PONE-D-24-31966Monitoring bay-scale bivalve aquaculture ecosystem interactions using flow cytometryPLOS ONE

Dear Dr. Sharpe,

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PLOS ONE

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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

Reviewer #2: Yes

Reviewer #3: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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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

Reviewer #3: Yes

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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

Reviewer #3: Yes

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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: Review of PONE-D-24-31966

This study used flow cytometry to investigate natural spatial and temporal drivers of plankton dynamics in coastal embayment’s with shellfish aquaculture leases. Although environmental forcing appeared to be a strong driver of plankton dynamics there was some evidence that bivalve aquaculture may reduce near-lease phytoplankton abundance and favor bacterial growth.

General comments

I want to first acknowledge that this paper is not exactly within my areas of expertise so please take my comments accordingly.

I accepted the review because I was interested in the topic with regards to the impacts of shellfish aquaculture on marine nearshore habitats. Although the first two paragraphs of the introduction focused on shellfish aquaculture including effects on plankton dynamics that may affect other commercially important species like lobster, the effect of shellfish aquaculture on plankton dynamics was only tangentially assessed.

Given this, I suggest the authors re-work their paper to emphasize their study addresses some of the environmental drivers that may confound investigating the effect of shellfish aquaculture on plankton dynamics, as they didn’t explicitly test for a shellfish effect.

Reviewer #2: This manuscript investigates the spatio-temporal variations in bacteria (i.e., bacteria and archaeal communities) and pico- and nanophytoplankton composition in four contrasted bivalve aquaculture embayments using flow cytometry.The proposed work is a solid attempt to show that bivalve aquaculture may reduce near-lease phytoplankton abundance and favor bacterial growth, even if environmental forcings appeared to be a strong driver of spatio-temporal trends of these communities. In addition, this manuscript provides an interesting and complete discussion on aquaculture monitoring and their effects on more globally benthic-pelagic coupling processes and microbial biogeochemical cycling.

Overall, this manuscript is well written and is an interesting contribution that is wholly suitable with minor revisions for publication in PlosOne. There are indeed few areas of the manuscript that could be improved or clarified.

First, the number of items (13 figures + 2 tables) in the main text is a bit disproportional. Not all the figures are needed to be placed in the main text and the authors may consider to move some of them as supplementary material or detail them a bit more than in the current version of the manuscript in the results section, or even redo some of them. For instance, the three NMDS plots could be eventually placed in a single figure.

L87-90: Another non-negligeable limit of the technics is the low taxonomic resolution that can be obtained from it. With flow cytometry, researchers can only quantify bacteria and phytoplankton at a wide taxonomic level (i.e., assemblages based on size, pigmentation or level of nucleic acid content) while it has been shown for instance that bivalves may alter the relative biomass of certain phyla/species such as diatoms by their selective grazing activity (Lucas et al., 2016, doi: 10.3389/fmars.2016.00014).

L190: Was there a prefiltration on 20 um to only catch and measure the < 20 um planktonic community?

L220-222: The authors have studied the planktonic community composition (abundance of planktonic taxa) using flow cytometry. It is yet unclear what the authors meant by taxa here (which suppose a taxonomic resolution close to the genus level) and how they have achieved this taxonomic identification thanks to the glow cytometry scatters they obtained... More details need to be given before in the M&M section on how the authors have categorized the studied planktonic community at the taxa level. However, if by “taxa” the authors meant, for instance for bacteria, the differentiation between HNA and LNA, the term “assemblage” will be more adapted throughout the text, I guess.

L255-258: Please specify in the legend caption the bacterial and phytoplanktonic composition were assessed by flow cytometry. As mentioned above it is unclear what the authors meant by “community composition” and what are the “taxa” they have considered in their analysis. It is then difficult to judge on the pertinence of the NMDS analysis done in that figure.

Reviewer #3: Review of PONE-D-24-31966

This article reports on spatio-temporal variability in planktonic bacteria and phytoplankton communities in 4 Eastern Canadian embayments with a link with aquaculture intensity. The ultimate goal of the article is to inform on proper sampling design to detect impacts of aquaculture activities on bay-scale pelagic primary producers. I feel the article is well written (I added several minor comments and edits in the manuscript file attached), is easy to follow and the statistical analyses and result presentation are generally adequate. However, I have more severe criticism about the sampling design and the link between the ultimate objective and how the study was designed.

First, I appreciate the challenges of designing a sampling scheme that allows comparisons among very different environments. It is clear that one cannot sample plankton at 3 different depths if the water depth does not allow it. However, the variations in the sampling design among the different embayments reported here is huge (different number of samples, different number of sampling dates, different years, different sampling locations in relation to position of leases, different depths, different tidal cycles, and different variables measured). In my opinion, it would be important to discuss why this final design was chosen and why, for example, it was decided not to take routine measurements (such as Chl a, POM, SPM in Nova Scotia) or sample the two New Brunswick sites on two different years rather than simultaneously.

From the last two sentences of the abstract, I was expecting the discussion to focus on details pertaining to aquaculture impacts and how to detect them. I found that a large part of it instead attempted to identify causes (other than aquaculture) for the patterns observed. In my opinion, the discussion should be framed to discuss, in light of the levels of spatio-temporal variability observed in the present study, how do we move forward to better understand the impact of aquaculture on ecosystem functioning using monitoring programs. There are parts of the discussion that do so, but in my opinion the topic could be expanded to better fit the stated aim of the article. I see value in the data presented in terms of a baseline to build on to come up with a proper design to detect bay-scale effects if they indeed exist. Reading the title and the abstract, I was expecting the article to describe a sampling design, that uses flow cytometry, that was capable of separating the farm effects from all other factors shaping planktonic communities. Clearly, this is not the case, and the article could be re-framed with a ‘lessons learned’ format.

I have on concern about the statistical analyses. From my understanding, Mann-Withney U tests are pairwise tests. When comparing multiple levels of a factor (for example, multiple dates within an embayment) the type-1 error rate is inflated. I think a Bonferonni correction or others methods should be used to keep the error rate constant.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Attachments

Attachment

Submitted filename: PONE-D-24-31966.pdf

Monitoring bay-scale ecosystem changes in bivalve aquaculture embayments using flow cytometry

PONE-D-24-31966R1

Dear Dr. Sharpe,

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.

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Kind regards,

Qing Wang

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 #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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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 #2: Yes

Reviewer #3: Yes

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

Reviewer #2: Yes

Reviewer #3: 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 #2: Yes

Reviewer #3: Yes

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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 #2: Yes

Reviewer #3: 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 #2: The authors have done a thorough work with the revised version and addressed and/or defended many of the issues that I have raised. I was pleased to see that they have followed most of my recommendation and I believe their manuscript has greatly gained in clarity. I am therefore pleased to recommend this revised version to be accepted for publication in PlosOne. Looking forward to see it online!

Reviewer #3: I feel that all comments from all reviewers have been adequately addressed and the manuscript is therefore suitable for publication

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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.

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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 #2: No

Reviewer #3: No

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