PONE-D-19-22269

A scalable culturing system for the marine annelid Platynereis dumerilii.

PLOS ONE

Dear Dr. Ozpolat,

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.

Both reviewers considered this work valuable and containing information useful to the research community. Both reviewers (as well as myself) have provided comments for improving the clarity of the manuscript, and I hope the authors would follow those suggestions to revise the manuscript. 

We would appreciate receiving your revised manuscript by Nov 11 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,

Jr-Kai Sky Yu, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

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

1. We note that Figure(s) [7] in your submission contain copyrighted images. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:

1.    You may seek permission from the original copyright holder of Figure(s) [7] to publish the content specifically under the CC BY 4.0 license.

We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:

“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”

Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission.

In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].”

2.    If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.

Additional Editor Comments (if provided):

Additional comments from editor:

Line 194-195: What kind of algal culture medium was used? It would be useful to include the recipe or citation of the medium.

Line 198: Need to provide a better specification about the desired density of the algal cultures, for example, cell numbers/ml.

Line 203: How long can this algae stocks last at this refrigerated condition?

Line 209-213: How often do you conduct this check-up?

Figure 9 and its legend text, line 704- 714: I had a hard time understanding this figure; I cannot find the “black and white bars” from D-G. In addition, would it be possible to further calculate the percentage of total mature worms found in each month?

Line 723-724: It would be helpful to indicate the color (cyan and magenta) for respective mRNA injection results. Confocal “microscope”.

[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 manuscript “A scalable culturing system for the marine annelid Platynereis dumerilii’ by Kuehn et al. describes in detail the set up of small culturing units for this versatile annelid system. This enables any research group with access to natural seawater to rear these animals through the entire life cycle, and to control critical parameters like day/night cycles, moon cycles, light intensities, and test various feeding regiments. Thus, this set up may also open up this animal to new areas of research including various physiological and nutritional studies.

The description of the culturing method is detailed, and mostly sufficient, and the critical areas are pointed out. Although the described culturing method reaches a higher level of flexibility and control for several parameters, the authors point out where more control could/should be achieved in the future. Thus, this set up also offers a good entry point for critical improvements that can not be tackled in current larger, less flexible systems. I recommend potential publication of the manuscript after a few concerns have been addressed.

1. One parameter that is not sufficiently defined is the seawater described in the Methods as ‘full strength natural sea water’ (lane 140). Some of the experiments were performed in a laboratory in Vienna with apparently somewhat different ‘natural sea water of lower salinity’ which the authors acknowledge in lane 501, and it is important to include better definitions for the seawater here.

2. The authors also mention the important observation that the jelly production by fertilized eggs at the MBL culture to be much longer compared to eggs produced by the ‘European’ cultures (lane 502). As this narrows the window for injections of zygotes significantly, it should be discussed that this might be a critical issue if the main purpose of an investigation of this annelid includes injection of zygotes. It would be important to include data that documents the extension of jelly production which seems quite dramatic (from 1h to 1.5h).

3. ‘unknown gender worms’ (lane 224 and Figure 9): Not sure why the authors created this category as these unknown worms will mature into females and males a little later, and could be added to these gender categories accordingly? Seems unnecessary or requires better explanation.

4. Figure 4: Black out curtain construction: As light contamination is a critical issue, I noticed that there are no Velcro strips along the bottom of the curtain (Fig 4A). Are these curtains so fitting that no light can penetrate from the bottom? How reliable is the fitting of the curtains under daily operations?

5. Figure S4: Maybe this supplemental figure would be more useful by adding also the number of maturing worms or successful fertilizations each month to the figure?

Lane 118: .. that are affected

Lane 119: rephrase sentence

Reviewer #2: General comments: This is an all-round very detailed and in-depth resource for the culturing of the important model organism Platynereis dumerilii. The paper provides a novel design for small-scale cultures that can be varied in terms of environmental conditions etc.

Overall, this manuscript provides a lot of data to support the successful establishment of a small-scale culturing system which is able to produce mature individuals in good numbers. I believe it to be an excellent starting place for those looking to establish cultures of their own at whatever scale and it provides important data to support future efforts for standardisation of culturing techniques across laboratories working with this species.

Minor comments:

1) I feel that the standardisation of feeding method by using the spirulina diet shows promise however the authors should make it clear that this method is not yet fully optimised. The results presented do not provide enough information to give a comparison between this and traditional feeding methods due to feed volumes which may or may not contribute to inferior growth rates and the different sites of the experiments. The animals on the spirulina diet were not fed ad libitum.

2) It would be useful if the authors mentioned the potential impact of several ‘unseen’ water quality parameters such as pH, nitrites, nitrates, ammonia; all of which would impact the long term health and prosperity of the culture. These parameters will potentially all be influenced by the feeding regime.

3) Do the authors also have data about the size of the worms at maturity? It would be useful to add this to the paper, if such data exist.

100 "transgenesis and genetic tools" - it would be good to mention that homozygous knockout lines have been established, first with ZFN, then TALEN, and more recently with CRISPR, and cite the relevant papers

101 "behavioral tracking [8,16]" - under behavioral tracking, it would be good to mention work also on the larval stages

101 "and live imaging [5] - please cite also work on neuronal activity imaging

118 “This is particularly important for studying biological processes are affected dramatically by nutrition” incomplete sentence

177-185: What is the size / what are the dimensions of a large box? And what volume of water is added?

182-184: What bubbling rate was used as this could impact water quality or organism behaviour. If this was not measured directly, was it gentle / vigorous etc?

223-231: Please define ‘mature worms’ here

288-290: You mention that you ‘stack’ boxes on a shelf – do you have any information as to whether this impacts culture conditions, for example light levels for the top box in the stack vs the bottom?

325: Please reference who the ‘others’ are that you followed a standardised protocol from.

423-427: Do you have any additional information or opinions on why the algal growth may have been different when comparing the two locations? For example, different sources of seawater / different filtration etc?

455-456: Do you have any data or quantification to reinforce the ‘appearance’ of a larger size of larvae?

466-467: In how many boxes / what culture size are this number of mature worms being produced?

470-473: What lighting regimen were the initial boxes on if they were not on the one you have detailed?

474-475: For these 22 mature worms, what timescale is this over? A day? A cycle?

501-503: The authors mention differences in salinity but what salinities are these and how large is the difference between the two?

511-514: I feel this needs to be discussed in a little more detail as the authors haven’t mentioned the inability to culture in artificial sea water before this point. Is there any data / information / references which compare natural and artificial sea water or discuss not being able to culture Platynereis in artificial sea water?

**********

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.

A scalable culturing system for the marine annelid Platynereis dumerilii.

PONE-D-19-22269R1

Dear Dr. Ozpolat,

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,

Jr-Kai Sky Yu, Ph.D.

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

**********

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: Kuehn et al. address every of my previous minor concerns in the revised manuscript “A scalable culturing system for the marine annelid Platynereis dumerilii’ providing a detailed valuable description of a set up for small culturing units for this versatile annelid system. For all the reasons that I mention in my previous review I recommend publication of the manuscript in PloS ONE.

**********

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