PWAT-D-22-00091

Analysis of Microcystis aeruginosa physiology by spectral flow cytometry: Impact of chemical and light exposure

PLOS Water

Dear Dr. Zucker,

Thank you for submitting your manuscript to PLOS Water. After careful consideration, we feel that it has merit but does not fully meet PLOS Water'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 Feb 04 2023 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 water@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pwat/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

We look forward to receiving your revised manuscript.

Kind regards,

Ghaffar Ali, PhD

Academic Editor

PLOS Water

Journal Requirements:

1. Please send a completed 'Competing Interests' statement, including any COIs declared by your co-authors. If you have no competing interests to declare, please state "The authors have declared that no competing interests exist". Otherwise please declare all competing interests beginning with the statement "I have read the journal's policy and the authors of this manuscript have the following competing interests:"

2. Please amend your detailed Financial Disclosure statement. This is published with the article. It must therefore be completed in full sentences and contain the exact wording you wish to be published.

a. Please clarify all sources of funding (financial or material support) for your study. List the grants (with grant number) or organizations (with url) that supported your study, including funding received from your institution. 

b. State the initials, alongside each funding source, of each author to receive each grant.

c. State what role the funders took in the study. If the funders had no role in your study, please state: “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

d. If any authors received a salary from any of your funders, please state which authors and which funders.

3. Please provide a complete Data Availability Statement in the submission form, ensuring you include all necessary access information or a reason for why you are unable to make your data freely accessible. If your research concerns only data provided within your submission, please write "All data are in the manuscript and/or supporting information files" as your Data Availability Statement.

4. We noticed that you used “data not shown”/"unpublished data" in the manuscript. We do not allow these references, as the PLOS data access policy requires that all data be either published with the manuscript or made available in a publicly accessible database. Please amend the supplementary material to include the referenced data or remove the references.

5. We have noticed that you have uploaded Supporting Information files, but you have not included a list of legends. Please add a full list of legends for your Supporting Information files after the references list. 

Additional Editor Comments (if provided):

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does this manuscript meet PLOS Water’s publication criteria? Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe methodologically and ethically rigorous research with conclusions that are appropriately drawn based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

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

Reviewer #1: No

Reviewer #2: No

**********

3. Have the authors made all data underlying the findings in their manuscript fully available (please refer to the Data Availability Statement at the start of the manuscript PDF file)?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception. 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 Water 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: Review for PLoS Water

This research describes the use of spectral flow cytometry for evaluating physiological changes in Microcystis aeruginosa. The authors show clear changes in fluorescence using spectral cytometry.

General comments

This is an interesting study. A lot of work has been done and in a logical clear manner. It is a strong study. The study would benefit from some major revisions.

The power of spectral cytometry is not fully harnessed. The authors describe the data density of the Aurora and then use biplots of two band passes to describe changes.

This study is a thorough investigation to the spectral changes of M. aeruginosa after chemical and light treatment. It is an interesting study and patterns are clear. The study suffers from a few of major issues that can be addressed.

Major issues

The study suffers from lack of statistical analyses clarifying the strength of the relationships for all treatments.

Complementary measures of the process described to be affected would strengthen the conclusions of the article. Primary production and measures of electron transport are both measurable. The authors address the use of PAM at the end of their results and state that there are issues with PAM. Their statement for non-inclusion is that it doesn’t directly measure PBS and chlorophyll fluorescence. However, the conclusions throughout point to changes in the photosynthetic vitality as the reason for changes in various levels of fluorescence. A complimentary measure of primary production would further support their conclusions.

In addition to the qualitative data, quantitative changes in the populations/concentrations would be beneficial to show growth/death due to treatment.

The authors indicate changes in size as well as fluorescence but size normalised fluorescence was never shown. They do acknowledge the potential issue but do not address it using data.

If the ultimate goal is to demonstrate irrecoverable damage to HABs it would be nice to see an attempt to regenerate the cultures after exposure.

Specific comments

Lines 52-59 Please specify the excitation laser when discussing changes in “red and green” fluorescence. The following sentence that is remains unclear what the increase in green from the UV, Violet and blue lasers is due to exemplifies the requirement. I cannot say for certain but it looks like the emission curves for each laser are very similar i.e they appear to peak and nearly the same wavelength. The difference in apparent shape may be due to the differences in bandpasses for the various lasers. As such the causal change for one excitation wavelength may be the same for another wavelength, the same fluorescent particle is similarly excited by the various lasers. It would be interesting to note if the emission peaks from each laser are similar across the 5 lasers.

Line 262 and Figure 6 It is my understanding that a 5 laser Aurora was used, inclusion of the signature across all detectors would be better than the selection. It would also be nice to see the control and treatment curves normalised to see if there is a difference in their emission shapes as well as their intensity.

Line 273 It is indicated that there is a change in FSC and SSC in response to exposure. This change in relative size could have affects on the fluorescence. Was the fluorescence normalised to cell size using FSC or SSC?

Line 278 and Figure 7 When comparing measures it would be nice if they were overlaid on the same figure, it makes direct comparison easier for the reader and will save space.

Lines 345-348 Again the authors describe the data density and potential benefits of using spectral cytometry but their analyses depend mostly on biplots.

363-365 Measurement of photosystems and use of fluorescence is possible using a FIRe or FRRF or PAM system. If these measurements weren’t made in comparison to the Spectral cytometry measurements I would not go so far as to conclude that the cause of the change in fluorescence is something in the electron transport system of the M. aeruginosa.

392 If the hypothesis of the study is that H2O2 inhibits photosynthetic electron transport that should be directly measured.

Line 504 It is concerning that there was a marked decrease in size in addition to the decrease in fluorescence. It could be that normalised fluorescence remained the same. In which case the conclusions about the changes in the movement of electrons etc are not supported. This is another reason that direct measure of the electron transport to support their conclusions would be advantageous.

Lines 526-530 Address the above concern. However, they do not do so quantitatively. The numbers would be convincing.

Line 546 “Indicating that photosynthesis was inhibited” it would be advantageous to have a corresponding measure of photosynthesis to validate this conclusion.

Line 607 “… the data presented in this manuscript suggest that this increase in green fluorescence emissions is indicative of irreversible damage to M. aeruginosa” This statement is not supported. In particular the “irreversible” part. The treatments were not removed and the cultures allowed to recover.

Line 613 “inform water treatment methods” The authors have clearly demonstrated fluorescent changes in M. aeruginosa cultures with different chemical and light treatments. While these results align with previous results causality is not demonstrated and I would not yet indicate that these are sufficient to inform water treatment methods alone.

Figure 1 is not necessary for understanding this study.

Figure 3 It is slightly misleading to compare directly the flow cytometry and microscopy as they are different band passes and therefore allow more/less light through. The comparison of dose and green and red fluorescence is nice. A plot of dose vs fluorescence and a regression with error bars would be a nice way to show this relationship statistically. That could also be done for the fluorescence measured using the spectral cytometer. Remember to normalise to relative cell size (using scatter) to ensure that the increases aren’t only due to changes in cell size.

Figure 4 is my favourite

Figure 5 could benefit from making gates on figure 4 and watching the cells progress through those gates in addition to the control region.

PLOS water criteria:

1. YES – Presents resuls of primary research

2. YES – results reported not published elsewhere

3. Yes and No – Experiments, statistics and other analyses are performed to a high technical standard and are described in sufficient detail.

The experiments are clear and valid, the standard of the work is very nice. It is only lacking in statistical analyses. This can be remedied.

4. Somewhat – The conclusions are regularly compared to measurements not taken directly in this study. The authors are putting the results in the larger context which is great and assigning them meaning, however the text comes close to overstating their claims.

5. Yes – the article is presented in an intelligible fashion and is written in standard English

6. Yes – the research meets all applicable standards for the ethics of experimentation and research integrity

7. Yes - The article adheres to appropriate reporting guidelines and community standards for data availability. There are separate flow cytometry reporting standards, these are outlined for mammalian cytometry, but some of the requisites

Reviewer #2: Overall good study, I enjoyed reading the draft. However, at present some major issues exist that deter the quality to publish. My quick comments and suggestions are following:

1. Fig.1 can be deleted.

2.In Fig 6. include the signature across all detectors and that can be preferable than selection.

3. Some information mentioned in the conclusions should be addressed in the results/analysis clearly such as complimentary measure of primary production, that could enhance the vigor of results and conclusions.

4. Fig. 3 can also be further modified, thus far it is not presenting the clear picture, confusing.

5. Authors mentioned changes in size as well as fluorescence but size normalized fluorescence is not there. It would be better to address them with data, if possible.

6. few more changes to be made while revising.

**********

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.

Do you want your identity to be public for this peer review? If you choose “no”, your identity will remain anonymous but your review may still be made public.

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.

PWAT-D-22-00091R1

Analysis of Microcystis aeruginosa physiology by spectral flow cytometry: Impact of chemical and light exposure

PLOS Water

Dear Dr. Zucker,

Thank you for submitting your manuscript to PLOS Water. After careful consideration, we feel that it has merit but does not fully meet PLOS Water'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 May 29 2023 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 water@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pwat/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

We look forward to receiving your revised manuscript.

Kind regards,

Ghaffar Ali, PhD

Academic Editor

PLOS Water

Journal Requirements:

Additional Editor Comments (if provided):

[Note: HTML markup is below. Please do not edit.]

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. Does this manuscript meet PLOS Water’s publication criteria? Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe methodologically and ethically rigorous research with conclusions that are appropriately drawn based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

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

Reviewer #1: N/A

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available (please refer to the Data Availability Statement at the start of the manuscript PDF file)?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception. 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

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS Water 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: I appreciate the extensive revisions the authors have made. I am not sure that the changes have addressed the issues raised in the original review. It is a fine paper I just think that it could have been stronger had they dug into the data a bit more. I could have been more clear in my original review but below are things that I think could make this study more powerful.

In displaying the data I understand that the majority of people are used to bi-plots for flow cytometry. With spectral cytometry the power really comes in when you consider all of the channels. You can harness this power by using dimensionality reduction algorithms (Umap and optSNE). It might be interesting to look at your data using these techniques as it may more clearly pull apart the populations you are interested in.

If all experiments were conducted in triplicate with similar results that is great working up the statistics should be fairly easy. I understand that generally speaking non-mammalian cytometry does not include statistical description of the fluorescence of populations. However this study is specifically looking at changes in fluorescence with a treatment. Quantifying this change seems really important for repeatability of the method. MFI is a common tool for cytometrists to employ to describe the mean/median fluorescent intensity of a population. By defining that and then comparing across treatments the change can be quantified and statistical analyses done to verify the relative change currently described.

With regards to cell death. There are live dead kits, but you have something even better. You have measured changes in the concentration of the cells. The Aurora is volumetric, it should be fairly easy to export the concentration of the samples and then compare growth/death rate for the cultures. This is even better than the live/dead kits because it’s been shown that the cellular permeability may not be a great representation of who really is alive and who is dead.

The authors in their response indicate that they did try to recover the cells and that they were in fact not viable. It would be great if this were included in the manuscript. If they were to include growth/death rate over time they could then mark one time point as irrecoverable because they tried to bring it back. I think that this really demonstrates the power of the treatment.

Are the figure legends supposed to be interspersed in the results section? It makes it very hard to read as a reviewer because the fonts don’t change etc but the flow does. Just something to check prior to printing.

The authors have an entire section using scatter parameters to discuss the morphological response (cell size) to stress. Scatter is either good enough to talk about relative size or it is not. I fully understand that FSC and SSC are scatter parameters that are affected by a number of factors but 1) it is a culture so the changes should be consistent across the entirety of the culture and 2) the authors use it to describe relative size changes. I reaffirm that normalisation of the fluorescence to relative size would paint a clearer picture.

Reviewer #2: Changes are acceptable.

**********

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.

Do you want your identity to be public for this peer review? If you choose “no”, your identity will remain anonymous but your review may still be made public.

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.

PWAT-D-22-00091R2

Analysis of Microcystis aeruginosa physiology by spectral flow cytometry: Impact of chemical and light exposure

PLOS Water

Dear Dr. Zucker,

Thank you for submitting your manuscript to PLOS Water. After careful consideration, we feel that it has merit but does not fully meet PLOS Water'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 Aug 24 2023 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 water@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pwat/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

We look forward to receiving your revised manuscript.

Kind regards,

Ghaffar Ali, PhD

Academic Editor

PLOS Water

Journal Requirements:

Additional Editor Comments (if provided):

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

[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.

Analysis of Microcystis aeruginosa physiology by spectral flow cytometry: Impact of chemical and light exposure

PWAT-D-22-00091R3

Dear Dr Zucker,

We are pleased to inform you that your manuscript 'Analysis of Microcystis aeruginosa physiology by spectral flow cytometry: Impact of chemical and light exposure' has been provisionally accepted for publication in PLOS Water.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow-up email from a member of our team. 

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

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 water@plos.org.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Water.

Best regards,

Debora Walker

Executive Editor

PLOS Water

***********************************************************

Reviewer Comments (if any, and for reference):