PONE-D-19-17240

The effect of bacteria on settlement and metamorphosis in planula-larvae of the octocoral Rhytisma fulvum fulvum

PLOS ONE

Dear Prof Otero,

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.

As you can see both reviewers ask for some minor changes and correction, so please modify your manuscript according to these comments and resubmit it

We would appreciate receiving your revised manuscript by Sep 09 2019 11:59PM. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Hector Escriva, PhD

Academic Editor

PLOS ONE

Journal Requirements:

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

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

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. In your Methods section, please provide additional location information of the collection sites, including geographic coordinates for the data set if available.

3. 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.

4. Thank you for stating that “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript” in your financial disclosure.

Please also provide the name of the funders of this study (as well as grant numbers if available) in your financial disclosure statement.

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

5. 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.

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: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

**********

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: 1. While I wouldn't ask for repeat of these experiments, given the challenges of working with coral larvae, I'm a little concerned with the conclusions regarding vertical transmission of bacteria from the parental colony. While a previous publication of a co-author does show the inhibitory effects of settlement from the addition of antibiotics, given the annual variation in larvae, there's insufficient evidence to preclude the effects of settlement and metamorphosis resulting from vertically transmitted microbes.

2. While I believe the Mann-whitney test is appropriate for these data, please provide cutoffs in text and in the figures. There's often mention of significance, but statistical values are missing. And although there does appear to be some trends, values are necessary.

3. Please provide values for the statistics on figures, in text, and figure descriptions.

4. Grammatical suggestions, as well as specific points addressed above are provided by line.

Line 45: remove "a" before substantial

Line 46: There are several sentences within the first paragraph (including this line) that are rather long (run-on). Perhaps the paper will read better by separating the sentence? Another instance is Line 58/59.

Line 177: Perhaps it's beneficial to the reader to write out what BCCM/LMG stands for?

Line 184: Please clarify what is being exchange here. I understood it to be bacterial containing seawater, but are filtered fractions from the same batch used for all exchanges over the study period and stored? Or is it a new batch of water from the same source, which is filtered prior to the exchange?

Line 192: insert "of" between "comprised" and "coccoid"

Line 202: missing "the" before "data"

Line 208: remaining --> remainder

Line 225: Please indicate the statistical cut-off of significance. Figure S1 also lacks information regarding the cut-off. Also, what exactly is this line referring to? It seems all larvae died by day 12, regardless of the light condition, although those in light:dark cycle at a greater rate.

Line 230: Please provide the statistics. Were there any statistically significant differences in survival rates for data presented in Figure 2?

Line 238: Why is the advanced stage for Figure 3 not categorized as short and long tentacles? Please provide this data. Additionally, please provide statistics for figure 3.

Line 241: rate --> rates

Line 241: could be --> were

Line 241: It'd be great to have some values provided for this statement regarding survival, as in, what percentage did survive under stagnant conditions?

Line 264: This paragraph refers to light:dark but it's unclear until I read line 314 that light:dark in this section is referring to un-autoclaved SW. Please make this more clear.

Line 273: Is this line referring to figure 5?

Line 285/286: I don't see this is figure 4. Perhaps I should read it as "by" day 35? On day 35 for 2.7 uM-dark, metamorphosis is below 70%.

Line 293: Please provide statistics.

Line 330: Are there any statistics for this?

Given the complexity of the microbiome of the seawater, perhaps some of the observed effects of the fractions were due to competitive interactions between microbes within the seawater resulting in exclusion of pathogens. Therefore, while no "beneficial" bacteria directly interacted with the larvae, pathogenic/harmful bacteria were also prevented from affecting the larvae.

Line 348: insert "of" before 80-90%

Line 349: Is this line referring to Figure S4?

Line 352: remove "the" before "larval ..."

Line 353: I believe this is referring to Figure S5?

Line 356: insert space after "...P14)"

Line 359: Please provide method in more detail, preferably in methods section.

- What does this mean?

Line 361: Planule --> planulae

Line 349 - 351: Is this reduction in metamorphosis significant? Also, it seems that although more larvae do achieve the early metamorphosis stage in unchanged water, those under exchanged water conditions achieved advance metamorphosis. Can it really be considered retardation if some larvae are capable of reversion? Also, is the statement in line 352 contradictory to that in line 349-351?

Line 365: Insert "uM" after 0.22

Line 396: I think either "unraveling" or "revealing" was meant to be used here, rather than "unrevealing"

Line 407: As mentioned in the beginning, there's no evidence on what bacteria are vertically transmitted or horizontally acquired. I don't think it's appropriate to discuss acquisition, but rather potential interactions

Line 449: It would have been nice to have a negative control in which larvae were maintained with antibiotics.

Line 450: It's oogenesis and not embryogenesis?

Line 501: There seems to be some information missing after "addition of ..."

The authors investigated seawater filtered through various pore sizes on larval survival and settlement/metamorphosis in order to determine if microbes can have a beneficial role. The authors found survival rates and settlement/metamorphosis of larvae to be partially increased in some instances by the presence of microbes. It's somewhat unclear whether the claims made in the discussion regarding the benefits of particular bacterial species are real. However, there are interesting findings that could certainly provide avenues for further exploration.

Additional questions I had were whether the metamorphic stage of the polyp is a good indication of timing of initiation. Does development to the long tentacle stage occur at the same rate, or can some polyps remain at a short tentacle stage for longer periods?

Reviewer #2: In this manuscript, Freire et al. investigate the effect of bacteria on larval settlement and metamorphosis of the soft coral species Rhytisma fulvum fulvum sampled from the Red Sea. Using an in vitro cultivation system, they assess survival, settlement and metamorphosis performances of wild caught planulae, comparing different types of filtered, autoclaved and bacteria-enriched seawater. This study provides interesting data on octocoral larval settlement, for which little is known. I would suggest few changes in the representation and interpretation of data before publishing the manuscript.

1) In the Material and Method section, the Authors write: “Since data did not fulfill the conditions of normality and homoscedasticity, and could not be improved by transformation, they were analyzed using the non-parametric Wilcoxon-Mann-Whitney test” (line 202). In all graphs, however, spread of data is represented using the mean and standard deviation, generally a bad representation for non-normally distributed data, since they can be strongly affected by extreme values. A more appropriate alternative would be to use median and interquartile range as a measure of dispersion. If mean and standard deviation are to be kept, it should be demonstrated why they provide a good representation of the analyzed data.

2) One of the main conclusions of this study is that vertically inherited bacteria (acquired during the brooding period) are sufficient to induce settlement. This is based on the observation that wild-caught planulae put into sterile (autoclaved) water can still settle and metamorphose, while previous reports showed that planulae put in seawater containing antibiotics do not (Ben-David-Zaslow and Benayahu 1998). From this, the Authors conclude that “vertical transfer of the bacteria to the larvae during oogenesis is sufficient to support larval settlement and development in R. f. fulvum” (line 450). While the Authors are likely correct in their conclusions, experimental data proving this claim are still missing, and it is still possible that environmental bacteria acquired during the course of the experiments were involved in larval settlement. A key control would be to place the planulae first in autoclaved-seawater containing antibiotics and then to replace the medium with autoclaved-seawater, expecting that those planulae would never settle and metamorphose, contrary to those that have not been treated with antibiotics. Considering the difficulty in obtaining new biological material of this species I would not suggest the Authors to perform new experiments. Instead, I would suggest the Authors to be more cautious in the Abstract and Discussion.

3) The Authors make the interesting observation that planulae maintained in constant darkness have initially a higher chance to settle but then lower chance to complete metamorphosis, compared to planulae under a day-night cycle. They conclude that “Light is beneficial for planulae development after day 20 […] indicating the possible presence of beneficial phototrophic bacteria in the coral microbiome” (line 36). However the Authors do not provide any proof for the presence of those phototrophic bacteria and their role in this process. I think the Authors should be more cautious and discuss alternative scenarios – in particular the possibility that the metamorphosing planulae themselves positively respond to light. Many types of photosensitive cells have indeed been described in cnidarians, including at the planula stage in some species.

4) A few typos can be found throughout the text. Few examples (please correct):

Line 27 “in the absence nature reef substrata”

Line 129 “the resultis indicate”

Line 135 “(Forskål 1975)”

I would also suggest changing the title to: “The effect of bacteria on larval settlement and metamorphosis in the octocoral Rhytisma fulvum fulvum”.

**********

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 to be viewed.]

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 us at figures@plos.org. Please note that Supporting Information files do not need this step.

The effect of bacteria on planula-larvae settlement and metamorphosis in the octocoral Rhytisma fulvum fulvum

PONE-D-19-17240R1

Dear Dr. Otero,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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.

With kind regards,

Hector Escriva, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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

Reviewer #2: 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

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: 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

Reviewer #2: No

**********

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

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 authors addressed the issues brought up by the reviewers. While they acknowledge the issues in replicate numbers, the previous interpretations of these data have been addressed. As the authors point out, these findings can inform future experiments and the manuscript should be accepted for publication.

Reviewer #2: The Authors have appropriately addressed all the reviewer comments.

Related to 4. - According to the PLOS Data policy, the data point values should be made available. I could not find this information in the files provided by the Authors despite their claim that "All relevant data are within the manuscript and its Supporting Information files."

**********

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