PONE-D-19-22253

Insights on aquatic microbiome of the Indian Sundarbans mangrove areas

PLOS ONE

Dear Dr. Dhal,

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Jiang-Shiou Hwang, Ph.D.

Academic Editor

PLOS ONE

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1. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: Partly

Reviewer #2: Partly

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

Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: 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: PLOS ONE

Manuscript: PONE-D-19-22253

In this manuscript, the authors examined environmental parameters, microbial community composition in the island of Sundarban mangroves (ISM) and open marine water (OMW) using 16S rRNA gene-based amplicon, and attempted to assess the capability of environmental parameters for explaining the variation in microbial community composition. The authors reported that a higher eutrophic condition changes in bacterial communities in OMW. The authors did not make a solid conclusion. The authors concluded that multiple environmental/anthropogenic stressors (salinity, pollution, eutrophication, land-use) affect the estuary water and consequently the microbial communities in concert. Suggest also sample open seawater (or seawater standard), conduct examinations of water parameter and microbial community in open seawater, ISM, and OMW, and make comparison among three areas, and make full discussion. Suggest report clearly on pollution and eutrophication (Line 54). Examination on the environmental determinants that contribute to the microbial communities is unclear (Line 103-104). Suggest examine total phosphorus, organic phosphorus, total alkalinity, hardness, dissolved solids, and major ions and substances like sodium, potassium, calcium, magnesium, sulfate, carbonate, and chloride. There are many grammatical errors and typo errors.

1. Line 53: Rewrite the sentence.

2. Line 112, Line 117: Reference “Das 2011” is not listed in the Reference section.

3. Lines 117-119: Suggest examine total phosphorus (TP). Suggest examine orthophosphate-phosphorus (Orthophosphate-P, PO43--P).

4. Lines 127-136: Suggest rewrite the sentences. Suggest write detail about each parameter assay. It said “continuous flow analyzer (Flowsys by Unity Scientific, Brookfiels, USA)” (Line 129-130). It said “spectro-photometrical analysis with a Flows continuous flow analyzer (Systea, Anagi, Italy)” (Lines 133-134).

5. Line 129: It is “phosphate (PO43-)” or “orthophosphate (PO43-).

6. Line 128: Report content of dissolved inorganic nutrients (DIN).

7. Line 128: Report clearly on nitrate and nitrite (NOx).

8. Line 129: The statement “silicate (Si)” is not correct. Report it is silicon (Si), Silica (SiO2), or silicate.

9. Line 132: Suggest report examination of major water compositions like sodium, potassium, calcium, magnesium, sulfate, bicarbonate, carbonate, chloride.

10. Line 156: Reference “Maher et al 2014” is not listed in the Reference section.

11. Line 265: Suggest report clearly on high suspended matter.

12. Line 272: Reference “Eloc-Fadrosh et al. 2015” is not listed in the Reference section.

13. Line 273: Change to “Ghosh and Bhadury 2018, 2019”.

14. Lines 288-289: Change to “Basak et al. 2015, 2016; Ghosh and Bhadury 2017, 2018”.

15. Line 203: Reference “Buchan et al. 2010” is not listed in the Reference section.

16. Lines 302-306: Rewrite the sentence.

17. Lines 351-356: Rewrite the sentences.

18. Lines 251-358: The discussion is disjointed. Suggest link water parameters and microbial community composition in Family, make full discussion, and make discussion in these areas and open seawater.

19. Lines 359-377: The conclusion is lengthy. Suggest make a solid conclusion.

20. Lines 394-540: Suggest follow the Journal format, and write reference. Suggest write all authors.

Reviewer #2: In this study the authors have investigated aquatic microbial community structure from two estuaries of Sundarbans, world's largest contiguous mangrove ecosystem. While the findings of this study are potentially very interesting, I have highlighted some points that the authors need to address along with further strengthening of the discussion section.

The reference cited in the first line of Introduction is not correct. For example, Spalding et al (1997) and Gopal and Chauhan (2006) references would be more appropriate.

Introduction: However, sampling locations of those studies on microbial communities.... a single site specific with limited number of samples. This sentence need to be correct. The paper by Ghosh and Bhadury (2019) has looked in bacterioplankton community composition from four estuaries- Mooriganga, Thakuran, Matla and Harinbhanga.

Was the sampling undertaken during high or low tide? This should be clearly mentioned. What factors were taken into consideration for selection of sites? These points should be clearly stated.

How was the calibration of Eureka 2 Manta multiprobe undertaken?

Why V4-V5 region of 16S rRNA was targeted? This needs to be written upfront in introduction section given the importance of scales of taxonomic resolution among variable regions of this molecule.

The authors have reported dominance of Actinobacteria-like OTUs/sequences in their studied samples. I believe it is important to compare this finding with previous study undertaken in the same estuary as well as from other mangroves (e.g. Gong et al 2019, Scientific Reports). It is also important to highlight the specific roles of this group in terms of biogeochemical cycling (e.g. breakdown of complex forms of carbon and resulting role in remineralization).

One of the important groups that was reported from this mangrove is the presence and dominance of Firmicutes. However, I do not see any discussion on this point given Firmicutes signal were found to be much higher in Sundarbans compared to other mangroves.

It is really interesting to see the relative dominance of Thaumarchaeota Marine Group (MG I) and Euryarchaea MG II in this ecosystem. I am just curious if the authors can link this finding with reported dissolved ammonium concentration in this sector of Sundarbans given the availability of ammonia seems to be episodic in nature and thus there are consequences for nitrification rates. Also, the selection of primers improved the resolution in terms of encountering these groups? It is also important to highlight given the sampling has been focused only one time point, the observed community structure may also exhibit temporal variability.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-19-22253R1

Insights on aquatic microbiome of the Indian Sundarbans mangrove areas

PLOS ONE

Dear Dr. Dhal,

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.

We would appreciate receiving your revised manuscript by Mar 06 2020 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,

Jiang-Shiou Hwang, Ph.D.

Academic Editor

PLOS ONE

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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

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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 #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: No

Reviewer #2: 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

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: PLOS ONE

Manuscript: PONE-D-19-22253-R1

The authors have revised and improved the manuscript. Several parameters are concerned. Suggest conduct minor modification.

1. Lines 121-133: It said physicochemical parameters (salinity and pH), DOC, nutrient, nitrate, nitrite, combined nitrite and nitrate etc. However, they did not report measurements of TN and DIN (Table 1).

2. Line 131-132: It said nitrite, nitrate, phosphate, silicate were analyzed using continuous flow analyzer. Suggest report clearly the measurements.

3. Line 132: It said ‘phosphate (PO43-)”. In Table 1, it reported TN (total nitrogen), DIN (dissolved inorganic nitrogen), and PO4. Nitrogen and phosphorus are important nutrients. Suggest check the measurement is orthophosphate (PO43-), not including pyrophosphate and metaphosphate. It is fine to use phosphate (PO43-). The orthoposphate (PO43-) is commonly used and reported as phosphate (PO43-). However, suggest check the standard solution, and check the measurement is phosphorus or phosphate. Suggest change the term here to orthophosphate-P or phosphate-P (PO43--P) to match well with DIN. Suggest check the term PO4 in Table 1. It is not correct. Suggest use orthophosphate-P, or simply use PO43--P.

4. Line 132: It said silicate (Si). In Table 1, it said Si. They are not correct. The term of Si is silicon, not silicate. Readers know silicate is important, and the measurement of silica (SiO2) is used for evaluating content of silicate. Suggest check the standard solution and measurement of this parameter. I think it is silica (SiO2), not Si, not silicate. Please check it is silica (SiO2) or silicon (Si). Suggest report silica, not silicate, not silicon.

5. Line 125 and Table 1: It said DOC and said DOC (µM). Suggest report clearly the measurement. Suggest check the unit “µM”. Suggest check the standard and report which chemical is used as standard.

Reviewer #2: The authors have addressed the points that I raised earlier. The manuscript is now acceptable 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.

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: Yes: Punyasloke Bhadury

[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.

Insights on aquatic microbiome of the Indian Sundarbans mangrove areas

PONE-D-19-22253R2

Dear Dr. Dhal,

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.

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With kind regards,

Jiang-Shiou Hwang, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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