Phylogenetic evidence for the invasion of a commercialized European Phasmarhabditis hermaphrodita lineage into North America and New Zealand

Dana K. Howe; Anh D. Ha; Andrew Colton; Irma Tandingan De Ley; Robbie G. Rae; Jenna Ross; Michael Wilson; Jiří Nermut; Zhongying Zhao; Rory J. Mc Donnell; Dee R. Denver

doi:10.1371/journal.pone.0237249

Peer Review History

Original SubmissionMarch 4, 2020
2 Jun 2020 Decision Letter - Bi-Song Yue, Editor PONE-D-20-06376 Phylogenetic evidence for the invasion of a commercialized European Phasmarhabditis hermaphrodita lineage into North America and New Zealand PLOS ONE Dear Dr. Howe, 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. Please submit your revised manuscript by Jul 17 2020 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. 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Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide. 3. In your Methods section, please provide additional details regarding the collected slugs used in your study and ensure you have described the source. For more information regarding PLOS' policy on materials sharing and reporting, see https://journals.plos.org/plosone/s/materials-and-software-sharing#loc-sharing-materials. 4. In your Methods section, please provide additional location information of the collection sites, including geographic coordinates for the data set if available." 5. 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 collection sites access and, if no permits were required, a brief statement explaining why. [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: I Don't Know 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 have demonstrated similarities between Nemaslug and P. hermaphrodita collected in the USA and NZ. There were differences between the nematodes in this clade and another P. hermaphrodita clade constituted by collections from England and Czech Republic. For these reasons the authors suggest that study has “provided evidence that the nematodes deriving from the Nemaslug biocontrol product have invaded parts of the world where its use is prohibited by regulatory agencies and not commercially available.” The bacterium associated with Nemaslug was not detected in P. hermaphrodita strains collected in the USA. I tend to agree with the authors’ conclusion. However, I might have made the conclusion a bit more tentative. The sample size of P. hermaphrodita is small, with collections from only four countries. The absence from collections of P. hermaphrodita from the USA and NZ of the bacterium associated with Nemaslug weakens the conclusion. Is there any type of statistical test by which the authors could test their conclusion? The manuscript is refreshingly clear and therefore easy to read. Reviewer #2: This is a well written and presented manuscript on an important and critical topic related to the environmental and ecological impacts of deployment of non-native biological control agents of slug pests. The literature is rife with the unexpected and deleterious consequences of deployment of non-native organisms for control of pest species. Here the authors present compelling phylogenetic data to support the idea that Phasmorhabditis hermaphrodita has been spread from its native region (Europe, registered as NemaSlugTM) to New Zealand and North America by deployment of a commercial product unregistered in these areas. This is not only worrisome from a regulatory standpoint, but could have grave ecological impacts on native, non-target gastropods species. The methods employed by the authors are robust and the use of mtDNA as a tool was a good choice for phylogenetic analysis. Particularly compelling was the observation that P. hermaphrodita has only recently appeared in Oregon (since 2007). Although the authors rightly point out that these data might have not detected a panmictic species, the data presented in this manuscript strongly suggest introduction rather than non-detection. Although the phylogenetic data strongly support the introduction hypothesis, the complete absence of the bacterial symbiont Moraxella is perplexing. Because the commercial NemaSlug products contain Moraxella, it is not clear why there is no evidence of this bacterium from any of the populations evaluated. The authors do explain that P. hermaphrodita can associate with multiple bacterial genera in the wild and in the lab, but this does not explain the total absence of Moraxella in the relatively short time period since putative introduction. In my opinion, the manuscript would be stronger with more discussion of this finding. For example, did the bacterial species found in the P. hermaphrodita samples simply outcompete Moraxella, or was Moraxella unsuited to survive the new environment? Having said that, I believe this is an important manuscript and should be published. ****** 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.0237249.r001
Revision 1
17 Jul 2020 Author Response Thank you for the opportunity to revise our manuscript and address these comments. Below I have included the editor’s and reviewers’ comments in black font with my responses in blue font, for clarity. Editor’s comments: 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 Thank you for the style templates. I corrected the title, indentations, subheading, and adjusted the formatting for Table 1 to match the examples. 2. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide. We have competed our GenBank submission and completed the sentence with the accession numbers: MT179848 – MT179854 for 18S sequences; MT472183 – MT472270 for mtDNA sequences. These will be “live” with the acceptance of this manuscript. 3. In your Methods section, please provide additional details regarding the collected slugs used in your study and ensure you have described the source. For more information regarding PLOS' policy on materials sharing and reporting, see https://journals.plos.org/plosone/s/materials-and-software-sharing#loc-sharing-materials. Thank you for the suggestion about sharing materials. The collected slugs don’t culture well in the lab and are generally not maintained. Most of the nematodes are available from each individual’s labs (identified in Table 1), but there isn’t a central repository for natural nematode collections. 4. In your Methods section, please provide additional location information of the collection sites, including geographic coordinates for the data set if available." Many of our collection sites were businesses or private farmers. We would prefer not to include the business names or exact addresses/coordinates of these places surveyed because there is a certain amount of trust involved in these entities allowing us to survey, and we don’t want to betray that by “outing” them. 5. 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 collection sites access and, if no permits were required, a brief statement explaining why. We appreciate you pointing out this oversight to us. We added a sentence about permits in the methods. Some locations required them while other locations did not. 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: I Don't Know Reviewer #2: Yes ------------------------- 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy [1] 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 have demonstrated similarities between Nemaslug and P. hermaphrodita collected in the USA and NZ. There were differences between the nematodes in this clade and another P. hermaphrodita clade constituted by collections from England and Czech Republic. For these reasons the authors suggest that study has “provided evidence that the nematodes deriving from the Nemaslug biocontrol product have invaded parts of the world where its use is prohibited by regulatory agencies and not commercially available.” The bacterium associated with Nemaslug was not detected in P. hermaphrodita strains collected in the USA. I tend to agree with the authors’ conclusion. However, I might have made the conclusion a bit more tentative. The sample size of P. hermaphrodita is small, with collections from only four countries. The absence from collections of P. hermaphrodita from the USA and NZ of the bacterium associated with Nemaslug weakens the conclusion. Is there any type of statistical test by which the authors could test their conclusion? The manuscript is refreshingly clear and therefore easy to read. Thank you for your helpful comments. We agree that we have a limited number of sample locations where P. hermaphrodita is found, so we revised our abstract to not extend our findings too broadly. As for the lack of Moraxella osloensis, multiple studies have found that it is quickly lost in culturing. Unlike entomopathogenic nematodes, it is not an obligate symbiosis. We added a paragraph with more explanation in the discussion (see pg 19). Reviewer #2: This is a well written and presented manuscript on an important and critical topic related to the environmental and ecological impacts of deployment of non-native biological control agents of slug pests. The literature is rife with the unexpected and deleterious consequences of deployment of non-native organisms for control of pest species. Here the authors present compelling phylogenetic data to support the idea that Phasmorhabditis hermaphrodita has been spread from its native region (Europe, registered as NemaSlugTM) to New Zealand and North America by deployment of a commercial product unregistered in these areas. This is not only worrisome from a regulatory standpoint, but could have grave ecological impacts on native, non-target gastropods species. The methods employed by the authors are robust and the use of mtDNA as a tool was a good choice for phylogenetic analysis. Particularly compelling was the observation that P. hermaphrodita has only recently appeared in Oregon (since 2007). Although the authors rightly point out that these data might have not detected a panmictic species, the data presented in this manuscript strongly suggest introduction rather than non-detection. Although the phylogenetic data strongly support the introduction hypothesis, the complete absence of the bacterial symbiont Moraxella is perplexing. Because the commercial NemaSlug products contain Moraxella, it is not clear why there is no evidence of this bacterium from any of the populations evaluated. The authors do explain that P. hermaphrodita can associate with multiple bacterial genera in the wild and in the lab, but this does not explain the total absence of Moraxella in the relatively short time period since putative introduction. In my opinion, the manuscript would be stronger with more discussion of this finding. For example, did the bacterial species found in the P. hermaphrodita samples simply outcompete Moraxella, or was Moraxella unsuited to survive the new environment? Having said that, I believe this is an important manuscript and should be published. Thank you for your thoughtful comments. We added a paragraph with more explanation in the discussion (see pg 19) to help clarify the lack of Moraxella osloensis in the newly acquired samples. Multiple previous studies have found that it is quickly lost in culturing because, unlike entomopathogenic nematodes, it is not an obligate symbiosis. https://doi.org/10.1371/journal.pone.0237249.r002
23 Jul 2020 Decision Letter - Bi-Song Yue, Editor Phylogenetic evidence for the invasion of a commercialized European Phasmarhabditis hermaphrodita lineage into North America and New Zealand PONE-D-20-06376R1 Dear Dr. Howe, 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, Bi-Song Yue, Ph.D Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0237249.r003
Formally Accepted
4 Aug 2020 Acceptance Letter - Bi-Song Yue, Editor PONE-D-20-06376R1 Phylogenetic evidence for the invasion of a commercialized European Phasmarhabditis hermaphrodita lineage into North America and New Zealand Dear Dr. Howe: 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. Bi-Song Yue Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0237249.r004

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