Sodium molybdate does not inhibit sulfate-reducing bacteria but increases shell growth in the Pacific oyster Magallana gigas

Roxanne M. W. Banker; Jacob Lipovac; John J. Stachowicz; David A. Gold

doi:10.1371/journal.pone.0262939

Peer Review History

Original SubmissionSeptember 13, 2021
5 Nov 2021 Decision Letter - José A. Fernández Robledo, Editor PONE-D-21-29631Sodium molybdate does not inhibit sulfate-reducing bacteria but increases shell growth in the Pacific oyster Magallana gigasPLOS ONE Dear Dr. Banker, 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 Dec 20 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. 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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. Additional Editor Comments: Dear Dr. Banker, let me first apologize for the time that took to send you back the reviews, I have had difficult finding reviewers willing to review your manuscript. I have reviewed it my self as a non specialist on the subject and I have no major comments and some of the edits are already indicated for the other reviewer. Overall, I find the experimental procedures and presentation of results were very clear. The oysters spat was produced following hatcheries procedures. Still, the reviewer rises some questions for you to address regarding, e.g., "the experimental setup was not oxygen-deprived, and often sulfate-reducing prokaryotes thrive in anaerobic environments." Please, address the concerns. Line 188, add the reference for the primers used. Take also the time to review the references as some of them are not in the same format (e.g., capitalization of the title, journal abbreviation, italics in scientific names). -j [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: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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 ****** 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 ****** 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: Overall Comments: The main goal of this manuscript was to characterize the effects of sodium molybdate on the Pacific oyster M.gigas based on the fact that sodium molybdate would competitively replace sulfate ions, inhibiting activity by sulfate-reducing bacteria (SRBs) and potentially leading to decreased oyster growth due to the influence of these bacteria on calcification. But this manuscript lacks a critical component when it comes to sulfate-reducing bacteria and the experimental setup. The experimental setup was not oxygen-deprived, and often sulfate-reducing prokaryotes thrive in anaerobic environments. This is evident from the presence of SRBs in low abundances in the system. Since SRBs are present in such low abundances in the system, it is hard to reach any conclusion. Microbiome harvesting, DNA extractions, and sequencing depth will have a huge impact on these lower abundance data. The changes in different taxa can also be contributed by the presence of multiple copies of the 16S rRNA gene in a bacterium which was not accounted for in this study. Other critical questions are 1) Do all SRBs promote calcification? 2) Is there any other microbial population in the system which can affect oyster growth or promote calcification? Other microbial populations which were abundant in the system were not reported/discussed in the manuscript. Those microbial populations can also play role in promoting the growth of Oysters. So, the antagonistic behavior of sodium molybdate could have been countered by the activity of other microbial populations. I feel that the starting microbial community will also be a determining factor for supporting/contradicting the hypothesis. Specific comments: Line 49: “A previous study showed ……” in place of “[21] showed”. Then the reference should be added at the end of the sentence. Line 61: 1500 L Line 78: 20 µm Line 82: What were the criteria for selection based on egg quality? Line 145: 35 µm Line 181: Elaborate the method heading Line 181: S will be in uppercase for 16S; here and in other places. Line 205: Add reference for Decontam’s prevalence method. Line 210: >25,000 ASVs or a total of >25,000 ASV abundances? Line 223: Bray-Curtis dissimilarity Line 532: What does concentration of bacteria mean? Will it be abundance? ****** 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 [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. 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Revision 1
4 Jan 2022 Author Response Response to Reviewers Editor Comments: Line 188, add the reference for the primers used. Change made, Parada reference added. Take also the time to review the references as some of them are not in the same format (e.g., capitalization of the title, journal abbreviation, italics in scientific names). The references have been reviewed and fixed. Reviewer #1: Overall Comments: The main goal of this manuscript was to characterize the effects of sodium molybdate on the Pacific oyster M.gigas based on the fact that sodium molybdate would competitively replace sulfate ions, inhibiting activity by sulfate-reducing bacteria (SRBs) and potentially leading to decreased oyster growth due to the influence of these bacteria on calcification. But this manuscript lacks a critical component when it comes to sulfate-reducing bacteria and the experimental setup. The experimental setup was not oxygen-deprived, and often sulfate-reducing prokaryotes thrive in anaerobic environments. This is evident from the presence of SRBs in low abundances in the system. Since SRBs are present in such low abundances in the system, it is hard to reach any conclusion. The reviewer brings up an important caveat regarding the role of SRBs in animal biomineralization. However, there is ample evidence that SRBs are capable of precipitating calcium carbonate in aerobic environments, including oxygenated sediments and microbial mats (e.g. Visscher et al. 2000; Jørgensen BB 1994; Canfield D & Des Marais DJ 1991; Cypionka et al. 2000). There are also plenty of opportunities for anaerobic microenvironments to develop in the oyster shell, either in fluid-filled spaces within the shell or during times of oyster valve closure (Vermeij 2013; 2020). Our goal was to study the role of SRBs in shell formation under natural conditions, so artificially creating anaerobic environments would not make sense in this context. We have added a new paragraph in the introduction to bring up these relevant points and references. Microbiome harvesting, DNA extractions, and sequencing depth will have a huge impact on these lower abundance data. The changes in different taxa can also be contributed by the presence of multiple copies of the 16S rRNA gene in a bacterium which was not accounted for in this study. We acknowledge that 16S rRNA gene copy number variability could cause certain microbial taxa to appear more abundant than is actually the case. This is a challenge for all microbiome studies that use the amplicon sequencing approach. How to correct for gene copy number remains an open question and tools developed for this purpose have been unreliable thus far (e.g. Louca et al., 2018; Stark et al., 2021). For these reasons, we chose not to correct for 16S rRNA copy number in this study. We added text and references to the Methods subsection “Microbiome Analysis and Visualization” to note this concern and justify our decision to not perform corrections. Other critical questions are 1) Do all SRBs promote calcification? There is compelling evidence that SRBs as a group can promote calcification, but there is uncertainty in the extent to which particular clades do so, the environments in which they can do it, and their role in animal calcification. Our new paragraph in the introduction includes these points. 2) Is there any other microbial population in the system which can affect oyster growth or promote calcification? There are almost certainly other microbial taxa that affect oyster calcification. The entire microbial community dictates the alkalinity of the microenvironment and the subsequent probability that minerals will precipitate. While our focus in this project was on SRBs, we have added this caveat to the discussion. Other microbial populations which were abundant in the system were not reported/discussed in the manuscript. Those microbial populations can also play a role in promoting the growth of Oysters. So, the antagonistic behavior of sodium molybdate could have been countered by the activity of other microbial populations. We do provide a summary report of the microbial clades that exhibit significant differences between treatments (see section “Patterns and differences in microbial community composition”, Figure 7, and Table S6). As mentioned in the previous response, it is certainly possible that some of these non-SRB clades play a role in calcification. While we have no direct evidence of that in this study, we allude to the possibility in the discussion (see our response to the previous point). We are also preparing a follow-up study that will allow us to compare the microbial community in our experimental oysters to animals in other environmental conditions that produce similar changes in shell structure. That has the potential to reveal other candidate microbes that are broadly associated with shell growth. We appreciate the Reviewer’s suggestion for this analysis. I feel that the starting microbial community will also be a determining factor for supporting/contradicting the hypothesis. We do have microbiome data from pre-settlement larvae. However, the microbial community in larvae is dramatically different from post-settlement oysters, and provides little information on “starting” conditions relevant to the interpretation of the experiment. Transcriptome and 16S data from the larval treatment are publicly available through our NCBI Bioproject, and we anticipate discussing this data in a follow-up manuscript. Despite these limitations, we feel the control population provides sufficient insight for us to test the impact of sodium molybdate on the oyster microbial community. If the reviewer wishes to expand on how they think our hypotheses could be strengthened with information from the “starting” community, we would be happy to consider it. Specific comments: Line 49: “A previous study showed ......” in place of “[21] showed”. Then the reference should be added at the end of the sentence. Change made. Line 61: 1500 L Change made. Line 78: 20 μm Change made. Line 82: What were the criteria for selection based on egg quality? Text added: Eggs were considered good quality if they had a regular, round shape with a plump appearance, as opposed to ovate or wrinkly. Line 145: 35 μm Change made. Line 181: Elaborate the method heading Changed to “16S rRNA Sample Preparation and Sequencing” Line 181: S will be in uppercase for 16S; here and in other places. Change made. Line 205: Add reference for Decontam’s prevalence method. Reference added: Davis NM, Proctor D, Holmes SP, Relman DA, Callahan BJ (2017). “Simple statistical identification and removal of contaminant sequences in marker-gene and metagenomics data.” bioRxiv, 221499. doi: 10.1101/221499. Line 210: >25,000 ASVs or a total of >25,000 ASV abundances? Text changed from “but all remaining samples had >25,000 ASVs” to “but each remaining sample had >25,000 ASVs” Line 223: Bray-Curtis dissimilarity Change made. Line 532: What does concentration of bacteria mean? Will it be abundance? Response: “concentration” changed to “abundance” Attachments Attachment Submitted filename: Gold-Response-to-reviewer.pdf https://doi.org/10.1371/journal.pone.0262939.r002
10 Jan 2022 Decision Letter - José A. Fernández Robledo, Editor Sodium molybdate does not inhibit sulfate-reducing bacteria but increases shell growth in the Pacific oyster Magallana gigas PONE-D-21-29631R1 Dear Dr. Gold, 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, José A. Fernández Robledo, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Thank you for addressing the reviewer's comments including mine. Stay healthy and safe. -j Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed ******** 2. 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 ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes ****** 4. 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 ****** 5. 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 ****** 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: (No Response) ****** 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 https://doi.org/10.1371/journal.pone.0262939.r003
Formally Accepted
25 Jan 2022 Acceptance Letter - José A. Fernández Robledo, Editor PONE-D-21-29631R1 Sodium molybdate does not inhibit sulfate-reducing bacteria but increases shell growth in the Pacific oyster Magallana gigas Dear Dr. Gold: 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. José A. Fernández Robledo Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0262939.r004

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