Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: DETRIMENTAL EFFECTS OF HEAT STRESS ON GRAIN WEIGHT AND QUALITY IN RICE (ORYZA SATIVA L.) ARE AGGRAVATED BY DECREASED RELATIVE HUMIDITY
Review round: 3
Reviewer: 1

Output:
Basic reporting: I acknowledge the authors have revised substantially to my and other reviewers' suggestion on the presentation of the introduction and discussion.
Experimental design: Methods are satisfactorily revised.
Validity of the findings: Discussion has been satisfactorily revised.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: OPTIMAL EXTRACTION, PURIFICATION AND ANTIOXIDANT ACTIVITY OF TOTAL FLAVONOIDS FROM ENDOPHYTIC FUNGI OF CONYZA BLINII H. LÉV
Review round: 1
Reviewer: 1

Output:
Basic reporting: The PeerJ Manuscript 55486v1. A conceptual mistake, the fungus is not a plant as described in section 2.1 (lines 62 and 63). The species name of the fungus must be provided (line 19); it’s ambiguous in some of the Methods described that make impossible to repeat: …was higher than before… (line 32), the meaning it´s not clear; all the Keyword are included in the title (line 39); …grew to a certain size (lines 75 and 76), a wet or dry weight must be provided; a small amount (line 80) specify a volume value; …and repeated three times like that (line 82), if this is an extraction, it is not clear how did they do the separation; …with slight modifications (line 86), without a explanation of the modifications; …with certain… an amount must be provided (line 91); it´s not clear the meaning of 2.3.5 or 2.4 on line 105 and 106; need the Absorption rate units in figures 1 to 6; need the Elution rate and Purity units in figure 7; in figures 9 to 11 the Clearance rate units must be given.
The English must be improved: …the regeneration speed.. (line 18), must be the production speed…; …is an Flavonoid… (lines 19 and 52), must be …is a flavonoid…; 60% ethanol was the best elution volume (line 26) must be …elution solvent; there are several type writing spaces, like in line 31, 39, 44, 87, 90, 177 and 186; …for further effect… (line 31), must be …for further uses…; …providing more theoretical basis for… (line 37) must be … evidence for…; …endophytic fungi… (line 52), must be endophytic fungus..; …total flavonoid… (line 58), must be …total flavonoids…; …then extract… (line 91), must be …then the mixture was incubated…; …and elution rate… (line 104) must be and elution time..; the Tables 1, 2 and 3 have two points instead of only one (lines179, 183 and 189); …optimiza… (line 299) ,,,optimize…; Figure 3 …under different temperature to liquid, must be …under different liquid temperature…
Experimental design: The paper is original and is within the aims and scope of the journal. The research question is well defined, but methods are not well described and ambiguous that make it hard to reproduce.
Validity of the findings: There is not data to support the purification times an important value to evaluate the good performance of the method.

The conclusion section is a result resume, don't have any conclusion
Additional comments: The English must be revised and more specific data have to be provided in order to be able to reproduce the method,


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: OPTIMAL EXTRACTION, PURIFICATION AND ANTIOXIDANT ACTIVITY OF TOTAL FLAVONOIDS FROM ENDOPHYTIC FUNGI OF CONYZA BLINII H. LÉV
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English should be carefully revised. Some sentences are hard-to-read. The other points are OK.
Experimental design: It is not clear the gap that is going to be filled with this research article. It is difficult to understand how the authors establish that the extracted content is mainly flavonoids.
Validity of the findings: Although it is an interesting research article, I think there are a few details that should be clarified, and some conclusions are not evident from the described results.
Additional comments: The manuscript is interesting, and new methodologies to extract flavonoids are welcomed. However, some points should be clarified before the article's acceptance.
 It is difficult to understand the evaluated extracts; for example, the L9F-E was not assed?
How was obtained the extract L9F-M?
The sentence from line 104 to 106 is confusing.
Why use ethyl acetate for the extraction? Some flavonoids are less soluble in this solvent.
How was the extract's flavonoid content established?
The authors should clarify how the research provides a more theoretical basis for producing flavonoids by biological fermentation method?


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: OPTIMAL EXTRACTION, PURIFICATION AND ANTIOXIDANT ACTIVITY OF TOTAL FLAVONOIDS FROM ENDOPHYTIC FUNGI OF CONYZA BLINII H. LÉV
Review round: 2
Reviewer: 1

Output:
Basic reporting: All my comments or answers were addressed in the file peerj-55486-Response_letter_to_peerj.docx, but some are deficient in the paper peerj-reviewing-55486-v1.pdf.
For example:
The keywords were substituted for similar others, that still in the title.
There still some mistakes as in Figure 7: The data point indicate the Indicate ….
Point 10 of my letter, if Elution rate is a %, they do not express that in the figure.
Response 10, It represent the proportion of sample in the population. This can not be an Elution rate.
Experimental design: Still not clear:
line 70: .. until the mycelial pellets grew to a certain condition (Bacteria is not growing and The fermentation broth is dark enough). I still thinking that a mycelium dry weight must be provided.
line 75: A small amount of concentrated fermentation broth (mL) was mixed… The amount must be given.
Validity of the findings: Table 3, they do not give the meaning of SS, DF, MS, F and >F, so for the Table is not clear.
Additional comments: I still thinking that the manuscript could be improved English and some data must be provided.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: INFLUENCE OF SEX AND MATURITY STATE ON TRACE ELEMENTS CONTENT IN LIVER AND MUSCLE OF THE SCIAENIDAE TOTOABA MACDONALDI
Review round: 1
Reviewer: 1

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: Data obtained in this study is worthwhile but not exhaustively interpreted.

Authors should have used Principal Component Analysis to model the risk assessment.

Monte Carlo modeling should also be used.

Include LOD & LOQ  and information on QA/QC of data.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: INFLUENCE OF SEX AND MATURITY STATE ON TRACE ELEMENTS CONTENT IN LIVER AND MUSCLE OF THE SCIAENIDAE TOTOABA MACDONALDI
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The reviewer has carefully read through and reviewed the manuscript. Although the manuscript reports the concentrations of several elements in the liver and muscle of fish species, the reviewer could not find the importance and significance of this regional study as a research publication for The PEERJ Journal. Therefore, the manuscript was judged as Rejection.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: INFLUENCE OF SEX AND MATURITY STATE ON TRACE ELEMENTS CONTENT IN LIVER AND MUSCLE OF THE SCIAENIDAE TOTOABA MACDONALDI
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: good results obtained
Additional comments: General comment:
 the results obtained are good and important for society, scientists and industry thus for publication. congratulation.

Introduction
Line 56: antioxidant and lipid peroxidation is not necessary as it will appear in the second paper

Material and Methods
- Line 108: CIBNOR, what is this about and is the permits number available?
- Line 109-110: “...and alway conducting...endangered species.” is not necessarily needed. “This study was performed following…” is enough.
- Sample collection: i think this paragraph needs to be restructured in a better order: where, institution for legal permission, the number of samples, the types, and the groups of obtained samples, and the instruments followed by the muscle collection. For example: totoaba individuals were collected from … under/following regulation of Mexican government (mention all institutions). There are three types of habitats…. A total of 142 were obtained between May 2010 - .
- Trace element analysis: muscle and liver preparation, is there any methods reference?
- Line 144-145: last sentence is not necessarily needed, or if needed, put it in the discussion part.
- Statistical analysis: it would be better to move this section after nutritional and risk assessment.

Discussion
- Line 249: the first sentence is not necessary
- Line 281: the standard for Cu level for human health refer to?
- Line 351-353: the sentence “SOD activity can be …” is not necessarily needed.

Figure:
- figure 2 is not needed. SOD analysis was not conducted on this paper. Citing and referencing SOD data in the discussion is possible, but the figure is not necessarily needed.
- Comparison with SOD results, in the discussion section is good, however, please check to make sure if this is allowed or legal to include them in Table 3, Table 4, figure 3. Maybe it is important to check the permission from the journal cited publisher to include this data in the paper.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: INFLUENCE OF SEX AND MATURITY STATE ON TRACE ELEMENTS CONTENT IN LIVER AND MUSCLE OF THE SCIAENIDAE TOTOABA MACDONALDI
Review round: 2
Reviewer: 1

Output:
Basic reporting: The revised version has taken care of the concerns. I recommend acceptance  at this stage.
Experimental design: The revised version has taken care of the concerns. I recommend acceptance  at this stage.
Validity of the findings: The revised version has taken care of the concerns. I recommend acceptance  at this stage.
Additional comments: The revised version has taken care of the concerns. I recommend acceptance  at this stage.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: INFLUENCE OF SEX AND MATURITY STATE ON TRACE ELEMENTS CONTENT IN LIVER AND MUSCLE OF THE SCIAENIDAE TOTOABA MACDONALDI
Review round: 2
Reviewer: 2

Output:
Basic reporting: Clear and well described
Experimental design: Well described
Validity of the findings: well described
Additional comments: Thank you very much for the revision. Only some minor comments to clarify:

Materials and Methods
Line 113: please clarify the word “filed” or field?

Results
Line 261-262: 30% of the samples were detected for Cd level. If I get it correct, from Table 3, there are only 14 samples (7 in autumn C, UN and 7 samples in Spring R, MF) 14 out of 144 muscle samples is about 10%. Please clarify.

Line 267: the same, 40% and 70%. There are only 12 samples (Spring R , IF) which were not undetectable out of 144. Please check and clarify


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: ASSOCIATION BEHAVIOR BETWEEN SAND TIGER SHARKS AND ROUND SCAD IS DRIVEN BY MESOPREDATORS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors present a unique and well designed study based on field observations of species interactions with an apex predator.  They do a great job placing their work in a broader ecological context, in a subject area ripe for greater attention in ecological research with application in he conservation arena.  That said, there are multiple but mostly minor revisions below that would improve this draft.  Ultimately, this will make a fine contribution to PeerJ and will be widely cited in the ecology and ocean conservation literature.
Line 29.  Suggest “… likely possibility …” is redundant.  Perhaps “These interactions potentially yield additional trophic consequences to this unique association and highlight the value …”
Line 34 – paragraph.  The authors should include a bit more detail describing hard bottom reefs.  These are sandstone and other sedimentary and biogenic materials, generally surrounded by fine-grained sediments, principally sand.  These emerge from the rippled sand.  Heights above the surrounding seafloor vary, with some a meter or more high, some undercut, some partially buried with sand veneer.  Inverts colonize these sites adding to spatial complexity.  A few more terms to better describe the geologic and ecologic setting would better inform readers.
Line 48.  Not all fishes are pelagic.  Dense schools and aggregation of 0-yr tomtate are significant members of schools over reefs, in addition to round and mackerel scad.
Line 51.  Suggest specificity and state hard bottom or live bottom “reefs” or ledges.
Line 56.  Auster et al. 2009 fine but also see for additional detail:  Campanella, F., Auster, P.J., Taylor, J.C. and Muñoz, R.C., 2019. Dynamics of predator-prey habitat use and behavioral interactions over diel periods at sub-tropical reefs. PloS one, 14(2), p.e0211886.
Line 67.  See Campanella et al reference above.  Changes in forage fish distribution based on predator behavior over 24 hr periods.
Lies 106-113.  See ASMFC Coastal Sharks Review for 2017 (http://www.asmfc.org/uploads/file/5d00081cCoastalSharksFMPReview_2018.pdf)  and NOAA Fisheries SAFE report (https://www.fisheries.noaa.gov/resource/document/2018-stock-assessment-and-fishery-evaluation-report-atlantic-highly-migratory) for most recent status.  Perhaps worth noting that this species is a prohibited species to catch and must be released when encountered as bycatch in fisheries on the US east coast.
Line 114.  Are the authors sure these are all round scad?  Live bottom reefs at 20-30-ish meters off Georgia often have a mixture (in mixed species schools or segregated within single schools) of both round and mackerel scad (Decapterus macarellus).  For example, the Auster et al papers cited within report both species of Decapterus in schools of prey.  This issue is not fatal at all for the paper, just a need to acknowledge both species present or possible based on the video observations, especially if IDs were from video only.
Line 161.  Usually referred to as “wasp-waist” web or system, not “waisted” - at least in my experience.
Line 184.  Campanella et al. reference apropos here too.
Line 201.  Suggest “ … using video records from a unique …”
Line 227.  Provide finer biogeographic resolution here?  This area represents the boundary between the Virginian and Carolinian provinces.
Line 274. Suggest “ … six MPs selected a priori …” or something similar.
Line 291.  Why square root transform?  Need some brief rationale for this versus no transform at all or any of the multitude of others.
Line 335.  Is “entity” the right term?  Suggest moving as a “highly coordinated group” or similar?
Line 352.  N=?  How many are rare?
Line 388.  What was the p value?  It is useful to report the actual p value to let the reader know the actual level of significance.  That is, was p=0.08, or 0.11, or 0.68?
Line 407.  Motivation is not what was addressed here, but perhaps “rationale” or similar term(s) to explain association in ecological terms.
Line 474.  See Price, V.E., Auster, P.J. and Kracker, L., 2013. Use of high-resolution DIDSON sonar to quantify attributes of predation at ecologically relevant space and time scales. Marine Technology Society Journal, 47(1), pp.33-46.
Experimental design: Well designed.  See above.
Validity of the findings: Yes.  See above.
Additional comments: Yes.  See above.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: ASSOCIATION BEHAVIOR BETWEEN SAND TIGER SHARKS AND ROUND SCAD IS DRIVEN BY MESOPREDATORS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors mostly use clear and unambiguous language in their manuscript. There are several instances, which I outline below where clarification would be helpful. See general comments below.
Experimental design: I recommend that the authors reorganize or rework the introduction to more clearly identify the knowledge gap that seek to fill. The methods require some additional details to ensure reproducibility. See general comments below.
Validity of the findings: See general comments below
Additional comments: Coleman and Burge provide compelling evidence for association behavior between a large coastal shark (sand tiger shark) and baitfish (round scad). Their evidence stems from review of videos from a long term monitoring dataset collected off of Cape Fear, North Carolina. They also demonstrate that the association between the large sharks and small baitfish may be related to mesopredators, including piscivorous fish, such as jacks. The manuscript will make an important contribution to the peer-reviewed literature because it advances current discussions of predator-prey interactions.

Despite the value of the manuscript, I have several concerns that I would recommend the authors address prior to publication. I would recommend that this be conducted through a major revision. I outline my broad feedback below and then provide more detailed feedback.

Broad comment 1: I recommend that the authors rework the organization of the introduction. As written, the introduction provides an incredible amount of detail, but I think that reordering of certain paragraphs so that the first paragraphs are broader and the later paragraphs more study/system/organism specific (e.g., hourglass formation) may facilitate reader understanding and also frame the research more broadly within the field of marine ecology. For example, the current paragraph 3 could become paragraph 1 because it is broadly about multispecies interactions and theoretical ecology. The current paragraph 1 focuses on hard bottom reefs of the Carolinas and seems too detailed and system-specific to occur so early on in the introduction. Theoretical and broad ecological content are mixed into the sections on sand tiger sharks, round scad, and mesopredators. I recommend making these sections parallel to one another (e.g., open with broad content then narrow down) or potentially moving the broad content up in the introduction. There are many ways to accomplish this. To me, it would make sense to open broadly about multispecies interactions, narrow into marine systems, all while setting up the knowledge gap that your study will address. Then, with the knowledge gap identified, you can describe that you require a system and accompanying species to help fill the knowledge gap, which is where the sand tigers, round scad, and mesopredators come in, along with the descriptions of hardbottom reefs (e.g., the system).

Broad comment 2: While there are plenty of details in parts of the methods, other details are lacking or deserve clarification to provide full understanding of the methods. For example, it would be helpful to clarify how videos were selected and retained (e.g., shark presence/absence, mesopredator presence/absence, scad presence/absence). Please also clarify which data were used for which analyses. For example, how were mesopredator abundance values used in the analyses (or were they?)? Was scad abundance incorporated or only presence absence? These details would help me assess the validity of the analyses more thoroughly.

Broad comment 3: Sand tiger sharks are described as apex predators, but I think they should be referred to as large predators instead of apex predators because they likely aren’t filling the role of apex predators (e.g., white sharks, tiger sharks) in this system (see Heupel et al. 2014 in Marine Ecology Progress Series – “Sizing up the ecological role of sharks as predators”).

Broad comment 4: It seems that many of the results are correlational, but in some circumstances are interpreted as causal. Please check language throughout to address this.

Detailed feedback

Abstract

Line 16: Suggest adding an overarching background sentence or two at the start of the abstract to establish the general knowledge gap and broader impact of the study. This would assist readers in understanding the relevance of the study.

Line 16: Explain in the abstract what Frying Pan Tower is. Perhaps you could say “Frying Pan Tower, an artificial tower that rests on natural, hard bottom off Cape Fear, North Carolina…”

Line 25: Perhaps consider a different word for “important” to describe scad. Perhaps, commonly occurring, ubiquitous, abundant, or something similar?

Line 28-29: Could rephrase to be “… round scad association conceals and attracts mesopredators, enhancing predation opportunities” to reduce wordiness.

Line 29: Remove “likely” as it is implied with “possibility.”

Introduction

Line 44 & 48: Reconsider use of word “attract” given that this word is often used in the attraction vs. production debate regarding artificial structures.

Line 56-59: Elaborate on what the effects of the associations are thought to be.

Line 83: Please define “polarized aggregations,” if appropriate.

Line 129: How have the round scad schooling behaviors been described?

Line 130-131: Something seems off about this sentence. The observations don’t really accentuate the affinity but maybe they suggest an affinity. I’d also like to see a citation for the affinity to “physical objects” including the “larger fishes and elasmobranchs,” if possible, or you could qualify the statement to say that the observations suggest an affinity for physical objects, which could (instead of can) include the larger critters.

Line 135-137: This needs clarification. Plenty of studies report on in situ sand tiger shark observations. Do you mean that there are few studies that report on sand tiger and scad associations?

Line 182-184: This sentence seems out of place.

Line 196: Add “stationary” so reads “underwater stationary video.”

Methods

Line 226-227: It seems like a stretch to say that Frying Pan Shoals is so close to the Cape Hatteras biogeographic transition zone. Either drop this sentence or qualify it by providing the distance of the tower from Cape Hatteras.

Line 232: Describe the anthropogenic debris.

Line 245: I am confused about the video clips used for this research versus the larger project database. Please clarify. For example, for the clips used for this MS, were they 20 min clips? Were there 1024 video clips in the overall larger project but then 216 were used for this study? I know this is explained in other parts of the manuscript, such as with the results, but a central place (or maybe a table?) containing the video clip information would be helpful. If a round scad was not present were the clips excluded even if a sand tiger shark was present? What about if a mesopredator was present but not scad and vice versa?

Line 250: Is there any possibility that the round scad could have been misidentified as other types of scad using video footage?

Line 259: Five body lengths of what? Scad body lengths?

Line 267: From what point were the 10-min intervals assigned? For example, were they assigned based on when the shark was first visible in the video clip?

Line 273: “Species composition” doesn’t seem to be the correct term here. Species identity, perhaps? Composition would imply the community composition and thus the abundance and identity of mesopredators? Or, maybe just drop “species composition” as it is implied with the phrase “of six selected MPs.”

Line 290: Presence / absence doesn’t usually get transformed in multivariate statistics, right? If you had abundance, it could be transformed using a square root transformation. Was the mesopredator abundance transformed?

General: What happened if the interaction continued outside of the clip interval?

General: Were videos with sand tigers but without scad excluded, even if they contained mesopredators?

Results

Line 323: I’m unclear on how this statement is supported. I thought videos were excluded if they didn’t include sand tiger sharks but did include round scad?

General: Should the YouTube video links be removed and these videos added as supplementary videos? I’m not sure on journal standards here.

Line 346: What was the number of recorded predation attempts?

Line 349: What does “LT” refer to?

Line 352-356: Wow!! This is fascinating!

Line 358-359: Is this frequency of occurrence for all months or winter months?

Line 385: p = 0??

Line 388-389: Here, you state that the association between sand tigers and round scad was not significantly different in the presence of mesopredators. However, in the first sentence of the discussion, you state that this association is significantly different in the presence of mesopredators? Please clarify this.

Discussion

Line 402: “the ecosystem as a whole depends on round scad…” seems too strong. This study did not examine energy transfer.

Line 404: Similar to comment above, this seems too strong given the correlational evidence. Perhaps “relate to” instead of “effects on.”

Paragraph beginning line 414: This seems redundant with the intro paragraph on underwater video. Perhaps keep the paragraph here in the discussion and remove from intro.

Paragraph beginning line 480: Seems to relate to ideas presented elsewhere in the discussion, so perhaps consider combining with another paragraph.

Line 485-486: Suggest rewording this sentence. I don’t fully understand it, as written.

Line 493-495: Yes! Agreed that this would be very interesting.

General: Sand tiger sharks are assumed to mostly forage at night. Can you please reflect on this? How would this influence the interpretation of your results?

Line 506: Reconsider use of term “apex predator” to describe sand tiger sharks (see broad comment above).

Figures

Figure 2: It would be helpful to know how many videos are from each month. The figure likely isn’t the place for this, but consider including this information in a supplementary table, as it is important for interpreting Figure 2 and statements that sand tiger sharks aren’t observed in some summer months, when I’m wondering if it was actually a detection issue.

Figure 3: Are the data that went into this presence/absence data?

Figure 4: Could this be scaled per clip to be more standardized and aid in comparisons between the left bars (no mesopredator) and right bars (mesopredators)? For example, if we assume (this is my visual estimation…) that the “no mesopredator” bar values are 8, 10, and 9 (from left to right), then the total number of clips without mesopredators is 27. You could then divide the values by 27, so for the third bar it would be 9/27 = 0.33, which would be the scaled occurrence of no mesopredators for tightly associated. You could then repeat this process for all bars. These data could also be displayed in a table of the treatments (NVI, LA, TA) crossed with MP absence and MP presence, if preferred over a figure.

Thank you for the opportunity to review your manuscript. It is a fascinating topic and a compelling dataset. I know your manuscript will be an important contribution to the literature, and I hope that my feedback is helpful.

-Avery Paxton


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: ASSOCIATION BEHAVIOR BETWEEN SAND TIGER SHARKS AND ROUND SCAD IS DRIVEN BY MESOPREDATORS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors use clear and unambiguous language and have addressed previous concerns.
Experimental design: The authors have reworked the introduction substantially and added additional details to the methods resolving my previous concerns.
Validity of the findings: No comments to add.
Additional comments: Thank you for the opportunity to re-review this fascinating manuscript. Coleman and Burge have done a commendable job revising their manuscript. They have addressed all of my prior concerns, and I recommend that the manuscript be accepted for publication. This manuscript will make an important contribution to the peer-reviewed literature.

-Avery Paxton


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: BIOINFORMATIC PREDICTION OF IMMUNODOMINANT REGIONS IN SPIKE PROTEIN FOR EARLY DIAGNOSIS OF THE SEVERE ACUTE RESPIRATORY SYNDROME CORONAVIRUS 2 (SARS-COV-2)
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors performed an epitope level analysis of the spike protein in novel coronavirus or SARS-CoV2. The authors selected these epitopes based on the predictions but did not include the peptides or epitopes that are already confirmed. These epitopes could be easily retrieved from sources like the IEDB.
Experimental design: While the authors concluding the manuscript with the following remark "Our next move is to express and purify the recombinant protein in a suitable expression system, followed by immunizing the mice with purified immunogen to obtain specific antibodies", that shows the interest only in the B cell response. In such a case, prediction to only B cell epitope could have been enough, why did they choose to predict the T cell epitopes too?
Validity of the findings: The authors present preliminary findings or concluded that the validation will be performed in their next move. In the present scenario, the manuscript offers no other validation except for using multiple tools to go with the consensus.
Additional comments: The authors have performed an analysis using a standard set of tools. There are many papers published with pipelines. The authors should consider providing data or discussions on how their analysis/results/interpretation is different from others.

Many of the relevant papers and tools are not referred in the manuscript.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: BIOINFORMATIC PREDICTION OF IMMUNODOMINANT REGIONS IN SPIKE PROTEIN FOR EARLY DIAGNOSIS OF THE SEVERE ACUTE RESPIRATORY SYNDROME CORONAVIRUS 2 (SARS-COV-2)
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English language is clear and professional. Also the tables and figures are understandable. The repetations in material/methods and results should be revised.
Minor revisions for introduction;
* Line 45 : Accession date should be added.
* Line 47-48 : Use passive voice in sentence starting with ‘Nevertheless..’
*** Line 84 : ‘The whole flowchart of our work is depicted in Fig. 1.’  It should be added to Material and Methods section.
Experimental design: Experimental design and the selection of bioinformatics tools which made to achieve the aim of the study are sufficient and appropriate to the literature.
Minor revisions for Materials and Method;
* In ‘Data retrieval and sequence alignment’ section, it should be mentioned that alignments were done to find out the SARS-CoV-2 specific genomic regions.
* The accession numbers of sequences (Fig 2) should be added to ‘Data retrieval and sequence alignment’ section.
*The detailed information about servers is not necessary.  For example Line 139: VaxiJen was the first server for alignment-independent prediction of protective antigens OR Line 143-145: . ProtScale was one of the protein identification and analysis tools in the ExPASy server, which could calculate hydrophilicity based on amino acid scales.
*The websites of servers and the references should be added.
Validity of the findings: The results of the study were well presented and answered the research question.
Minor revisions for Results;
*Do not repeat the same information which was mentioned in materials and methods. For example Line 177-178 : ‘The antigenicity was calculated via VaxiJen v2.0 with the given cutoff of ≥0.40’
*Line 162: There is no information about performing phylogenetic tree in materials and methods section even a result was mentioned.
*Line 259-261: The result of blast which written as ‘little similarity’ should be mentioned clearly.
*Line 302: ‘an’ instead of ‘a’
Additional comments: This is a well-organized in silico study which needs minor revisions. These preliminary studies are very important for producing early diagnosis approaches in struggling against SARS-CoV-2 pandemic. The authors analysed the coronavirus sequences to find the regions specific to SARS-CoV-2. Then epitopes were predicted in S1 and epitopes out of the glycosylation and highly mutated regions were selected as candidates. Finally, the 3 epitopes were linked with linkers.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: BIOINFORMATIC PREDICTION OF IMMUNODOMINANT REGIONS IN SPIKE PROTEIN FOR EARLY DIAGNOSIS OF THE SEVERE ACUTE RESPIRATORY SYNDROME CORONAVIRUS 2 (SARS-COV-2)
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have reported a set of epitopes from SARS-CoV2 and suggested a recombinant peptide to elicit the desired immunity.
Experimental design: The experimental design is somewhat questionable. In a computational study, the authors have the choice to work on immune reactivity in humans, and they have chosen a mouse model.
The authors are justifying that choosing a mouse model is one of their novelties. It would be worthwhile to compare the study against human results.
Validity of the findings: The results are computationally predicted and offer no wet lab validation. I appreciate that the authors browsed known epitopes from reference databases like and they found 4 peptides to be matching.

I would suggest authors to expand the search space a little bit and provide a nice comparison between their set of peptides versus epitopes retrieved from the IEDB.
Additional comments: No other comments.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: POTENTIALLY TOXIC METAL(LOID) DISTRIBUTION AND MIGRATION IN THE BOTTOM WEATHERING PROFILE OF INDIGENOUS ZINC SMELTING SLAG PILE IN CLASTIC ROCK REGION
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript prsents a study determined the concentrations of PTMs and pH values and analyzed PTM distribution in two weathering profiles (slag-covered and slag-absent) with small horizontal distance in clastic rock region in the indigenous zinc smelting area.
     Generally speaking, the research content of this study is detailed,  and it is very interesting.The manuscript is wirtten clearly and unambiguously. Obviously,the authors have done a lot of work in writing this manuscript.However, there are still some problems need to be considered before acceptance.
Experimental design: It is suggested that the author should refer to more recent references and explain clearly in introduction how this research  fills the identified knowledge gap.
   In part 2.1 of the paper, it is suggested to describe in more detail the sampling method and the principles adopted in the sampling process, as well as how to sample at the bottom weathering profile.
Validity of the findings: It is suggested that results and discussion should be written in a chapter, or a section on migration of potentially toxic metals (PTM) should be added to the discussion part separately.
   It is suggested that more detailed supplementary support should be given to support the conclusion that “the downward migration of Cd and Zn from the covered slag might finally contaminate to groundwater through the bottom weathering profile in the clastic rock region”.
Additional comments: Line71 to line 72. The site location information for lead, zinc and arsenic contaminated subsoil within 40 to 50cm depth of soil is required to be added when comparing with the one in Poland.
    Please check the details of the article carefully and pay attention to the writing and drawing specifications of the paper. For example,the unit of flagis suggested to be added in Figure 1.The unit of flagis suggested to be added in Figure 1.
    Therefore,the manuscript may be reviewed after major modification.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: POTENTIALLY TOXIC METAL(LOID) DISTRIBUTION AND MIGRATION IN THE BOTTOM WEATHERING PROFILE OF INDIGENOUS ZINC SMELTING SLAG PILE IN CLASTIC ROCK REGION
Review round: 1
Reviewer: 2

Output:
Basic reporting: I have completed the review of the manuscript “Potentially toxic metal(loid) distribution and migration in the bottom weathering profile of indigenous zinc smelting slag pile in clastic rock region” by Peng et al. This is a very interesting manuscript that provides pedogeochemical data of two weathering profiles (slag-covered and slag-absent) in an indigenous zinc smelting slag site located in Guizhou Province, China. However, the discussion section must be significantly improved before publication. In particular, the relevance of the clastic region in the potential dispersion or stability of potentially toxic metal(loid)s is not discussed.

General
I strongly advise to use professional English editing services to improve the grammar in your manuscript. Examples are lines 40-41, 54-55,

The introduction section needs significant improvement. I suggest to include a brief description of indigenous metal smelting slag and its environmental significance.

Table 1. It is unclear what do the authors mean with “a minimum of soil”, “maximum of soil”, “mean of soil”.

Figure 1 repeats information from Table 1. I suggest to include the table in a Supplementary Material file.

Line 48: Peng et al., 2018b, it should be 2018a.
Please provide a description of the study area located at the beginning of the Introduction section. Lines 47 to 51 are unclear to the unfamiliar reader.
Experimental design: Lines 116-118. Please provide the sample digestion method, as well as detection limits for all studied metal(loid)s.

Line 124. The pollution index does not specifically determine the degree of harm. Please re-phrase. To accurately estimate the Pollution Index (PI), authors should provide a discussion of the local geochemical background, if such background is not used in the calculation of PI, then using risk-based screening values for the soil contamination of agricultural land should be supported by a discussion.
Validity of the findings: The discussion section needs improvement. The impact and novelty of this research is not assessed.
Additional comments: I have completed the review of the manuscript “Potentially toxic metal(loid) distribution and migration in the bottom weathering profile of indigenous zinc smelting slag pile in clastic rock region” by Peng et al. This is a very interesting manuscript that provides pedogeochemical data of two weathering profiles (slag-covered and slag-absent) in an indigenous zinc smelting slag site located in Guizhou Province, China. However, the discussion section must be significantly improved before publication. In particular, the relevance of the clastic region in the potential dispersion or stability of potentially toxic metal(loid)s is not discussed.

General
I strongly advise to use professional English editing services to improve the grammar in your manuscript. Examples are lines 40-41, 54-55,

Lines 116-118. Please provide the sample digestion method, as well as detection limits for all studied metal(loid)s.

Line 124. The pollution index does not specifically determine the degree of harm. Please re-phrase. To accurately estimate the Pollution Index (PI), authors should provide a discussion of the local geochemical background, if such background is not used in the calculation of PI, then using risk-based screening values for the soil contamination of agricultural land should be supported by a discussion.

The introduction section needs significant improvement. I suggest to include a brief description of indigenous metal smelting slag and its environmental significance.

Table 1. It is unclear what do the authors mean with “a minimum of soil”, “maximum of soil”, “mean of soil”.

Figure 1 repeats information from Table 1. I suggest to include the table in a Supplementary Material file.

Line 48: Peng et al., 2018b, it should be 2018a.
Please provide a description of the study area located at the beginning of the Introduction section. Lines 47 to 51 are unclear to the unfamiliar reader.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EFFECT OF LEAF POSITION AND DAYS POST-INFILTRATION ON TRANSIENT EXPRESSION OF COLORECTAL CANCER VACCINE CANDIDATE PROTEINS GA733-FC AND GA733-FCK IN NICOTIANA BENTHAMIANA PLANT
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript is well written and the introduction is comprehensive. The materials & methods section needs a minor update but results and discussion summarizes the body of work clearly. Details provided in the figures are quite helpful.
Experimental design: In general, the experimental design was good but a minor clarification is needed.
A.	Line227-228: The statement does not clarify the procedure for extraction. Is the sample used for western-blot analysis a crude extract or purified preparation? Please provide the details.
Validity of the findings: 1) Line 229-231: Please clarify: Does the author is intending to say in the context of 70 kDa band only?
2) Figure 4. The differences observed in the protein band position between + (mammalian expressed GA733-Fc) and Nicotiana benthamiana expressed proteins is due to differences in the glycosylation due to different expression platforms?
3) Can an explanation be provided for the differences observed in the molecular weight of the + control and the test samples? In the western-blot (Figure 3), a band corresponding to 70 kDa is seen whereas in Figure 4 and Figure 5 A, the band lies in between 50 – 70 kDa for + control as well as the test samples. What is the reason for this shift observed between the figures?
4) Line355-356: In my opinion, the study does not support the claim that KDL tagging of GA733-Fc improved its biological activity. This claim (biological activity) needs further support by performing cell or animal-based studies.
5) Line359: I would recommend removing the word “efficiently” from the conclusions as the productivity of the protein has not been reported in the manuscript.
6) It will be beneficial for the reader to and promote this expression system, the author could compare productivity (total product/gm of leaves) of this protein achieved in this study with other expression systems (mammalian).
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EFFECT OF LEAF POSITION AND DAYS POST-INFILTRATION ON TRANSIENT EXPRESSION OF COLORECTAL CANCER VACCINE CANDIDATE PROTEINS GA733-FC AND GA733-FCK IN NICOTIANA BENTHAMIANA PLANT
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript was written clearly and is easily understandable. The goal of this study was mentioned with supporting background information. The introduction was concise leading directly to the gap in the knowledge and the goal of the study is to fill that gap. The article was written strictly according to guidelines from standard sections. My only suggestion for the results section is to increase the resolution of figure 4. The raw data from the manuscript is very useful to me in assessing the quality of results from well-defined experiments. All results support the main point of this manuscript which is to identify the spatial and temporal expression of tumor-associated antigen GA733 in a plant-based transient expression system.
Experimental design: The question that was answered in this manuscript is relevant to the scope of PeerJ. Recombinant protein production using a plant-based system is well-known. However, generating a transgenic plant with stably expressing recombinant protein is a time-consuming process with unpredictable results. Therefore, this manuscript addresses these issues using a transient plant-based expression system.  Besides, the authors screened the spatial and temporal variables for maximum expression of tumor-associated antigens GA733-Fc and GA733-Fck in a plant-based transient expression system.  Overall, this will be very useful in bypassing the mammalian cell expression system and significantly reduces the recombinant protein production expenses.
The methods were described in great detail and also referenced to obtain the materials required for performing reproducibility studies. However, additional details would have assisted in the context comparing the expression of GA733-Fc and GA733-FcK:
1)	The manuscript concluded that GA733-FcK was expressed in higher amounts when compared to GA733-Fc. However, the exact amount of agrobacteria injected into bethamainan leaf was not provided. The small differences in the number of agrobacteria injected between GA733-Fc and GA733-FcK could lead to a difference in the protein yields as mentioned in this manuscript.
2)	The authors compared expression from leaves at different positions on the plant and concluded that top leaves produced more protein than base leaves. However, the study did not clearly state if the agroinfiltration had been performed on the same age of the leaf from different positions of the plant? Based on the text from results and discussion sections, it looks like the authors didn’t consider the age of leaf at a particular position (Top, Middle, and Base) before agroinfiltration and protein expression analysis.
Validity of the findings: Previous publications explored the plant-based expression of GA733. Also, these studies provided evidence that the recombinant GA733 expressed in the plant-based system was functional and economical to produce. However, the novelty of this study arises from two parts. First, use of a transient system instead of a laborious stable expression system and second, establishing the spatial and temporal conditions for maximum yield of recombinant GA733.
The presence of GA733-Fc and GA-733-FcK genes in benthamiana leaves was analyzed with PCR and protein expression was shown using SDS-PAGE. The specificity of expressed proteins was analyzed using the western blot technique. A critical step in quality control of a recombinant protein is its functional assessment. This manuscript provided ELISA data supporting 50 % of the answer to its functional assessment. In my opinion, an in vivo functional assessment of recombinant expressed GA733-Fc and GA733-FcK proteins would have been a significant boost to the impact of this manuscript.  The data provided in this manuscript is robust and proper controls had been used in all the experiments. For example, the authors assessed data from 4 leaves for calculating relative density results in figure 3B. However, a statistical analysis would have been useful in comparing the relative temporal expression of proteins. Also, supporting data comparing the spatial expression of proteins could have been generated by analyzing the absolute amount of protein produced from the same quantity of starting material. For example, the absolute total amount of proteins can be obtained by performing size-exclusion chromatography on Protein A elution material. The authors made conclusions based on results from well-designed experiments. Speculation had been made in the discussion section to answer the lower expression from base leaves. No supporting data is shown to compare either chlorosis or necrosis between leaf positions (top vs middle vs base).
Additional comments: 1) In figure 3B, it was shown that the expression of GA733-FcK was decreased from day 5 to day 7 and increased again. Did you get a chance to examine the reason for these changes?
2) What are the 240 and 30 kDa bands that showed up in SDS-page and western blot analysis?


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EFFECT OF LEAF POSITION AND DAYS POST-INFILTRATION ON TRANSIENT EXPRESSION OF COLORECTAL CANCER VACCINE CANDIDATE PROTEINS GA733-FC AND GA733-FCK IN NICOTIANA BENTHAMIANA PLANT
Review round: 1
Reviewer: 3

Output:
Basic reporting: The article uses clear, unambiguous, and professional English.
Literature references are sufficient with field background/context provided.
Article structure, figures, tables, etc. are professional.
Article is self-contained with relevant results to hypotheses.
Experimental design: Original research within the scope and aim of the journal.
Research question well defined and meaningful. Knowledge gaps explained and filled.

Investigation could have been more rigorous or at least the methods described more clearly in some cases. See the below comments for details.

Line 68-70: According to Peyret & Lomonossoff 2013 the pEAQ-HT vector allows for protein production at a maximum reported amount of ~0.5g/kg. However, Diamos et al 2020 reports production of monoclonal antibodies at “3-5 g/kg leaf fresh weight or ~50% total soluble protein.” Using the geminiviral expression system. This holds true with both VLPs and Toxoid Fusions produced using the same expression system. While it is perhaps not as easily utilized, this data shows the below statement to be of issue

“This deconstructed vector lacks the viral coat protein and has been shown to allow higher and faster recombinant protein production than other plant expression systems.”

Line 113-115: It is customary to report final OD600 of the infiltrated solution as was done in Sainsbury et al. 2009. While an OD range of 0.3-0.4 was measured before cells were spun down and resuspended inaccuracies during this step could lead to inaccuracies in the final amount of protein expressed.

Line 328-330: “except for 7 dpi” Protein production does not generally fall off during the expression period and then increase again according to all data I’ve gathered when doing time courses. It was my assumption that all samples were taken in triplicate, however this is not clearly stated in Figure 3 or in the text. If this was not done, a major short coming of this study could be that there is a large difference in protein expression among leaves/plants even if samples are taken from the same area (top, middle, or base). While the general trend is evident and supported, if the samples were not taken in triplicate, or were not taken from the same leaf, on the same plant, for each DPI, then I highly suggest noting these shortcomings in the paper or repeating this part of the experiment. While repeating the experiment would take a lot of time, and the benefit would be minimal (due to the general trend) it is important that this question of replication in the samples is addressed.

I suggest minor revisions be made to fix this issue.
Validity of the findings: The findings from this article are novel and well-articulated both through the text and figures.

Most data have been provided and is robust in general. See the below notes.

Line 328-330 as stated above.

Line 308-310: Is there a good reason why this data is not shown? If so, please state why (such as being part of a data set to be published in the future).

Conclusions are well stated, connected to the hypothesis, and limited to supporting results.

All speculation is based on the experimental evidence at hand and is clearly stated as such.
Additional comments: Line 32: The statement of difference in expression between GA733-FcK and GA73-FcK would benefit from the mg/g (or mg/ml) leaf tissue being reported, or a percentage.

Line 238-241: The experimentation used to determine the protein production in each major leaf group (top, middle, base) is quite robust from a design point of view (even if there is the issue of multiple samples being tested) and confirms what has been a general consensus among several plant-based protein expression groups using N. Benthamiana known to myself.

The paper is very clear throughout with the only errors being what I’ve noted herein. It is a very good paper and was worthwhile to delve into.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EFFECT OF LEAF POSITION AND DAYS POST-INFILTRATION ON TRANSIENT EXPRESSION OF COLORECTAL CANCER VACCINE CANDIDATE PROTEINS GA733-FC AND GA733-FCK IN NICOTIANA BENTHAMIANA PLANT
Review round: 2
Reviewer: 1

Output:
Basic reporting: I thank Authors for addressing reviewer's comments and the manuscript is well written and the introduction is comprehensive.
Experimental design: Once again, I thank Author's considering my comments and addressing the issues.
Validity of the findings: Author's did addressed my concerns with the claims and happy with their response.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EFFECT OF LEAF POSITION AND DAYS POST-INFILTRATION ON TRANSIENT EXPRESSION OF COLORECTAL CANCER VACCINE CANDIDATE PROTEINS GA733-FC AND GA733-FCK IN NICOTIANA BENTHAMIANA PLANT
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Thank you for taking the time to correct all the issues I had with the manuscript. It is a fine paper.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: DIVERSITY STRUCTURE OF THE MICROBIAL COMMUNITIES IN THE GUTS OF FOUR NEOTROPICAL TERMITE SPECIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1. The authors should clearly state the aim of this study in the Introduction.

2. Fastq files of sequenced amplicons must be publicly available. Please provide the accession numbers.

3. L55: “lower” and “higher” termites is not defined based on their phylogenetic positions, not the presence/absence of flagellates.

4. Please rephrase L78 to “prokaryotic 16S rRNA gene and fungal ITS...” and remove the duplicated sentence “The analysis was based on ...” at L86–87.

5. L128, L188: Use lower case for “unweighted”.

6. L133–L135: I recommend describing the value of sequences and ASV counts in the Result section.

7. L175: Remove the parentheses.

8. L181 and elsewhere: “Eveness” and “Observed” should be rephrased to other terms such as “Pielou’s evenness” and “number of observed ASVs”, respectively.

9. Figure axis labels: SVSs -> ASVs

10. Because Fig. 3A and Fig. S1A are indicating substantially same things, Fig. 3A can be removed.

11. Table 2: Number of OTUs -> Number of ASVs
Experimental design: 1. How did the authors identify the species of the termites?

2. L107–111: 515F and 806R primer pairs are for V4 region. Please cite the references of primers.

3. DADA2 have provided the official tutorial specific to ITS sequences (https://benjjneb.github.io/dada2/ITS_workflow.html). Did the authors conduct the ITS amplicon analysis in accordance with this tutorial? I am afraid that some artifact such as remaining primer sequences would affect the amount of the ASV-assigned reads. In addition, I ask the authors to show the read counts of raw fastq, quality-filtered fastq, and ASV-assigned reads in each sample.

4. L128: Which method was applied for the FDR correction?

5. L125–L128, L135–L139: It is confusing that methods for the community diversity calculations and statistic tests have been described in the two separate sections. Why did the authors apply two different approaches? Please provide a brief explanation.
Validity of the findings: 1. Because as few as one or two termite species were examined for each diet group, the results shown in this study cannot exclude the possibility that not diet but host phylogeny is the major factor determining the structure of gut microbial community. In order to justify the authors’ argument “diet-driven microbial community assembly in the termite gut (L39)”, gut community of several more termite species should be simultaneously compared. Thus, I request the authors to compare the results from this study with those from previous researches, e.g., Mikaelyan et al., 2015; Rahman et al., 2015; Bourguignon et al., 2018. Especially, in addition to the discussion stated between L236–L256, I request a supplemental figure in order to prove that the results shown in this study are truly consistent with the previously published ones. I understand that it is often difficult to compare the results from different research projects due to the different methodologies, but it may be possible to preliminarily compare taxonomic compositions as shown in Fig. 1.

2. Given that each ASV represents a unique amplicon sequence and that the four examined termite species are affiliated with the distinct clades, it is quite surprising that 38 ASVs, representing as much as nearly 30% of total sequences, were shared among all of the four termite species. Rather, I suspect that these “core ASVs” are the results from DNA cross-contamination and/or cross-talk during sequencing (Wright and Vetsigian, 2016). To clarify the abundance of these core ASVs within each of 12 samples, I request the authors to provide a heatmap-style figure.
Additional comments: In this manuscript, Virkram et al. investigated the prokaryotic and fungal community structures in the gut of four higher termite species with different food preferences. 16S amplicon analysis revealed that prokaryotic taxonomic composition was distinct between soil-/grass- feeding termites and wood-feeding ones, and that species evenness and richness of prokaryotes were higher in the soil-/grass- feeding species than wood-feeding ones. Although most of fungal ITS amplicons were taxonomically unclassified, alpha-diversity indices suggest that fungal community is also distinct between soil/grass feeders and wood feeders. The authors concluded that diet is the major factor to determine the microbial community structures in the termite gut.
As referred in this manuscript, several studies had already investigated the relationship between gut microbial community, phylogeny, and diet of termites. These researches pointed out that both host termite phylogeny and diet can influence the community structures of gut microbiota. Thus, the discussion provided in this manuscript is not such novel; nevertheless this research may provide novel information on the gut microbial communities of not-yet-studied termite species, which would facilitate our deeper understanding on the ecology and evolution of termites and their gut symbionts. I would like to ask the authors to clearly explain the value and limitation of this study in the revised manuscript.

Mikaelyan et al. (2015) Mol. Ecol. https://doi.org/10.1111/mec.13376
Rahman et al. (2015) Microbiome https://doi.org/10.1186/s40168-015-0067-8
Wright and Vetsigian (2016) BMC Genomics https://doi.org/10.1186/s12864-016-3217-x
Bourguignon et al. (2018) Curr. Biol. https://doi.org/10.1016/j.cub.2018.01.035


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: DIVERSITY STRUCTURE OF THE MICROBIAL COMMUNITIES IN THE GUTS OF FOUR NEOTROPICAL TERMITE SPECIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The bacterial community structures in several higher termites including the investigated genera have already been studied elsewhere (such as Calusinska et al. (2020) and Marynowska et al. (2020) in addition to Dietrich et al. (2014) and Mikaelyan et al. (2015) in the references). These literatures should be considered in Introduction.

Calusinska et al. (2020) doi: 10.1038/s42003-020-1004-3
Marynowska et al. (2020) doi: 10.1186/s40168-020-00872-3
Experimental design: The compositions of the gut bacteria in the termites in this study were more or less different from the previous studies, while the authors assumed it partly due to variations in DNA extraction methods (L254-L255). The method described that the authors employed the DNeasy Blood and Tissue kit, but this kit does not contain reagents for lysis of the fungal and bacterial cell walls nor those for removal of humic compounds. The authors should clarify methodological descriptions of pretreatments to lyse the fungal and bacterial cell walls and remove humic compounds from soil/humus-feeders that inhibit PCR.
Validity of the findings: The conclusion from the results on the bacterial community structures was awkward because the present study analyzed the community structures with only four termite species, which belong to three different subfamilies and also have incongruous feeding preferences, making unlikely to be grouped to each other. In this context, I encourage the authors to analyze alpha- and beta-diversities shown in Fig, 3 with the bacterial community structures previously reported from several higher termites to obtain more a rigid conclusion.
Additional comments: This study analyzed the gut microbiomes of four Algentinian termite species and confirmed that diet determine the microbial community structure in termite gut. Their finding on the fungal phylum that was shared among the investigated termites seems novel and sound except for the procedure of DNA extraction mentioned above. However, the analyses on the bacterial community structures were somewhat awkward considering the current situation of this study area and should be improved taking the previous similar studies into consideration.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: DIVERSITY STRUCTURE OF THE MICROBIAL COMMUNITIES IN THE GUTS OF FOUR NEOTROPICAL TERMITE SPECIES
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript describes a 16S rRNA/ITS based analysis of bacterial, archaeal and fungal gut communities associated with higher termites Cornitermes cumulans, Microcerotermes strunckii, Nasutitermes corniger, and Termes riograndensis. The primary hypothesis appears to have been to understand the role of diet in determining the gut community structure of these termite species, which has been addressed several times before, either using the exact termite species used in the current study or very closely related ones.

The manuscript is written clearly and I was able to follow the authors methodology. Most of the relevant literature has been cited, however, the authors do not cite it sufficiently in the introduction, which ends up overemphasizing the knowledge gap they are addressing. The article is clearly structured. Figures and tables have legible labels and captions. The results address the hypotheses adequately. However, as I remarked earlier, these hypotheses have been adequately tested in previous studies, which makes the results in the current study repetitive and unremarkable.
Experimental design: The research question:
The authors test the hypothesis that diet plays an important role in the determination of gut community structure in higher termites. This is a hypothesis that has been tested several times before with much larger datasets and using more sophisticated methods (1,2,3). While it may technically fulfill PeerJ's criterion for primary research, I would consider the study repetitive on account of selecting the exact same species used before and repeating a previously used methodology with a much smaller dataset.

Although the authors cite these and other studies as a token, they do not cite them in the right context in the introduction, which results in overemphasizing the knowledge gap. Moreover first-description statements as in 226-228 are highly inaccurate and misleading. N. corniger has the best assessed microbiomes among higher termites -not only do we know the presence and abundance of bacterial taxa in the hindgut (4), but in every gut section (5,6). It has been metagenomically (7) and metaproteomically (8) analyzed. We even know what bacteria are associated with wood fibers in the termite's hindgut (9). Moreover, C. cumulans has also been analyzed in great detail - taxonomically (10) and metagenomically (11.) We even know the microbiota associated with its mound (12). As for the remaining species, close relatives have been studied (1,3,6) in enough detail to give us an idea of at least their prokaryotic gut communities (which are by far the most ecologically relevant guild within the microbiome). In short, given all this published information, I am not sure a clear knowledge gap is being addressed.

However, despite the poor experimental design, the study is technically sound and has used state-of-the-art tools for processing sequencing data and statistical analysis. The methods described have been done so in enough detail that they can be replicated.

1. Mikaelyan, A., Dietrich, C., Köhler, T., Poulsen, M., Sillam‐Dussès, D. and Brune, A., 2015. Diet is the primary determinant of bacterial community structure in the guts of higher termites. Molecular Ecology, 24(20), pp.5284-5295.
2. Rahman, N.A., Parks, D.H., Willner, D.L., Engelbrektson, A.L., Goffredi, S.K., Warnecke, F., Scheffrahn, R.H. and Hugenholtz, P., 2015. A molecular survey of Australian and North American termite genera indicates that vertical inheritance is the primary force shaping termite gut microbiomes. Microbiome, 3(1), p.5.
3. Bourguignon, T., Lo, N., Dietrich, C., Šobotník, J., Sidek, S., Roisin, Y., Brune, A. and Evans, T.A., 2018. Rampant host switching shaped the termite gut microbiome. Current biology, 28(4), pp.649-654.
4. Dietrich, C., Köhler, T. and Brune, A., 2014. The cockroach origin of the termite gut microbiota: patterns in bacterial community structure reflect major evolutionary events. Applied and Environmental Microbiology, 80(7), pp.2261-2269.
5. Köhler, T., Dietrich, C., Scheffrahn, R.H. and Brune, A., 2012. High-resolution analysis of gut environment and bacterial microbiota reveals functional compartmentation of the gut in wood-feeding higher termites (Nasutitermes spp.). Applied and environmental microbiology, 78(13), pp.4691-4701.
6. Mikaelyan, A., Meuser, K. and Brune, A., 2017. Microenvironmental heterogeneity of gut compartments drives bacterial community structure in wood-and humus-feeding higher termites. FEMS Microbiology Ecology, 93(1).
7. Warnecke, F., Luginbühl, P., Ivanova, N., Ghassemian, M., Richardson, T.H., Stege, J.T., Cayouette, M., McHardy, A.C., Djordjevic, G., Aboushadi, N. and Sorek, R., 2007. Metagenomic and functional analysis of hindgut microbiota of a wood-feeding higher termite. Nature, 450(7169), pp.560-565.
8. Burnum, K.E., Callister, S.J., Nicora, C.D., Purvine, S.O., Hugenholtz, P., Warnecke, F., Scheffrahn, R.H., Smith, R.D. and Lipton, M.S., 2011. Proteome insights into the symbiotic relationship between a captive colony of Nasutitermes corniger and its hindgut microbiome. The ISME journal, 5(1), pp.161-164.
9. Mikaelyan, A., Strassert, J.F., Tokuda, G. and Brune, A., 2014. The fibre‐associated cellulolytic bacterial community in the hindgut of wood‐feeding higher termites (N asutitermes spp.). Environmental Microbiology, 16(9), pp.2711-2722.
10. Grieco, M.A.B., Cavalcante, J.J., Cardoso, A.M., Vieira, R.P., Machado, E.A., Clementino, M.M., Medeiros, M.N., Albano, R.M., Garcia, E.S., de Souza, W. and Constantino, R., 2013. Microbial community diversity in the gut of the South American termite Cornitermes cumulans (Isoptera: Termitidae). Microbial ecology, 65(1), pp.197-204.
11. Grieco, M.B., Lopes, F.A., Oliveira, L.S., Tschoeke, D.A., Popov, C.C., Thompson, C.C., Gonçalves, L.C., Constantino, R., Martins, O.B., Kruger, R.H. and de Souza, W., 2019. Metagenomic Analysis of the Whole Gut Microbiota in Brazilian Termitidae Termites Cornitermes cumulans, Cyrilliotermes strictinasus, Syntermes dirus, Nasutitermes jaraguae, Nasutitermes aquilinus, Grigiotermes bequaerti, and Orthognathotermes mirim. Current Microbiology, 76(6), pp.687-697.
12. Costa, P.S., Oliveira, P.L., Chartone-Souza, E. and Nascimento, A.M., 2013. Phylogenetic diversity of prokaryotes associated with the mandibulate nasute termite Cornitermes cumulans and its mound. Biology and fertility of soils, 49(5), pp.567-574.
Validity of the findings: Linking back to my comment in section 2 for an inadequately defined knowledge gap, I am not sure if the replicated sampling of the microbiomes of two well-studied species here is justified. Given that it has been shown that diet is the primary determinant of gut community structure in higher termites, I am not sure what contribution the current study makes (outside of some information on fungal communities) to literature.

The discussion is extremely rudimentary and superficial, and does not go into any detail about why they expect certain microbes to be dominant in a diet group. For instance, we know a lot about the microbial ecology of the guts of higher termites (1,2,3,4) and about how this may influence the gut community structure, but these valuable discussions have been left out.

1. Bignell, D.E. and Eggleton, P., 1995. On the elevated intestinal pH of higher termites (Isoptera: Termitidae). Insectes Sociaux, 42(1), pp.57-69.
2. Brune, A., Emerson, D. and Breznak, J.A., 1995. The termite gut microflora as an oxygen sink: microelectrode determination of oxygen and pH gradients in guts of lower and higher termites. Applied and Environmental Microbiology, 61(7), pp.2681-2687.
3. Köhler, T., Dietrich, C., Scheffrahn, R.H. and Brune, A., 2012. High-resolution analysis of gut environment and bacterial microbiota reveals functional compartmentation of the gut in wood-feeding higher termites (Nasutitermes spp.). Applied and environmental microbiology, 78(13), pp.4691-4701.
4. Mikaelyan, A., Strassert, J.F., Tokuda, G. and Brune, A., 2014. The fibre‐associated cellulolytic bacterial community in the hindgut of wood‐feeding higher termites (N asutitermes spp.). Environmental Microbiology, 16(9), pp.2711-2722.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: DIVERSITY STRUCTURE OF THE MICROBIAL COMMUNITIES IN THE GUTS OF FOUR NEOTROPICAL TERMITE SPECIES
Review round: 2
Reviewer: 1

Output:
Basic reporting: No additional comment.
Experimental design: No additional comment.
Validity of the findings: No additional comment.
Additional comments: I appreciate the authors’ efforts to improve the manuscript, however there remains some points that should be addressed.

Fig. 5 BC and beta-diversity analysis
(i) Although the authors imply the influence of not only food habit but also host phylogeny on termite gut microbiome, the present manuscript lacks the analyses on the differences of gut microbiome in terms of host phylogeny. The authors should provide some statistical comparisons between host phylogeny (i.e., subfamily or genera) as did between the food habit.
(ii) It will be more convenient for readers if each sample point shown in the NMDS plots (Fig. 5BC) is categorized (shaped or colored) by host genera or subfamilies in addition to the food habit.
(iii) At least the result of unweighted UniFrac (Fig. 5B), I can find at least two clusters separated along the NMDS1 axis. Could the authors provide some interpretation of these clusters?

L140: Remove “V3”. 515F-806R primer pair is for the V4 region.

L181–L201: The methods described here are almost identical to what described in the section “Bioinformatic analysis of 16S rRNA and ITS sequences”. Please simplify them.

L196: due “to” the low number

L233: It is somewhat odd to mention Methanimicrococcus (Archaea) in the context of comparison between bacterial genera.

Fig.2 labels: “SVSs” have not been corrected to “ASVs” yet.

Fig. 6
(i) (A)Shared ... “four gut microbiomes” -> “four termite species”.
(ii) Remove “(B)Shared prokaryotic ASVs between four gut microbiomes”

Supplemental Figures: Numbering of supplemental figures is inconsistent with the main text.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EFFECT OF COTTON RESIDUES INCORPORATION ON SOIL PROPERTIES, ORGANIC NITROGEN FRACTIONS, AND NITROGEN-MINERALIZING ENZYME ACTIVITY UNDER LONG-TERM CONTINUOUS COTTON CROPPING
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1. There are some grammar errors through the manuscript, authors should improve the wriiten English. If available, please ask a native speaker to help.
2. Not enough background and knowledge gap, authors should improve the introduction section.
3. The structure, figures and tables are fine.
4. Last but not least,nitrate reductase, and nitrite reductase are not N-mineralizing enzymes. This makes this manuscript not suitable to be published.
Experimental design: The experiment was designed reasonable. However, the authors classified nitrate reductase, and nitrite reductase into wrong group.
Validity of the findings: The results are meaningful to understand how carbon and nitrogen change after straw return. But from the title, the results of carbon took too much pages. "Soil N-mineralizing enzyme activities " included two unrelated enzymes. The wrong classification of enzymes directly resulted wrong conclusions.
Additional comments: 1. Please note that nitrate reductase, and nitrite reductase are not N-mineralizing enzymes. They are denitrification enzymes.
2. Try to find a native speaker to improve the written English.
3. The disscussion section should be clearer, it is hard to read now.
4. Try to include carbon in the title or delete some results on carbon.
5. Please re-draw figure 2. Do not merge the sub-figures.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EFFECT OF COTTON RESIDUES INCORPORATION ON SOIL PROPERTIES, ORGANIC NITROGEN FRACTIONS, AND NITROGEN-MINERALIZING ENZYME ACTIVITY UNDER LONG-TERM CONTINUOUS COTTON CROPPING
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is well organized and unambiguous. Language used in the manuscript is up to professional standards. Introduction is oriented in towards specific goals of the study.  Results are structured properly with matching figures and tables. Manuscript is written with proper references.
Experimental design: Experimental methods are thoroughly described and well within standards of the field. Methods used in the investigation are well known in the field to address the questions of the agriculture related study. Overall the investigation is supported by well documented methods.
Validity of the findings: Conclusions drawn in the study are supported by results described in the manuscript. Conclusions are made from statistical significant results.
Additional comments: The manuscript entitled “Effect of cotton residues incorporation on soil organic nitrogen fractions and nitrogen-mineralizing enzyme activity under long-term continuous cotton cropping” shows interesting results and presents important agricultural study of soil organic nitrogen, organic carbon and nitrogen-mineralizing enzyme activities.

Authors are requested to explain following results.

Line 245-246: The numbers described for protease, nitrate reductase and nitrite reductase do not match those described in Figure 2.

Figure 2: Significant increase in 15a compared to 10a has not been observed for protease, urease, nitrate reductase and nitrite reductase activities. While significant increase has been observed for 10a and 20a compared to 5a for all enzyme activities except nitrate reductase. Is there a specific reason for this discrepancy for 15a results?

Typological corrections:
Figure 2: Two panels are labelled as Protease activities. On of them has to be for nitrite reductase.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EFFECT OF COTTON RESIDUES INCORPORATION ON SOIL PROPERTIES, ORGANIC NITROGEN FRACTIONS, AND NITROGEN-MINERALIZING ENZYME ACTIVITY UNDER LONG-TERM CONTINUOUS COTTON CROPPING
Review round: 2
Reviewer: 1

Output:
Basic reporting: The written English is clear and professional English used throughout the article. However, more recent literatures (recent 5 years) should be cited to replace old references.
Experimental design: The experimental design was reasonable and could support the research goal.
Validity of the findings: There are some new findings in the result. Conclusions are clear and related to objectives.
Additional comments: 1. Please verify why 0-40 cm soil layer was selected in this study。
2. Please include important numbers in the abstract.
3. The methods to analyze N-mineralizing enzyme activities should be stated more, only one sentence is not enough to gain efficient knowledge.
4. Authors should note that the year when the experiment was started should be listed and the basic soil properties were lacked, please supply these data.
5. Try to discuss with recent literatures, i.e., published from 2016.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EFFECT OF COTTON RESIDUES INCORPORATION ON SOIL PROPERTIES, ORGANIC NITROGEN FRACTIONS, AND NITROGEN-MINERALIZING ENZYME ACTIVITY UNDER LONG-TERM CONTINUOUS COTTON CROPPING
Review round: 2
Reviewer: 2

Output:
Basic reporting: Manuscript language has been improved with review.
Experimental design: Necessary changes in experimental results have been made according to suggestions from reviewers. Reviewed manuscript fits desired objectives of the investigation.
Validity of the findings: No comments
Additional comments: No comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: LIMITED INFLUENCE OF MARINE SEDIMENT LYOPHILIZATION ON PROKARYOTIC COMMUNITY STRUCTURE ASSESSED VIA AMPLICON SEQUENCING: AN EXAMPLE FROM ENVIRONMENTALLY CONTRASTED SEDIMENT LAYERS IN TOULON HARBOR (FRANCE)
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors present an interesting manuscript whereby they investigated the effects of freeze drying (lyophilization) of samples for DNA based microbial community analysis.  This is important because as the authors point out in their introduction, freeze drying is used to determine the metal burden per dry mass sediment, and it is important to know whether those same freeze dried samples could be used for DNA analysis.  There are also other geochemical analyses that use freeze drying, so it is a very good idea to test the effects of lyophilization on the DNA protocols.  The authors appear to have done a good job in producing their data, and use the up -to-date 16S primers with the Y ambiguity from Parada et al (modified EMP primers).
Experimental design: My main suggestion would be for the authors to do a qPCR of the 16S rRNA genes from the lyophilized and non-lyophilized samples, to check whether there is a quantitative difference in the amount of DNA extracted from both methods.  If you do not have access to a qPCR, then at least you could present the total amount of DNA measured after the extraction?  This is really important to know I think, in addition to the diversity effects shown in the sequencing data.
Validity of the findings: My main criticism is that looking at Figure 2 and Figure 3, it seems that in the 19-20 cm sample the lyophilized samples have a lower richness compared to the fresh samples.  This should be stated in the text, and conclusions/discussion.  If you just read the text you get the impression there is no difference , which is clearly not the case looking at Figure 2 in the deep sample.  Also, in the 0-1 cm sample there is a huge difference between the lyophilization replicates.  How do you explain that?  Nevertheless, if you look at Figure 5 and Figure 6 there indeed does not appear to be a big difference between the treatments which is a good sign and indeed supports the authors conclusions.
Additional comments: General comment on the results: can you please add more information here?  More details on sequencing and diversity statistics would be appreciated.  Currently this is very sparse and general information that does not help the reader figure out what the really important trends are in the data.  Also, please do not make paragraphs from a single sentence.

Discussion: please add to the discussion how well the DADA2 pipeline captures the real diversity.  You are getting around 2000 ASVs which is realistic, but prior programs like UCLUST inflated diversity 10 to 100 times which was then corrected with USEARCH and confirmed using mock community controls (Edgar 2013).  Might be worth commenting on this.

Additionally, it would be appreciated if in the discussion the authors could expand the comparison between the lyophilized and non-lyophilized samples.  How many OTUs were found in one and not the other?  Which groups were enriched in one and not the other?  Was there a difference in the relative abundance of archaea, or bacteria, that are known to have different membrane structures that probably play a role in their being more or less easily lysed during the extraction procedure?

line 166: what do you mean "taxonomic affiliation precision", and how do you know what is "best"?

It is nice to see that microbial ecologists are starting to submit their work to PeerJ , and getting away from profit based journals (Nature, Frontiers, etc).  This is a good sign for our field!


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: LIMITED INFLUENCE OF MARINE SEDIMENT LYOPHILIZATION ON PROKARYOTIC COMMUNITY STRUCTURE ASSESSED VIA AMPLICON SEQUENCING: AN EXAMPLE FROM ENVIRONMENTALLY CONTRASTED SEDIMENT LAYERS IN TOULON HARBOR (FRANCE)
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors aimed at comparing the microbial community composition in lyophilized and fresh marine harbor sediments from different depths. They hypothesized that lyophilization does not substantially affect cell lysis or DNA accessibility during extraction or PCR.
To this end from three sediment cores slices from three selected layers were split into halves and lyophilized or not. DNA extraction, PCR and MiSeq sequencing were performed using identical methods.
Based on rarefaction analysis, Alpha diversity, “taxonomic precision”, phylum/class level community structure and a PCoA of BC dissimilarity of samples the authors conclude that the sediment treatment does not appear to affect microbial community structure.
Experimental design: The scientific question addressed in this manuscript is interesting and relevant to many scientific sample sets. However, based on the presented data I do not concur with the authors conclusion that sediment treatment has low effects on MC structure. The sample size is to low, split samples should be compared (presented)in a pairwise manner and a statistical analysis should be performed. Figure 2c suggests a somewhat higher ASV richness in lyophilized sediments? In particular, the data depicted in Figure 5 question the conclusions made. MC structure depicted in stacked bars #2 (fresh) and #12 (lyophilized) seem not to have a good match to their corresponding samples. The authors should also show in more detail, which samples come from the same core/slice.
I also suggest performing a weighted UniFrac analysis to take phylogenetic distances between ASV into account.
Validity of the findings: In summary, the data do not justify the conclusion that intersample differences are the same or larger than between pretreatments.
Additional comments: Specific comments:
Ln61 and elsewhere: Please check formatting of in-text citations. Does it meet PeerJ requirements?
-	Ln92: which instruments was used?
-	Ln108 and elsewhere: check for spaces following numbers, 1000 ppm
-	Ln113: three sediment slices
-	Ln¬¬117 and elsewhere: PeerJ format? May read Fig.1 !?
-	Ln117: 150 years old
-	Ln136: trimmed
-	Ln147: give details on used packages
-	Ln160: 2,396
-	Ln170 and elsewhere: check for italics
-	Ln170: among 49 phyla
-	Ln189: check for double spaces


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: LIMITED INFLUENCE OF MARINE SEDIMENT LYOPHILIZATION ON PROKARYOTIC COMMUNITY STRUCTURE ASSESSED VIA AMPLICON SEQUENCING: AN EXAMPLE FROM ENVIRONMENTALLY CONTRASTED SEDIMENT LAYERS IN TOULON HARBOR (FRANCE)
Review round: 1
Reviewer: 3

Output:
Basic reporting: The English is generally well written although the redaction of some sentences can be improved and have been indicated in the pdf file.

The introduction cover basically all the aspects required although some more details are needed and more clear text is necessary to convey some points. However, no basis is given on why the effect specifically of Hg can be potentially important.

Figures are generally of good quality although fig 4 can be improved.

Sequence data have been uploaded to a database.

Figures should be relevant to the content of the article, of sufficient resolution, and appropriately described and labelled.

The results are relevant to the hypothesis put forward but are not sufficient.
Experimental design: The subject is of interest and is still a matter of debate given the limited studies.

However, neither the questions are properly defined neither the design is adequate.

This is not a well designed looking into the effect of freeze drying vs fresh samples.

First, there are only two replicates which prevents any statistical analysis that will confirm the spurious observations made. Therefore, conclusions are a matter of personal perception. I actually interpreted the data in a different than the authors. In addition, taking into account that this is a potentially methodological paper, few details are given for the procedures. Finally, on this matter, authors used an additional pre-treatment for the fresh vs  freeze dried sediment without explaining why and invalidating the comparison. I would also like to point out that what the authors label as fresh sediment is frozen sediment, it has not simple been freeze dried. This is very important as any freezing effect to the cells/sediment structure is similar.

Second, the authors intend to identify an effect of high Hg content in the sediment, however, they are unable to discern the effect of Hg from the effect of sediment depth. Unless they show that the OM content and quality and biogeochemical conditions are the same at 10 and 20 cm depth. Hg and depth are confounded.
Validity of the findings: Given the above, despite the potential interest of the study, given the lack of robustness in the design of the study, unless more replicate samples are used and a valid control site, conclusions are not verified.

More details are given in the attached pdf.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: LIMITED INFLUENCE OF MARINE SEDIMENT LYOPHILIZATION ON PROKARYOTIC COMMUNITY STRUCTURE ASSESSED VIA AMPLICON SEQUENCING: AN EXAMPLE FROM ENVIRONMENTALLY CONTRASTED SEDIMENT LAYERS IN TOULON HARBOR (FRANCE)
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have addressed all my comments.
Experimental design: The authors have addressed all my comments.
Validity of the findings: The authors have addressed all my comments.
Additional comments: The authors have addressed all my comments.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: LIMITED INFLUENCE OF MARINE SEDIMENT LYOPHILIZATION ON PROKARYOTIC COMMUNITY STRUCTURE ASSESSED VIA AMPLICON SEQUENCING: AN EXAMPLE FROM ENVIRONMENTALLY CONTRASTED SEDIMENT LAYERS IN TOULON HARBOR (FRANCE)
Review round: 2
Reviewer: 2

Output:
Basic reporting: ok
Experimental design: ok
Validity of the findings: ok
Additional comments: The manuscript has been greatly improved and additional analysis has been peroformed (uniFrac, qPCR). I support publication.