to comments from Reviewer 2 Reviewer #2: The authors did several different experiments attempting to identify feasible dsRNA candidates for pest control in the Polistes dominula invasion. They used both injected and oral delivery, with and without protective substances for the dsRNA, all in adult P. dominula. They found that both the injected and oral dsRNA (at least when protected with lipofectamine) were able to significantly reduce expression of some genes, but that none of them increased mortality. I think this is a thorough and well-designed set of experiments. I appreciate the variety of approaches that were taken to investigate this question. I’m glad to see negative results like this coming through. This is the sort of thing that can easily be written off as a failed experiment, so it’s good to see the negative results being reported. I think that this article adds a very important contribution to the work being done with dsRNA in hymenoptera and to the work on the Polistes dominula invasion. Thank you so much for such encouraging words. Indeed, these are not the results we expected when we planned to develop an alternative to insecticides as a control method to manage the paper wasp invasion in New Zealand. However, we also see value in communicating these ‘negative results’ to colleagues to avoid repetition and time consumption. Thank you very much for your time and suggestions. Major issues: 1. Please include the method used to produce dsRNA. We have included the methods and details of dsRNA production in this revised version of the manuscript. Minor issues: 1. Figure 1. The gene names trickling onto the picture makes it less clear. This would just benefit from some visual clean up. Both reviewers and the editor suggested separating the photographs from the graphs so that has been done, thanks! 2. Line 114: I think this should be “colonies were collected” Indeed. Thanks for finding this typo. 3. Given that previous studies have focused on targeting larval development with dsRNA and whole colonies were given dsRNA I find it disappointing that no data on larval development were reported and no measurements of gene expression were done in the larvae. I don’t think another experiment is required, but some discussion of this would be beneficial. In our trials, we focused on adult wasps as in the field, should we provide dsRNA to wild wasps, we will be targeting the adult forager wasps to carry the dsRNA back to the colonies. We did collect data on larval mortality for the whole nest feeding trials but as most larvae emerged, we didn’t include these data in the manuscript. We have now included a short paragraph discussing this topic, lines 530-536 of the Discussion. Thank you again for your time and help, Mariana Bulgarella, on behalf of all co-authors Attachments Attachment Submitted filename: Response-to-reviewers.docx https://doi.org/10.1371/journal.pone.0279983.r002
20 Dec 2022 Decision Letter - Volker Nehring, Editor Gene silencing for invasive paper wasp management: Synthesized dsRNA can modify gene expression but did not affect mortality PONE-D-22-26973R1 Dear Dr. Bulgarella, 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, Volker Nehring Academic Editor PLOS ONE Additional Editor Comments (optional): It seems that the authors have made all info regarding the dsRNA available in the supplement. From what I can tell, the raw data obtained from the experiments (i.e. individual measures of gene expression levels, survival numbers for wasps) are still missing and I would suggest to add them as supplementary files. Reviewers' comments: https://doi.org/10.1371/journal.pone.0279983.r003
Formally Accepted
22 Dec 2022 Acceptance Letter - Volker Nehring, Editor PONE-D-22-26973R1 Gene silencing for invasive paper wasp management: Synthesized dsRNA can modify gene expression but did not affect mortality Dear Dr. Bulgarella: 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. Volker Nehring Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0279983.r004

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