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: WITHASOMNIFEROL C, A NEW POTENTIAL SARS-COV-2 MAIN PROTEASE INHIBITOR FROM THE WITHANIA SOMNIFERA PLANT PROPOSED BY IN SILICO APPROACHES
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1.1. Please revise the writing of the entire manuscript before resubmitting it.

1.2. In the abstract, name the compounds WS54 (withaphysalin D) and WS81 (withasomniferol C).

1.3. Some unnecessary hyphens in words are found, e.g.: com-pounds (line 25),  in-volved (line 76), hydro-gens (line 167), con-formation (line 183),  re-ported (line 424), and so on.

1.4. Different paragraphs are concentred in a unique paragraph in lines 97-121, making it too long. Please revise it.

1.5. "According to the Lurie et al., 2021" (line 421), change to "According to Lurie et al. (2021)";
"Saggam et al., 2021 suggested" (line 425), change to "Saggam et al. (2021) suggested"; "Kushwaha et al., 2021," (line 432) change to "Kushwaha et al. (2021), and so on.

1.6. The figures must be improved. Revise the resolution and the size of words and number of graphs in figures (they are too short to visualize, mainly figures 2, 5, and 7), according to the dimensions of the manuscript page.

1.7. The captions must be improved and self-explanatory to the best comprehension (e.g. see figure 2).

1.8. The 2D structures of compounds from Table 1 are too short. Please look for a better way to show these structures in the manuscript.

1.9. Improve the title of table 2 (Where these ΔGBIND were calculated? What methodology was employed?).

1.10. Titles from figures and tables must be objective but self-explanatory.

1.11. To meet the standard spelling of amino acid residue and number position in the primary protein sequence, avoid space between amino acid code and number position. Write "Thr25" instead "Thr 25", Cys145 instead "Cys 145", and so on. Revise the entire manuscript, figures, and tables.
Experimental design: 2.1. In the "Molecular docking" section, detailed information about the construction of 3D structures of ligands in detail is missed. How were the geometry of the structures optimized? Quantum (semiempirical or DFT) methods, classical method (force field)? What are the procedures adopted by the authors concerned with the accuracy of 3D structures with global minima of energy? Accurate ligand structures are essential for the subsequent docking and MD studies.

2.2. Despite the information is presented in tables and figure 7, the description of the interactions shown in lines 403-409 must be improved and detailed between each compound and residues.
Validity of the findings: 3.1. Despite the interactions of WS69 (somniferine), WS42 (withanoside II), WS34 (withanolide J), WS33 (withanolide F), and WS75 (withanolide L) with the 3CLpro were already reported previously, it is important to compare the inhibitory activity of these compounds with the two new promissory compounds presented by this study WS54 (withaphysalin D) and WS81 (withasomniferol C). Are they present the same potency or more inhibitory activity?

3.2. The authors also excluded common phenolic, organic acids, and other natural compounds from the analysis with known activity against SARS-CoV-2 3CLpro in vitro (lines 443-450). Have some molecular modeling studies of these excluded compounds been reported? Explain this better in the manuscript. If no, present a good justification to explain why they were excluded.

3.3. It is important to compare these results with previous in vitro data (e.g. IC50 values, Km or others from isolated compounds or W. somnifera extract), to infer the involvement of the compounds in the activity.
Additional comments: In the introduction section (lines 117-121), the authors highlighted that "no studies have been performed to test all the reported WS phytoconstituents against SARS-CoV-2. In this research, all the reported WS phytoconstituents were collected through manual literature search, and their binding efficiency against SARS-CoV-2 3CLpro was evaluated using in silico molecular docking and MD simulation studies." In the way it is set out, the understanding is that all the 81 compounds were submitted to molecular docking and MD simulation, but that is not so. Compounds were excluded from the analysis (see comments here in section 3 "Validity of the findings"). The authors must revise and correct this fragment.

A set of reports concerning the inhibition of SARS-CoV-2 main protease by compounds from Withania somnifera has been recently published:
Tripathi, M. K., Singh, P., Sharma, S., Singh, T. P., Ethayathulla, A. S., & Kaur, P. (2021). Identification of bioactive molecule from Withania somnifera (Ashwagandha) as SARS-CoV-2 main protease inhibitor. Journal of Biomolecular Structure and Dynamics, 39(15), 5668-5681.
Dhawan, M., Parmar, M., Sharun, K., Tiwari, R., Bilal, M., & Dhama, K. (2021). Medicinal and therapeutic potential of withanolides from Withania somnifera against COVID-19. J. Appl. Pharm. Sci, 11, 6-13.
Sudeep, H. V., Gouthamchandra, K., & Shyamprasad, K. (2020). Molecular docking analysis of Withaferin A from Withania somnifera with the Glucose regulated protein 78 (GRP78) receptor and the SARS-CoV-2 main protease. Bioinformation, 16(5), 411.
Khanal, P., Chikhale, R., Dey, Y. N., Pasha, I., Chand, S., Gurav, N., ... & Gurav, S. (2020). Withanolides from Withania somnifera as an immunity booster and their therapeutic options against COVID-19. Journal of Biomolecular Structure and Dynamics, 1-14.
Kushwaha, P. P., Singh, A. K., Prajapati, K. S., Shuaib, M., Gupta, S., & Kumar, S. (2021). Phytochemicals present in Indian ginseng possess potential to inhibit SARS-CoV-2 virulence: A molecular docking and MD simulation study. Microbial pathogenesis, 104954.

The methodology employed and the results obtained are adequate and relevant. However, considering the works previously published, the authors must rethink the title, justification, and objective of the work, focusing on the novelty and the relevance of the data to the readers of PeerJ.


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: WITHASOMNIFEROL C, A NEW POTENTIAL SARS-COV-2 MAIN PROTEASE INHIBITOR FROM THE WITHANIA SOMNIFERA PLANT PROPOSED BY IN SILICO APPROACHES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors made the corrections from the first version of the manuscript, according to the reviewers comments, improving the quality of the study.
Experimental design: The authors made the corrections from the first version of the manuscript, according to the reviewers comments, improving the quality of the study.
Validity of the findings: The authors made the corrections from the first version of the manuscript, according to the reviewers comments, improving the quality of the study.
Additional comments: Given the data presented, the title can be modified to "Withasomniferol C, a new potential SARS-CoV-2 main protease inhibitor from the Withania somnifera plant identified by in silico studies".


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: WITHASOMNIFEROL C, A NEW POTENTIAL SARS-COV-2 MAIN PROTEASE INHIBITOR FROM THE WITHANIA SOMNIFERA PLANT PROPOSED BY IN SILICO APPROACHES
Review round: 2
Reviewer: 2

Output:
Basic reporting: The title must be improved to: Withasomniferol C, a new SARS-CoV-2 main protease inhibitor from the Withania somnifera plant proposed by in silico approaches.

line 162: Improve the sentence to (dimensions: x = 13.3, y = 58.2, z = 45.4).

Figure 1: The resolution of the figure is now satisfactory, but the font size of captions highlighted amino acids of the active binding site still remains too short to read in the figure 1. You must edit the image in an other image editor program to increase the font size to improve the visualization to readers.

Figures 5 and 7: the font size of information from the y-axis and x-axis in figure 5; and captions of interactions in figure 7 still remains too short to read in the final version of the manuscript (consider the final manuscript, where the size of figures will be adjusted to the manuscript page, they probably will diminish the figure size). The font size must be also increased.
Experimental design: No comments.
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: A NEW SPECIES OF DEEP-SEA GRUNT, RHONCISCUS PAUCO (LUTJANIFORMES: HAEMULIDAE), FROM PUERTO RICO
Review round: 1
Reviewer: 1

Output:
Basic reporting: NC
Experimental design: fails to meet standards, see additional comments
Validity of the findings: fails to meet standards, see additional comments
Additional comments: This ms describes a new species of deep water grunt in the caribbean, the sister species of the eastern Pacific species R. branickii.

It is a likely new and technically valid species, however the ms compares it to mainly the clearly unrelated Caribbean congener, instead of the relevant comparison to its sister species. Unfortunately, the sample size is so small that the question is moot, I have no idea how pauco is different from branickii.

The emphasis of the ms, that pauco is not crocro or bayanus, is unneeded, they are completely different and separated in a simple two couplet key. The comparison to the sister species is what is relevant.

The next emphasis of the ms is that pauco is an unusual deep water species in Rhonciscus- but "unusual" is barely applicable in a set of only 4. Then branickii, the fourth is actually found very deep, up to 500m in museum metadata, as well as estuarine. So the genus contains two shallow species, one deep and one both.

The definition of a valid species is whether it can be distinguished from its sister species reliably- not a statement that it is genetically isolated- there are hundreds of reef fish species isolated between sides of the Atlantic, or Caribbean and Brazil, or between the central and eastern Pacific, or between the Indian and Pacific Oceans that are considered the same species.

There are a number of transisthmic populations that are considered the same species (taxonomically if not practically) because there is no reliable diagnostic character separating them. In that case, one would not split the species based only on a genetic difference- people spend a lot of effort trying to find a difference that is persuasive and not an artifact that will disappear with adding a few more specimens. For example, Etropus crossotus is still considered the same species on both sides because after measuring and counting hundreds of fish in a huge study (long time ago), a clean persuasive difference cannot be found. There are others, like Hyporthodus mystacinus.

Thus the relevant comparison is what character distinguishes pauco from branickii- and that should not be done with 5 comparison fishes- I understand the limitations of access these days to museums, but there are large numbers of branickii specimens in major museums, such as SIO & USNM, from all depths from 0-500m. Perhaps a collaboration with a museum staff member who could do the relevant measurements on 100 fish would find the true difference.

The DNA comparison between the two sisters shows they are very close, making a morphological difference much harder to prove and that, by itself, means the sample sizes must be large. The DNA barcode database shows the two species are only 1.5% divergent (minimum interspecific distance, not mean.. two lineages that actually share haplotypes will have a mean difference, even a large one). The few sequences of branickii in the database already have a particularly high intraspecific variation, greater than 1.5% (maximum intraspecific; similarly the mean variation is irrelevant, since that can be low,  even in the case of intraspecific haplotypes more distant from each other than is the other species' haplotypes- meaning one cannot reject the hypothesis the two groups are the same). In this pauco/branickii case the result technically violates the "barcode gap"- i.e. min interspecific distance needs to be greater than max intraspecific, but that is not a disqualifier, just an indication one needs to be even more persuasive on the phenotypic comparison. In other words, when genetics do not provide much support for the split, one needs to document the morphological difference more completely.

Other than that overall issue, the methodology for comparing sets of fish specimens is unconvincing. We need to be assess whether measurements are 1) representative (covers geographic range, depth range, size range, etc.); 2) reliable (repeatable and comparable); and 3) robust (sample size).

It is obvious that comparing a set of morphometrics between 10 deep fish in the Atlantic and 10 freshwater fish in the Pacific is not useful. Similarly, 10 fish under 100 mm compared to 10 fish over 200mm, or 10 fish from California to 10 fish from Puerto Rico. Given the difficulty of collecting pauco, a small sample size is adequate if the set it is compared to is a reasonable sample of branickii to see if there is anything pauco have that can single it out from the specimens of branickii.
The main problem in grunts is the question of reliability. If there is unexpectedly more variation out there than is suggested by the few specimens in this study, then it means that feature cannot be established as a diagnostic character. For grunts, the variability in reported morphometrics in the literature is so extreme that either the guides and keys are mostly errors or the variation from one population to another is an order of magnitude more than from a small sampling.

For example, in this ms there are values that are in a different world from values in the guides and keys. The ms mentions that in passing on a comparison with crocro that literature guide has completely different values for crocro than the ms- not even overlapping- raising questions about consistency. But crocro is not an issue, it is different in many features. The pertinent comparison with branickii is needed, but the guide to eastern Pacific shorefishes is in a different world- the eye diameter, the main feature said to distinguish the species, of this ms' five branickii specimens is so different, it is hard to understand:   2.6-3.2 in HL in guide-- vs. 3.95 to 4.0 in HL in the ms (Table 2- HL=28.5-35.6 %SL divided by eye diam=7.1-9 %SL). For a grunt, a range of 3.95 to 4.0 is far too narrow a range to represent a species, and the 5 specimens range from 159 mm to 234 mm- that alone should increase the range of eye diameters (far) beyond 3.95 to 4.0 based on allometry (smaller fish have relatively larger eyes).

There are numerous other not-even-overlapping differences between meristic and morphometric values found in the ms and in the various literature, suggesting there is vastly more variability than encountered in the small samples here.

Note that gross morphometrics, such as eye size or body depth are known to be highly variable, so differences found must be approached with skepticism, especially when the populations are so close genetically. Typically differences between sister species are in color, markings, or meristics, at best.

To understand the enormous variation in reported eye sizes in rhonciscus, I researched a few photographs and find these photographs in the attached document- the eye size of pauco and branickii look the same. Four photos of the grunt P. macracanthus show huge variation in eye diameter, all among adults.

There also seem to be errors- these results seem impossible for the 4 species in Table 2:  these measurements below are first pauco range, and then branickii range: branickii have a first dorsal spine almost ten times smaller than pauco? Attached in the photographs one from the guide of a branickii with a normal length first dorsal spine.

First dorsal spine

20.93-25.04

2.84-4.81

Fourth dorsal spine

2.92-5.79

13.65-17.32

Fifth dorsal spine

12.06-19.07

13.02-16.24

Penultimate dorsal spine

14.69-19.12

4.06-6.66


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: A NEW SPECIES OF DEEP-SEA GRUNT, RHONCISCUS PAUCO (LUTJANIFORMES: HAEMULIDAE), FROM PUERTO RICO
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. BASIC REPORTING
-  Clear, unambiguous, professional English language used throughout.
Yes, very well written.
-  Intro & background to show context. Literature well referenced & relevant.
Yes, see general comments.
-  Structure conforms to PeerJ standards.
Yes.
-  Raw data
I may be me missing something but I cannot find the data deposition statement.
-  Figures are relevant, high quality, well labelled & described.
Yes.
Experimental design: -  Original primary research within journal scope.
Yes.
-  Research question well defined, relevant, meaningful. It is stated how the research fills an identified knowledge gap.
Yes to all of these issues.
-  Rigorous investigation performed to a high technical & ethical standard.
Yes, see general comments.
-  Methods described with sufficient detail & information to replicate.
Yes.
Validity of the findings: 3. VALIDITY OF THE FINDINGS
-  Impact and novelty not assessed. Meaningful replication encouraged where rationale & benefit to literature is clearly stated.
Yes, in terms of this as a new species description.
-  All underlying data have been provided; they are robust, statistically sound, & controlled.
Yes, as i can determine for a new species description.
-  Conclusions
Well written, supported by results, linked to original question.
Additional comments: Review comments from the manuscript:
The paper is more than a good new species description, it indeed reflects a very interesting and novel biogeographical issue in a solid fashion.
-  Line 24. After the phrase “sister species”, the authors may want to provide the characters that suggest this relationship, not essential.
-  29.  If the authors desired, they could end the abstract by saying that the species occurs in far deeper waters than Western Atlantic congeners.
-  44-46.  For the uninformed, this paragraph does not refer to Rhonciscus.  It refers to Pomadasys being split into lineages – seems like there should be a statement saying that 1 of those lineages was Rhonciscus.
-  76.  “Of” should probably be “on”.
-  165-167.  This is an extremely limited area. Have fishers elsewhere on the island been asked about this species?
-  168.  Is the phrase “ that suggests extending these limits.” really needed? Perhaps just take out the phrase in quotes, it seems a little early to imply this is the only population. I wonder if any are at Mona Island or the eastern Dominican Republic across the Mona Channel.
-  Discussion.  There are 2 long and dense paragraphs in this section. They are solid but would be easier to read  if split into smaller paragraphs, suggestions follow.
-  207.  Suggest inserting a new paragraph before “What is even more surprising…”
-  210.  After rivers suggest inserting: “… or slow creeks (in easter Florida).”
-  217.  After South America possibly insert: “… with it’s WA congener known primarily from coastal rivers, …”
-  229.  Suggest new paragraph before “It is common…”
-  Tables and captions are solid.
-  Figure 4 caption:  first line, “on” could be “of” or it could best, be deleted.
-  Figure 5 is valuable. Fyi, the top fish image is laterally out of alignment (a bit) with the three images below.


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: HIGH-THROUGHPUT SEQUENCING REVEALS RHIZOSPHERE FUNGAL COMMUNITY COMPOSITION AND DIVERSITY AT DIFFERENT GROWTH STAGES OF POPULUS EUPHRATICA IN THE LOWER REACHES OF THE TARIM RIVER
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: Is it possible for the author to validate and justify the work carried out in this study using the following reference, specifically with reference to the use of primers and the bias in sequencing outcome?

Bellemain et al. BMC Microbiology 2010, 10:189
http://www.biomedcentral.com/1471-2180/10/189

ITS as an environmental DNA barcode for fungi: an in silico approach reveals potential PCR biases (Eva et al. 2010).
Additional comments: The research paper titled "High-throughput sequencing reveals rhizosphere fungal community composition and diversity at different growth stages of Populus euphratica in the lower reaches of the Tarim River" explains the details of the rhizosphere fungal communities at the different growth  stages of P. euphraria.

The  outcomes of this piece of research  provide scientific basis for the detail study and importance of rhizosphere  fungal communities and their interaction with  plants in arid ecosystem.

This study provides a base for future investigation of  differences in the composition and structure of soil fungal community in the rhizosphere of diverse ecosystem.


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: HIGH-THROUGHPUT SEQUENCING REVEALS RHIZOSPHERE FUNGAL COMMUNITY COMPOSITION AND DIVERSITY AT DIFFERENT GROWTH STAGES OF POPULUS EUPHRATICA IN THE LOWER REACHES OF THE TARIM RIVER
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors are interested in the diversity, community structure, and relationship between rhizosphere fungi and environmental factors at different growth stages of Populus euphraria. The MS is basically well-written with the information clearly-presented.
Experimental design: Overall, the  experimental design and data analysis seem solid.
Validity of the findings: The Result part should not take the statistical method as the sub title, please reorganized the Result part based on scientific problems mentioned above.
Additional comments: 1.The Abstract needs to be rewritten, with the following parts for your reference:
Aim,
Methods,
Results, specially this part should be briefly presented important findings, not all your results.
Main conclusions

2.A figure about the location of study and samping sites is needed at the "Study sites and sampling" in M&M part.
3.Please provide higher resolution pictures in the MS.


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: HIGH-THROUGHPUT SEQUENCING REVEALS RHIZOSPHERE FUNGAL COMMUNITY COMPOSITION AND DIVERSITY AT DIFFERENT GROWTH STAGES OF POPULUS EUPHRATICA IN THE LOWER REACHES OF THE TARIM RIVER
Review round: 1
Reviewer: 3

Output:
Basic reporting: The hierarchical structure of the paper “High-throughput sequencing reveals rhizosphere fungal community composition and diversity at different growth stages of Populus euphratica in the lower reaches of the Tarim River (#69932) ” is clear, and the research results have certain reference value. But there are many problems that need to be modified. After minor revision, it can be accepted for publication in "Peerj."

1.In order to clarify the difference of fungal function of spesies level between different comparison groups, if it is better to add KEGG passway t test analysis?

2. The clarity of the drawing is too poor to see the details.

3. In the part of “Soil physicochemical properties”. Please add the number of replicates for the experiment.

4. Line 36: P. euphraria ? Moreover, the abbreviate of Populus euphraria in the manuscript should be unified.

5. Line 36 have-had; is-was. In the manuscript, there were a lot of such problems in the article. Please amend.
Experimental design: no comment
Validity of the findings: no comment'
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: HIGH-THROUGHPUT SEQUENCING REVEALS RHIZOSPHERE FUNGAL COMMUNITY COMPOSITION AND DIVERSITY AT DIFFERENT GROWTH STAGES OF POPULUS EUPHRATICA IN THE LOWER REACHES OF THE TARIM RIVER
Review round: 2
Reviewer: 1

Output:
Basic reporting: Overall, this version is better than before, but there are still some parts that need to be modified.
1.	Abstract: Methods, is more appropriate than Methodology.
2.	Results: There may be misunderstandings. My comment was “The Result part should NOT take the statistical method as the sub title”,the authors could read more papers and learn how to write a scientific paper, including the title of the results part.
Experimental design: no comment
Validity of the findings: no comment
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: HIGH-THROUGHPUT SEQUENCING REVEALS RHIZOSPHERE FUNGAL COMMUNITY COMPOSITION AND DIVERSITY AT DIFFERENT GROWTH STAGES OF POPULUS EUPHRATICA IN THE LOWER REACHES OF THE TARIM RIVER
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript is well written.  Besides the data support the conclusion and the research results have certain reference value. References are properly cited. The structure of the article and the pictures are suitable.
Experimental design: The experiment is designed rigorously. There are very interesting data in this manuscript.
Validity of the findings: The data support the conclusion and the research results have certain reference value.
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: GRAFTING-ENHANCED TOLERANCE OF CUCUMBER TO TOXIC STRESS IS ASSOCIATED WITH REGULATION OF PHENOLIC AND OTHER AROMATIC ACIDS METABOLISM
Review round: 1
Reviewer: 1

Output:
Basic reporting: In the manuscript entitled "Grafting enhances tolerance of cucumber to toxic stress by regulating phenolic acid metabolism" its authors focused on elucidating the phenolic acid  metabolism, using the method of qRT-PCR and HPLC,  using  Cucumis sativus cv. ‘Xinchun No.4’  which was treated with 0.5 mM of cinnamic acid (CA) in the hydroponic culture. The manuscript submitted to Peer J fits to the scope of journal, but need to be checked by a proficient English speaker because there are sentences that need  to be reworded, especially in Abstract, Discussion and Conclusions. Figures are relevant to the contents of the manuscript. Figure 1 needs improvement. here, the ratio of fresh and dry matter should be given. The manuscript include sufficient Introduction and relevant literature.
Experimental design: The experimental design is adequate to this type of  work. The phenolic acid metabolism of non-grafted (NG) cucumber seedlings and those grafted (RG) onto Cucurbia ficifolia presented different response to added cinnamic acid. Authors have ascertained  that exogenous supplementation of CA induced up-regulation of PAL, 4CL and COMT, and accumulation of cinnamic acid, benzonic acid and p-Hydroxybenzoic acid in RG cucumber plants. In the case of NG the  four key enzymes were all down-regulated , p-coumaric acid and sinapic acid content were decreased, but still  the level of ferulic acid was relatively high. T
Validity of the findings: The authors have suggested that either an unknown pathway could produce the ferulic acid or transformation of ferulic acid were inhibited. Moreover, the type and amount of phenolic acid in root exudates was improved by exogenous CA especially for RG cucumber. Their results point that the possible reason for possible resistance to CA of RG cucumber could be  rapid production of phenolic acid or its dynamic release from roots system to the rhizosphere.
Additional comments: Some detailed comments:
Lines 25-26: ..."we focused on the phenolic acid metabolism using the method of qRT-PCR and HPLC in hydroponic culture"... REALLY! HPLC in hydroponic culture ?
line 178: ..." 16-20min"... INSERT SPACE
line 263:  ... up- regulation" REMOVE SPACE
Dissussion: Not all results have been discussed!
line 269: ..."Phenolic acids are a common group of phenylpropanoids, consist of aromatic acid compounds"... THAT CONSIST
line 271-275: The begging of the sentence should be: They are  used distributed in human diet....
...and have been verified the activities of multiple nutrition and pharmacology, particularly 273 high anti-oxidant capacity - this is hard to understand what do you mean - please, reword this sentence!
Next sentence should probably be:: Phenolic acids exhibit diverse biological functions, including plant-microbe symbiosis, plant allelophatic activity, resistance to pathogen attack and abiotic stress, and allow cross-linking with lignin to act as components of cell wall.
And so on and the like
1ine: 290: were demonstrated
line 315: plant resistance/tolerance
line 219: ..."Similar results also were reported by Simek et al. (Simek et al., 2016), phenolic compounds rose under cadmium stress in cucumber plants"....  HAD ROSEN
line 333: ..." We deduce that it is another reason for grafting improve the resistance"... - Please write the sentence correctly


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: GRAFTING-ENHANCED TOLERANCE OF CUCUMBER TO TOXIC STRESS IS ASSOCIATED WITH REGULATION OF PHENOLIC AND OTHER AROMATIC ACIDS METABOLISM
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors investigate the effect of grafting on tolerance to toxic stress (autotoxins) in cucumber monoculture through the regulation of phenolic acid metabolism.This study builds on a previous study, including transcriptomics data, which sheds light on this graft-induced tolerance. The writing is clear, unambiguous and technically correct. The structure of the article has an acceptable format for PeerJ.

The introduction has sufficient and up-to-date background information. Only one thing needs to be clarified: "We found more phenolic acid..." (lines 91-93). I think this sentence does not belong in the introduction section.

The figures and tables chosen by the authors are adequate to show the results of the research. The graphs are of sufficient resolution (although the font size of the axis titles should be increased) and are correctly described and labelled. However, in all figure captions the authors did not indicate what each letter is (A, B, C, D). Although I can understand the figures with the axis titles, this should be corrected by including the meaning of the letters in the figure captions.
Experimental design: Manuscript clearly shows the research question in lines 85-87. Methods are well described. Experimental design is correct. I have some recommendations about Material and Methods section:

o I recommend the use of PPFD units (μmol/m2/s) and not lux (illuminance) units (line 98).
o When is the "graft union completely healed" (line 101)? How many days are necessary for this after grafting?
o Are the "shoot and root parts" (line 112) equivalent to the scion and rootstock parts in the case of grafted plants?
o Which part of the plant (first true leaf, second true leaf, whole shoot...) was selected for the enzyme activity assays?
Validity of the findings: The results are well described and correspond to the figures. The authors should correct the CA concentration (0.25 should be 0.50 mM) in lines 195 and 208. The discussion is of sufficient quality. The conclusions are adequate, connected to the original research question and supported by the results. However, I suggest that the authors send figure 7 to the discussion section.
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: GRAFTING-ENHANCED TOLERANCE OF CUCUMBER TO TOXIC STRESS IS ASSOCIATED WITH REGULATION OF PHENOLIC AND OTHER AROMATIC ACIDS METABOLISM
Review round: 1
Reviewer: 3

Output:
Basic reporting: The work of Xiao et al. deals with the potentially interesting topic of cucumber cultivation.
However, the work has a series of flawns that depreciate the value and lower the quality of the manuscript.
1. The language quality is low. The text lacks clarity; it's challenging to follow the author's way of thinking.
2. The references need to be tidied out. There are editorial problems. The background of the problem is not sufficiently presented.
3. There is a lack of sufficient justification of the novelty of work and importance of problem presented (ma by caused by the low quality of language)
4. There is an essential issue of phenolic acids. Please be informed that benzoic acid and cinnamic acids are not phenolic acids. Compound to be classified as the phenolic compound has to have.
Experimental design: There are severe allegations of to method used.
1. Plant vegetation conditions are insufficiently described. Some remarks:
 - lighting environment - what was a light source, light intensity reported as illuminance is not appropriate
Please be warned that lighting intensity has a significant impact on phenolics metabolism.
2. How to measure enzyme activity by sandwich ELISA???
3. Extraction of phenolic and benzoic acids from exudates has no sense. Liquid-liquid extraction (LLE) from acidic water solution followed by LLE from basic water solution is odd. What was the purpose?
Why is the extraction from plant material done another way?
Validity of the findings: Based on previous remarks findings cannot be validated.
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: GRAFTING-ENHANCED TOLERANCE OF CUCUMBER TO TOXIC STRESS IS ASSOCIATED WITH REGULATION OF PHENOLIC AND OTHER AROMATIC ACIDS METABOLISM
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have replied satisfactorily to my comments.
Experimental design: The authors have replied satisfactorily to my comments.
Validity of the findings: The authors have replied satisfactorily to my comments.
Additional comments: None


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: GRAFTING-ENHANCED TOLERANCE OF CUCUMBER TO TOXIC STRESS IS ASSOCIATED WITH REGULATION OF PHENOLIC AND OTHER AROMATIC ACIDS METABOLISM
Review round: 2
Reviewer: 2

Output:
Basic reporting: The work was substantially improved.
 The only obstacle to publication is for me that authors consequently use the terms "phenolic acids" or "phenolic compounds" regarding cinnamic acid and benzoic acid. Those are not phenolics, as they do not directly have a hydroxyl group attached to the aromatic ring. Generally, all compounds authors denote as phenolics are aromatic acids.
The use of "phenolics" regarding virtually any aromatic secondary metabolite seems to be a common mistake in the literature and promotes incorrect biochemical/physiological reasoning.
My compromise proposal is the term: "phenolic and other aromatic acids" or "phenolic and other related aromatic acids".
Experimental design: The work was substantially improved.
Validity of the findings: The work was substantially improved.
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: FERULIC ACID ATTENUATES HIGH GLUCOSE-INDUCED APOPTOSIS IN RETINAL PIGMENT EPITHELIUM CELLS AND PROTECTS RETINA IN DB/DB MICE
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1.	The English language should be improved to ensure that an international audience can clearly understand your text. It is advised that the authors seek help from a professional scientific writer to meet scientific and English language standards.
 Some examples where the language could be improved include:
Line 34: “using CCK-8 and Flow Cytometry.” Apoptosis is not detected by flow cytometry but by Annexin-5 staining that is visualized using flow cytometry.
Line 39: “blood samples were collected for detection” should be replaced by blood samples were collected to measured… levels
Line 48: “FA alleviatd HG-induced cell apoptosis in RPE cells,” should be FA alleviated HG-induced apoptosis in RPE cells
Line 56: “It is estimated that one third of patients with diabetes will eventually occur DR.” should be replaced by “…will eventually develop DR.”
Line 63: “is an integral component of the blood-retina barrier” should be “is an integral component of the outer blood-retina barrier”
Line 281: “Mechanically” should be replaced by “Mechanistically”
Line 131: “cells were seeded in a dish for cell climbing” does the author want to say that cells grow on glass coverslips?
Line 163: “Alcohol” should be replaced by “ethanol”
Line 172: “Immunofluorescence” should be replaced by “immunocytochemistry”
Line 182 “in order to nucleate” should be “in order to label the nuclei of the cells”

2.	Although the authors clearly demonstrated the effect of Ferulic acid on RPE and retinal cells. There are few weaknesses related to data gathering, unsupported conclusion and figure quality that need to be improved before acceptance. It is unclear how protein expression was quantified. Moreover, the retinal layer thickness should be quantified by measuring the length of the different layers at different regions of the retina and presented using a spidergram. Adding to the manuscript the quantification of tunnel or cleaved caspase-3 positive cells in retinal sections would substantially support the authors´ claims.

3. Figures
-	Figure 1 on panel B “Apoptostic rate” should be “Apoptotic rate”; on panel C “viability(%)” should be “viability (%)”
-	Figure 2 panels A, B and C “expersion” should be replaced by “expression”; panels E, F and G “experssion” should be “expression”. It is not clear how protein expression was measured and what is depicted on the graphics.
-	Figure 3. It is not clear how protein expression was measured and what is the unit of the values.
-	Figure 4 and 5. Should be combined to see side-by-side the effect of the treatments on the wildtype and db/db mice. All retinal sections should be presented with the same orientation, e.g., photoreceptors in the top part. Labels should be added to easily identify the different retinal layers, e.g., ONL, INL, GCL. Arrows or arrowheads should be used to pinpoint the alteration mentioned in the text. Scale bars should be made visible.
-	Figure 6. Similar as before, it should be clear how the protein quantification was performed, and the units of the values should be added. Retinas should be presented in the same orientation in a straight position and retinal layers should be properly labelled. Scale bars should be made visible.
-	Figure 7. Similar as indicated to figure 6.

4. Raw data
-	Images of the full western blot membrane should be provided.
Experimental design: Materials and methods
-	Line 97: The concentration of FA used on the in vitro studies should be indicated in mol/L?
-	There is no information about the cDNA synthesis and the PCR reaction condition. This information should be added to the manuscript.
-	The author should indicate how the protein extraction was performed and how protein expression was measured.
-	Information about the DAB staining should be added.
-	It is extremely important to make clear if animals were injected daily with FA for 2 months. This is not very clear on the material and methods section.
-	Did the authors use dissected retinas or all eyes for the analysis? They should indicate how tissues were processed.
-	Line 166: Please indicate the type of serum and the concentration used.
-	Please indicate the mounting medium used.
-	Line 187: which enzymatic methods were used?
Validity of the findings: Results:
-	Line 230-234: To support the authors´ statement retinal thickness should be measured. Moreover, cell edema and edema of ganglion cell layer are not obvious and therefore the small alterations on the morphology should be indicated by arrows on the figure.
-	Line 253–259: the author measured a range of different biochemistry parameters, however maybe the most important one glucose was not measured.
Additional comments: The manuscript by Zhu et al demonstrated that Ferulic acid protects RPE cells from high-glucose induced apoptosis. Moreover, experiments done using db/db mice, a widely used model of type 2 diabetes mellitus, suggested that Ferulic acid protects retinal cells from high-glucose induced apoptosis. The authors should better connect the two parts of the manuscript. The title just reflects the in vitro part of the work, once on the mouse studies changes in the RPE were not analysed.


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: FERULIC ACID ATTENUATES HIGH GLUCOSE-INDUCED APOPTOSIS IN RETINAL PIGMENT EPITHELIUM CELLS AND PROTECTS RETINA IN DB/DB MICE
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript entitled “Ferulic acid protects retinal pigment epithelium cells from high glucose-induced apoptosis” by Zhu et al. looks to expand upon the known role of Ferulic acid (FA) in ameliorating apoptosis in response to high glucose (HG) in an ARPE-19 model and a db/db mouse model. The authors present some interesting data on a relevant topic and provide a balanced discussion. The manuscript, in some areas, is underdeveloped and the flow between the use of the ARPE-19 line and the db/db mouse model needs to be clearer. In particular, as the authors chose not to analyses the RPE from this model but only the retina.
The authors should refrain from make statements such as “as expected” and “obvious”. If a statement such as “It has been widely confirmed” is used (line 198), please include some citations to this information. A number of small changes to improve flow of text are required. As an example, line 203 “Above findings indicated” should be changed to “The above findings indicate”. Line 204 “the apoptosis of RPE cells by exposure to HG” should be changed to “the apoptosis of RPE cells caused by the exposure to HG”. Line 207 “As shown in Flow Cytometry” should be “As shown in the Flow Cytometry”. Line 223 “changes in retina” to “changes in the retina”. Line 278 “Except ARPE-19: to “Besides ARPE-19”. Line 312 “In this study, both in vivo and in vitro models, our findings revealed” to “In this study, the data from both our in vivo and in vitro models revealed that:.
Discuss in the introduction the role of RPE in diabetic retinopathy (DB) in more detail. As an RPE in vitro system is implemented, clarification of its relevance in DB and thus to the subsequent experiments carried out in the db/db mouse model are required. Also discuss further the roles of FA in attenuating induced damage models of RPE and Retina. As an example, recently FA was found to attenuate induced damage in an ARPE-19 and mouse retina (Kohno et al. 2020, Scientific reports). There are also a number of papers which discuss the role of FA in treating HG, the introduction should include more information on this already well-established role (Sompong et al. 2015, PloS ONE). Lastly, He-Ying-Qing-Re Formula contains FA and has been found to ameliorate the phenotype in a diabetic model, reducing apoptosis (Zhang  et al. 2018, Journal of Ethnopharmacology). Additions do not have to be extensive and if deemed more appropriate can be added to the discussion.
Experimental design: The manuscript by Zhu et al. brings together a spontaneous diabetic mouse model of type 2 diabetes and a high glucose induce RPE cell apoptosis model. However, the authors do not adequately link these two models together.
-The title only focus on one aspect of the author’s paper (RPE) and does not tie in FA treatment in the db/db mouse model.
-Furthermore, only retinal tissue from the db/db mice are analyzed. In addition, collection and evaluation of the RPE would have helped link the data from the two models.
- Evaluation of the 16-week-old treated db/db retina needs to be done more thoroughly, compared to the controls, to assess the validity of the authors conclusion “FA attenuated pathological changes in the retina of diabetic mice”. I suggest the authors – Quantify total retinal thickness, ONL thickness, and INL thickness. Furthermore, the authors may wish to quantify the number of cells in the GCL, it should be reduced in the db/db mouse model (Bogdanov et al. 2014, PLoS One).
-The authors should strengthen their findings on the role FA pays in ameliorating apoptosis in the diabetic mouse retina by performing and quantifying Cleaved-caspase 3 staining and/or the Tunnel assay to show that in the FA treated retinal sections that apoptosis is reduced. A significant increase in Tunnel positive apoptotic cells is found in 16 week db/db mice (Bogdanov et al. 2014, PLoS One). Furthermore, the Bogdanov et al. paper highlights the role of lowering glucose in ameliorating the phenotype in db/db mice, finding lower apoptosis in the GCL of db/db mouse retina when mice were fed a restrictive diet. The authors may wish to further discuss this relevant finding in the context of their own work.
-The authors could strengthen their findings by carrying out a western blot for P53, BAX and BCL2 in the control, Model and FA mouse groups. To verify optical density quantifications.
-Please state in the results section and the figure legends the exact age at which mice were analyzed.  16 weeks db/db and 14weeks db/m+?
The authors provide no functional data e.g. ERG for A, B and C wave to show therapeutic efficacy. This would have greatly strengthened the authors findings.
Validity of the findings: Some statements made by the authors are not backed up by experimental findings. This is mainly in reference to the db/db mouse model and amelioration of its phenotype by FA.
-It is hard to clearly read the scale bar length from the figures, please state in the legends.
Figure 1. Please indicate that figure 1A is an example from one experiment, while Figure 1B is the average of all 3 experiments.
Figure 3: In the legend and results please make it clear that this is optical density quantifications. Please clearly state the method used to analyze the data. How were regions of interested created (ROIs), was background signal corrected for, were images taken at the same intensity, etc
-Figures 4, 5, 6, 7. The authors describe that the retinal thickness of each layer was slightly thinner and that the ONL thickness was significantly decreased. This general description does conform to the expected phenotype observed in the db/db mouse retina. However, the authors provide no quantifications for any of their statements. Furthermore, all their images of the db/db untreated mouse retina appear thicker than the control or FA groups, assuming images were taken at the same magnification and from similar areas of the retina. This may be due edema but then the number of nuclei in a row could be counted as an additional approach.
-Figure 5: Could the authors highlight the areas of edema in B. Please refrain from using statements such as “obvious”. Please quantity the number of cell nuclei in the ONL, do not just state that the number of nuclei are reduced without providing quantifications. “FA restored it to normal levels” What is “it”. Further in Line 292 the authors state that “FA significantly ameliorated retina edema” not quantification of this was done.
-Figure 6, 7: Could the authors clarify in text and in the legend that the quantification of protein expression levels is from analysis of immunohistochemistry (6) and fluorescence (7).  E.g. Please state optical density of images was quantified. Please include a thorough description of the methodology used to analyze this data. How were regions of interested created (ROIs), was background signal corrected for, were images taken at the same intensity, etc
-Figure 8B: The model and FA group have highly over lapping error bars. I performed an unpaired t-test using the author’s raw data, and found no significant difference.  Please correct.
Furthermore, could the authors double-check the statistics from other analysis.
-For clarity please include the Cat numbers of the Biochemical assays used from Stanbio.
-Please state exact information about equipment used. E.g. what type of fluorescence microscope.
-How were retina sections prepared. E.g. what percentage of PFA , were the cryoprotected with sucrose, were they stored in OCT compound.
-Consider using the wording attenuated or ameliorated instead of reversed throughout the manuscript.
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: FERULIC ACID ATTENUATES HIGH GLUCOSE-INDUCED APOPTOSIS IN RETINAL PIGMENT EPITHELIUM CELLS AND PROTECTS RETINA IN DB/DB MICE
Review round: 1
Reviewer: 3

Output:
Basic reporting: Overall Zhu and colleagues present a well-written manuscript in which authors introduce and discuss their work based on a list of recent literature references and show the beneficial effects of Ferulic acid (FA) in reverting diabetes-mediated retina effects. Concerning the figures, please consider to perform the following alterations:

1 - There are some grammar typos that must be corrected. Specifically, the Y axis of Figure 1B have "Apoptostic rate" instead of "Apoptosis rate". Also, on Y axis of Figure 2, "expersion" must be corrected to "expression" and the word "The" must be removed from all the Y axis of this Figure.

2- Figure 4 must be improved with a legend indicating the name of the different tissues that are observed on the figure to better understand the pathological alterations detected.

3 - The same for Figure 5.

4 - On Y axis of Figure 6A-C, remove the word "The".

5 - Regarding the figures with results obtained from animals, please remove the word "Model" when referring to the diabetic animals and simply use db/db or diabetes.

6 - In all figures legends authors must include the "n" in which were performed the experiments.

7 - Authors should improve the last paragraph of introduction. Presently is only focused in the in vitro study and do not mention the work that will be performed in animals.

8 - Also, authors should explain why the work was perform in this specific animal model of diabetes.

9 - Authors included a supplementary pdf file with representative western blots bands but do not explain what that means.
Experimental design: In the present manuscript, authors were solely focused on investigating the possible beneficial effects of FA on the apoptosis process occurring on a scenario of diabetic retinopathy. For that the study was conducted in both in vitro and in vivo models of diabetes. The experimental design was well thought and authors used appropriate methodologies to validate their hypothesis. Still, authors should consider the following comments:

1 - On materials and methods section, lines 97-98, authors say "Moreover, a negative control group was set up to evaluate the effect of DMSO". However, looking at figures, DMSO possible effects were only assessed when evaluating the mRNA and proteins expression levels of apoptosis-related proteins. It would be important to also evaluate DMSO effects on cells viability. Please consider to perform this additional experiment.

2 - Did authors performed any osmotic control to the glucose concentration used in the study? If not, please consider to perform this additional control on cells viability.

3 - Regarding the work with animals, authors must provide animals full characterization with body weight and glycemia values.

4 - In the abstract, authors say "...an HG+FA group (30mmol/L glucose and 2 mg/ml FA)", and in the material and methods section, line 100, we can read "...DMEM+30mmol/L HG+0.05g/kg FA." It seems that there is a mistake. Please verify this.

5- Please explain on the materials and methods section why the FA concentration used in the study was the same for the cells and for the animals and include a reference to justify it.

6 - Did authors used DMSO to dissolve the FA that was administered to the animals? Please include this information on materials and methods section.
Validity of the findings: Overall, authors validated their hypothesis by demonstrating the positive effects of FA in reverting the apoptosis process in both in vitro an in vivo models of diabetes. Authors do not propose any mechanisms.
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: FERULIC ACID ATTENUATES HIGH GLUCOSE-INDUCED APOPTOSIS IN RETINAL PIGMENT EPITHELIUM CELLS AND PROTECTS RETINA IN DB/DB MICE
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors were able to improve the manuscript, although the English levels can still be considerably improved. Although the authors did perform most of the requested changes, the manuscript still presents several issues:

Line 76/77: "Thus, inhibition of apoptosis in RPE cells has become an approach in the treatment of DR". Is inhibition of apoptosis in RPE cells an approved treatment for DR? If not sentence should be correct otherwise it might mislead the reader.

Related to the figures, as requested before the authors should increase the size of the lettering in the figures mainly of the scale bars. The spidergram presented now on figure 4G should be replaced by one similar to presented in Figure 3 of the following manuscript:  DOI: 10.1371/journal.pone.0154779 "Limited ATF4 Expression in Degenerating Retinas with Ongoing ER Stress Promotes Photoreceptor Survival in a Mouse Model of Autosomal Dominant Retinitis Pigmentosa." It should be clear that the thickness of the different retinal layers (ONL, INL, etc) are measured at different distances from the optic nerve.

It is not clear how many retinas/samples were used for the different analysis, eg, how many samples were on figure 5D. Accordingly to the raw data only 3. Moreover, the raw data is already pre-analysed, presenting the mean and the SD, and not the individual values.
Experimental design: The research question is clear and the material and methods were clearly improved. However, still not always clear how many samples/animals were used in each analysis.
Validity of the findings: no comment
Additional comments: The authors were able to improve the quality of the present manuscript however a few points still need to be clarified before publication.


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: FERULIC ACID ATTENUATES HIGH GLUCOSE-INDUCED APOPTOSIS IN RETINAL PIGMENT EPITHELIUM CELLS AND PROTECTS RETINA IN DB/DB MICE
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have, in part, addressed some of the points made in the first round of revision. In particular, the authors have improved the use of citations to back up some of the previous statements made in the first version of the manuscript, in addition to improving their introduction.
However, the authors still have not sufficiently addressed several points from the first round of review, including the following points:
•	Significantly the authors have not clearly shown apoptosis in retinal section and its amelioration upon FA treatment. They state they show this in Figure 2D in their rebuttal, but this addresses Cleaved –Caspase staining in ARPE-19 “Thanks. We have performed and quantified Cleaved-caspase 3 staining to show that in the FA treated retinal sections that apoptosis is reduced. The results have shown in Figure 2D.” The comments from the initial review report still stand “The authors should strengthen their findings on the role FA pays in ameliorating apoptosis in the diabetic mouse retina by performing and quantifying Cleaved-caspase 3 staining and/or the Tunnel assay to show that in the FA treated retinal sections that apoptosis is reduced. A significant increase in Tunnel positive apoptotic cells is found in 16 week db/db mice (Bogdanov et al. 2014, PLoS One).”

•	The authors state that they have “performed immunohistochemistry and immunofluorescence to examine and quantify P53, BAX and BCL2 proteins in the control, Model and FA mouse groups. We think that the results are reliable.” While the staining may be reliable, one way to significantly strengthen the data is to show this change in levels through western blotting for P53, BAX and BCL2.

•	The addition of Figure 4G is a step in the correct direction for showing the required thickness measurements, which would adequately clarify the differences between the Control, db/db and FA groups. However, these would be best shown as individual layer spider grams (as an example see Alves et al. 2019, IJMS Figure 5G-I). In its current format, Figure 4G is hard to read and also does not show the “significant” differences between the Control, db/db and FA groups the authors discuss between lines 260-274. The authors do not provide any statistics for these points. The comments from the initial review report still stand “Evaluation of the 16-week-old treated db/db retina needs to be done more thoroughly, compared to the controls, to assess the validity of the author's conclusion “FA attenuated pathological changes in the retina of diabetic mice”. I suggest the authors – Quantify total retinal thickness, ONL thickness, and INL thickness. Furthermore, the authors may wish to quantify the number of cells in the GCL, it should be reduced in the db/db mouse model (Bogdanov et al. 2014, PLoS One).”

•	Line 235: The above findings indicate that FA could alleviate the apoptosis of RPE cells by exposure to HG. Change to: The above findings indicate that FA could alleviate the apoptosis of RPE cells “caused” by the exposure to HG. – This was also commented on in the first round of review. RPE cells should be changed to ARPE-19 cells.

•	Figure 3J, please correct the overlap of the significant bars.

•	Discussion Line 336-341. Here the authors talk about the treatment of the db/db mice and the last sentence states “Therefore, we have reason to state that FA could suppress diabetic RPE cell apoptosis”. However, the authors do not analyze the RPE in the db/db mice and therefore can not make this statement. In turn, Line 357-359 also can not be said. As previously stated if you wish to make such statements, the authors should also analyze the RPE.
Experimental design: na
Validity of the findings: na
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: FERULIC ACID ATTENUATES HIGH GLUCOSE-INDUCED APOPTOSIS IN RETINAL PIGMENT EPITHELIUM CELLS AND PROTECTS RETINA IN DB/DB MICE
Review round: 2
Reviewer: 3

Output:
Basic reporting: No comment.
Experimental design: Authors revised the manuscript accordingly to the suggested comments but, it remains unclear the number of animals that were used to perform each experiment. The sentence "Each experiment was repeated three times" is not very clear and if it means that were only used three animals of each group, this is quite insufficient when it refers to animal studies. Please check this.
Validity of the findings: No comment.
Additional comments: Overall, authors followed the reviewers comments and suggestions and present an improved version of 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: FERULIC ACID ATTENUATES HIGH GLUCOSE-INDUCED APOPTOSIS IN RETINAL PIGMENT EPITHELIUM CELLS AND PROTECTS RETINA IN DB/DB MICE
Review round: 3
Reviewer: 1

Output:
Basic reporting: The authors should seek a professional scientific writer to help then with the editing of the manuscript.

The raw data presented are not truly raw but mean values. And the spidergram is not correctly presented, there is no mention in which area the measurements were performed. As indicated before the measurements should be performed in different areas/distances of the optic nerve of the retina.

The author did not provide a satisfactory reply to the previous comments.
The manuscript presents the (same) weakness as the lastest version.
Experimental design: Figure 6. No BCL2 is visible on B much likely because the retinal section misses the outer segments, the image should be replaced.

Clearly staining for apoptotic markers could be improved. They present a high background. Using fluorescence labelling coupled with confocal imaging will help the author to better present their data, and at the same time count the number of apoptotic cells (e.g. tunnel positive or cCasp3 positive)
Validity of the findings: It is not clear how the authors only used 3 retinas per analysis when then have a total of 15.
The raw data is not present, only the means and SD are presented.
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: FERULIC ACID ATTENUATES HIGH GLUCOSE-INDUCED APOPTOSIS IN RETINAL PIGMENT EPITHELIUM CELLS AND PROTECTS RETINA IN DB/DB MICE
Review round: 3
Reviewer: 2

Output:
Basic reporting: The authors made some effort to revise the comments from the last round of review. However, additional issues still remain:
•	I thank the authours for trying to carry out the Cleaved-caspase staining (Figure XQ-S). However, I am not fully convinced about the quality of this staining and would suggest the authours to carry out immunoflurecens. I would expect to see minimal Cleaved caspase staining in control compared to db/db mice, please see Figure 6 of the following manuscript: https://journals.plos.org/plosone/article/figure?id=10.1371/journal.pone.0097302.g006 Please also quantify the number of positive nuclei in the GCL.

•	The Authors still did not addressed the comments from the last review rounds and do not show any negative data that the tried. Previous comment “The authors state that they have “performed immunohistochemistry and immunofluorescence to examine and quantify P53, BAX and BCL2 proteins in the control, Model and FA mouse groups. We think that the results are reliable.” While the staining may be reliable, one way to significantly strengthen the data is to show this change in levels through western blotting for P53, BAX and BCL2.” The authours stated in their rebuttal to this comment “Reply: Thanks for the wonderful comment. Because the eyeballs of mice are so small that enough proteins cannot be extracted, it is difficult to detect the expression of P53, BAX and BCL2 proteins by western blot. Therefore, we performed immunohistochemistry and immunofluorescence. Our immunohistochemistry and immunofluorescence confirmed that FA could ameliorate the apoptosis of diabetic retinal tissues.” There reply is unfounded it is possible to westernblot for all of the indicated proteins in mouse retina. As an example see: https://www.jneurosci.org/content/33/5/2205#sec-2

•	Could the Authors state how the statistics were carried out for Figure 4G, Multiple T-Test? For clarity, the provide statistical comparisions mentioned in the legend are between the Control vs Model group and the Model vs FA group?

•	From my previous round of review “Discussion Line 336-341. Here the authors talk about the treatment of the db/db mice and the last sentence states “Therefore, we have reason to state that FA could suppress diabetic RPE cell apoptosis”. However, the authors do not analyze the RPE in the db/db mice and therefore can not make this statement. In turn, Line 357-359 also can not be said. As previously stated if you wish to make such statements, the authors should also analyze the RPE.” Thank you for removing the sentence started “Therefore, we have reason to state that FA could suppress diabetic RPE cell apoptosis”. However, the authours did not address the issue with the subsequent lines now 356-58. You did not look at the RPE from the in vivo model and there for cannont make this statement.
Experimental design: N/A
Validity of the findings: N/A
Additional comments: N/A


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: FERULIC ACID ATTENUATES HIGH GLUCOSE-INDUCED APOPTOSIS IN RETINAL PIGMENT EPITHELIUM CELLS AND PROTECTS RETINA IN DB/DB MICE
Review round: 3
Reviewer: 3

Output:
Basic reporting: The conclusion subsection of the abstract should be written using the present tense, i.e. "Our findings suggest that FA alleviates HG-induced apoptosis in RPE cells and protects
retina in db/db mice, which can be associated with P53 and BAX inactivation and Bcl2 activation."
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The paper was greatly improved after considering all the reviewers comments and suggestions.


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: FERULIC ACID ATTENUATES HIGH GLUCOSE-INDUCED APOPTOSIS IN RETINAL PIGMENT EPITHELIUM CELLS AND PROTECTS RETINA IN DB/DB MICE
Review round: 4
Reviewer: 1

Output:
Basic reporting: The manuscript and the writing were marginally improved. Although the authors replied to all my previous comments, they still do not provide robust and trusty data.
Experimental design: Authors should make very clear what is the "n" in each experiment. How many independent experiments and how many replicates were used. For example, the spidergram, how many mice were used and how many sections were counted.
It is quite a surprise that the authors have (only) 3 values for all the experiments/groups.

Please rotate the retinal pictures to have the same orientation and label the different layers.
Validity of the findings: Line 277/581 and Fig 4H, What are the "Positive cells"? These cells are positive for?

Lines 305: Remove "therapeutic effect". It is better to describe and discuss the data rather than have general statements.

Line 581-589: What was the number of mice/sections used?

Figure 5G, the middle picture seems to have excessive staining of cCasp3, the entire retina seems positive.
Additional comments: 'no comment


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: THE APPLICATION OF PLANT GROWTH-PROMOTING RHIZOBACTERIA IN SOLANUM LYCOPERSICUM PRODUCTION IN THE AGRICULTURAL SYSTEM: A REVIEW
Review round: 1
Reviewer: 1

Output:
Basic reporting: The title of the reviewed article suggests that it should be focused on Solanum lycopersicum production, problems, and the role of PGPR in addressing the issue and improving fruit yield. The majority of the study is devoted to generic rhizosphere and rhizobacteria information. All sections describe what PGPR can do, as well as their essential features and functions, however there is no correlation between tomato growth and mechanism of action.

The introduction is irrelevant, sentences are abrupt lack of consistency and not encouraging for the reader

The provided information is too general and basic , the author used obvious facts of filed as information e.g line 50-69, 149-158, 180-184, 239-242 and many others, Similarly, the subsections lack the focus of the main review aim, as it is stated that phytohomrnones play a function, but that it requires a certain mode of action and hormone contribution. Similarly, there is no information on how PGPR promotes plant growth and resistance through altering plant hormones, plant stimulants, or nutrient availability.

The ambiguous use of English language through out the document, sentences are poor in grammar and connection. The inappropriate use of words and terms in the manuscript as, 290“implement nutrient availability”, 294: plant to ingest nutrient, 311: tomato absorb phosphate quickly due to because of the high absorption surface area gradient, Line 282, “Biofertilizers are simply microbes that help the plant nutrient acquisition process through increasing surface areas like plant roots” not so correct,,, microbes are not synonym of biofertilizer these are min component of bio fertilizer and microbes do not increase surface area of plant root

It lacks the previous findings related to tomato plant and growth most of the cited literature is only about pgpr

The presented figures are too generalized to be presented in updated scientific review.
Experimental design: Article fails to meet the prospect aim of review.
The method opted for review selection should target to tomato plant association to PGPR or vice versa to improve plant growth resistance fruit yield and viablity rather than just PGPR qualities that are well-known presently.
Validity of the findings: As the review is too generalised lack the novelty, it brings no additional valid and comprehensive information to the reader.

Review must have latest information about specified topic, and have collection of valid findings in focused issue
Additional comments: The manuscript requires careful consideration in terms of English proficiency, connection, consistency and focus in content. It requires to remove so many redundant details and add precise and rational work from the 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: THE APPLICATION OF PLANT GROWTH-PROMOTING RHIZOBACTERIA IN SOLANUM LYCOPERSICUM PRODUCTION IN THE AGRICULTURAL SYSTEM: A REVIEW
Review round: 1
Reviewer: 2

Output:
Basic reporting: Is the review of broad and cross-disciplinary interest and within the scope of the journal?
Yes
Has the field been reviewed recently? If so, is there a good reason for this review (different point of view, accessible to a different audience, etc.)?
No comments
Does the Introduction adequately introduce the subject and make it clear who the audience is/what the motivation is?
no comments
Experimental design: Is the Survey Methodology consistent with a comprehensive, unbiased coverage of the subject? If not, what is missing?
Lack of novelty and presentation

Are sources adequately cited? Quoted or paraphrased as appropriate?
Almost yes

Is the review organized logically into coherent paragraphs/subsections?
Yes
Validity of the findings: Impact and novelty not assessed. Meaningful replication encouraged where rationale & benefit to literature is clearly stated.
No comments
Is there a well developed and supported argument that meets the goals set out in the Introduction?
no
Additional comments: Comments to Authors:
Manuscript ID: #70929
The manuscript titled “The application of plant growth-promoting rhizobacteria in Solanum lycopersicum production in the agricultural system: A review” submitted by Adedayo et al. summarizes the role PGPR application in tomato plant growth and development. They started with the introduction about the importance of soil health and quality on plant growth and development. Further they talked about pathogens such as Ralstonia solanacearum that limits the tomato yield. In next section they discuss the properties of rhizosphere. Here they talked about chemical derivatives and its negative impact on health followed by the mode of action of PGPR on tomato production. After that, availability and role of macroelements found in rhizosphere like nitrogen, calcium, magnesium, potassium, phosphorus, and sulphur has been explained. In the end they discussed about biological control technology to control bacterial and fungal infection on tomato plants.
	Overall, the concept of the manuscript is quite interesting but it is poorly presented. Also, it lacks novelty and there are several flaws in the manuscript that need to be resolved.

	In abstract section: lines 35-36; rhizobacteria that promote the growth and development of tomato plants are referred to as plant growth-promoting bacteria (PGPR). This sentence may confuse to the readers. Please remove tomato from this sentence.
	Lines 42-43: What about other essential nutrients?
	Please check and re-write the sentence of lines 43, 59-61, 73, 92-93, 120-121, 230.
	Lines 59-61: In addition, the following soil factors affect plant growth: water, phytopathogens and parasites, weed seed pools, water, nutrients and dissolved oxygen concentration (Patil & Fauquet 2021). Sentence is not clear please re-write it.
	Line 60: Repetition of “water”. Please remove it.
	Line 69: Figure 1 was cited in introduction section but its legend was presented in another section. Authors should move figure to introduction section after the paragraph where it was first cited.
	Figure 1 is very general representation. There is nothing new for the reader. Please provide the mechanism of action of plant growth promotion in tomato. Authors should show the production of hormones, nutrients by PGPR and their utilization by plants in schematic way. You may also present the name of stress responsive genes in tomato which are induced by PGPR. In this figure 1, abiotic factor: climate, sun, o2 level etc have been mentioned. There are other important abiotic factors such drought, salinity, high and low temperature etc that should be mentioned. Overall, figure should be improved. Also, authors need to properly elaborate Figure 1 in the legend section.
	In the abbreviation section, the authors need to capitalize the first letter and delete the other information. Lines 135-136 are not abbreviations. Also be consistent with the capitalization of the abbreviations used.
	Line 174: Authors wrote “some fungi, for example” but did not mention the name of fungi. What are those fungi?
	Line 187: Please replace “diffusing” with diffusion.
	Table and figures in the manuscript should be presented where authors cited them. Table 2 was cited in line 281 but presented after line 302. Same happened with table 3. Please check and fix it.
	The English language should be improved so that audience could understand the message. Please check and improve lines 104, 122-123, 149, 152-154, 164, 176-177, 192-195, 260, 264-265, 276-277, 284-285, 298, 307, 322-323,390, 425, 465-466, 473, 477,501-502.
	Line 306-308: Since this element is present primarily in insoluble and soluble organic forms, rhizobacteria in the soil help solubilize the phosphorus, making it usable by plants. If soluble is already present then what is the need of rhizobacteria? It would be great if author could explain how rhizobacteria help to solubilize the insoluble phosphate?
	Texts written in lines 462-466 is repeated in lines 489-493. Please check and correct accordingly.
	Authors need to double check all the references, for example, italicized the species (L-532, 577, 597, 732,) and remove extra brackets (L-550).
	In reference cites in lines 635-636 need to mention the location of the publisher.
	In table 2, somewhere authors have used bacterial name whereas somewhere they used only strain names. Please write bacterial names which their strain names properly.

Overall, the manuscript is poorly written and weak presentation. It may be consider after minor revision if authors would be able to justify the comments successfully and improved it.


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: THE APPLICATION OF PLANT GROWTH-PROMOTING RHIZOBACTERIA IN SOLANUM LYCOPERSICUM PRODUCTION IN THE AGRICULTURAL SYSTEM: A REVIEW
Review round: 1
Reviewer: 3

Output:
Basic reporting: The current review falls within the biological and important area of research now a days
Experimental design: Literature survey is ok
Validity of the findings: The findings from this review will be beneficial to the readers
Additional comments: The current study entitled “The application of plant growth-promoting rhizobacteria in Solanum lycopersicum production in the agricultural system: A review” is good. For a better understanding in-depth, it is a need for time to work on this topic. Furthermore, the achievement of potential benefits by using current technology is also dependent on the extensive research work for more exploration. Although the literature is well organized, yet I suggest modification due to the following deficiencies.
Major Concerns
•	Systematic abstract is missing. Introduce the need for study in 1-2 lines.
•	Please give a clear-cut point problem source as a problem statement that is tackled in the current study.
•	Give logical reason for the selection of current strategy.
•	Quantitative data is also important to support your conclusion. Would you please provide some quantitative data in terms of percentage significant increase or decrease in the abstract?
•	Please provide a conclusive conclusion with is withdrawn through research in a single line.
•	Please conclude with a statement that shows a knowledge gap covered, potential beneficiaries and specific recommendations as well.
•	Give future prospective in a single line.
•	As per standard suggestions, please avoid using title words as keywords
•	Also, provide a novelty statement at the end. What new things authors have done or correlated in this research compared to old ones?
•	Would you please give a single line about the knowledge gap which your research has covered along with the hypothesis statement?
•	If the authors are not sure, then give future recommendations for more research and investigation.
•	Add the targeted beneficiary audience who will get benefits from this research.
•	Also, give clear-cut recommendations and future prospective regarding this research.
This research shows the important scientific progress in content, some literature with related aspects should be cited for broader readership, such as

https://doi.org/10.1038/s41598-021-89103-9
https://doi.org/10.1016/j.jhazmat.2021.126493
https://doi.org/10.1016/j.envpol.2021.117854
https://doi.org/10.3390/ijerph17197251
https://doi.org/10.1038/s41598-021-01337-9


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: THE APPLICATION OF PLANT GROWTH-PROMOTING RHIZOBACTERIA IN SOLANUM LYCOPERSICUM PRODUCTION IN THE AGRICULTURAL SYSTEM: A REVIEW
Review round: 2
Reviewer: 1

Output:
Basic reporting: Is the review of broad and cross-disciplinary interest and within the scope of the journal?
Yes

Has the field been reviewed recently? If so, is there a good reason for this review (different point of view, accessible to a different audience, etc.)?
Yes

Does the Introduction adequately introduce the subject and make it clear who the audience is/what the motivation is?
Yes
Experimental design: Is the Survey Methodology consistent with a comprehensive, unbiased coverage of the subject? If not, what is missing?
Yes

Are sources adequately cited? Quoted or paraphrased as appropriate?
Yes
Article content is within the Aims and Scope of the journal.
Yes
Validity of the findings: Is there a well developed and supported argument that meets the goals set out in the Introduction?
Yes
Additional comments: The authors have addressed the comments and revised the manuscript successfully.


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: POPULATION GENETICS AND DEMOGRAPHY OF THE CORAL-KILLING CYANOBACTERIOSPONGE, TERPIOS HOSHINOTA, IN THE INDO-WEST PACIFIC
Review round: 1
Reviewer: 1

Output:
Basic reporting: Needs Genbank numbers for genetic data; figures and tables need a little improvement; a couple of citations to add, as detailed below.
Experimental design: I have some suggestions about statistics in the attached pdf, but I think they are fixable.
Validity of the findings: I commend the authors on their hard work. I'm sure that assembling this dataset was an enormous effort! Nice job! There is lots to like in this paper, and I think that it could be an important contribution.

However, to make sure the author's hard work is not wasted, this paper needs major changes to the analysis and how the results are discussed. First of all, the analysis of genetic variation needs to better take sample size into account. As shown below, doing so will change their conclusions significantly. Second, the authors need to let the data "speak". Instead, they are trying to find a way to force the data to support their a priori hypothesis: that the species has spread from Guam to other locations recently. A more balanced analysis of their data is unlikely to find support for this hypothesis, and I explain why below. There is no point in collecting data if you will say that it supports your a priori conclusion no matter what it looks like.

Please see the details in the attached pdf for more info.
Additional comments: Please see the attached pdf


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: POPULATION GENETICS AND DEMOGRAPHY OF THE CORAL-KILLING CYANOBACTERIOSPONGE, TERPIOS HOSHINOTA, IN THE INDO-WEST PACIFIC
Review round: 1
Reviewer: 2

Output:
Basic reporting: This study investigate the genetic variation of coral -killing Cyanobacteriosponge Terpion hoshinota, which is distributed widely in the Indo-west pacific. There results suggest H5 are most abundant haplotype in Indo-West pacific and H12, which originated from Guam, has invaded other regions with slight genetic change. It was found that some genotype contribute out-breaks of Terpios. Authors sampled a large number of Terpios over a wide area and the results showing the spread of certain genotypes were very interesting. One thing that should be pointed out throughout is that there should be some prior research on genetic variation in terpions, but this is not mentioned. Mentioning previous studies in DISCUSSION and INTRODUCTION, and discussing the differences between their results and the results here, is necessary to objectively evaluate the present data.
Experimental design: Another point of concern is the Terpios specificity of the primers used for genetic variation. The authors first used a universal primer for the ITS2 region （ITS４ and EF3RCNL）, but they need to explain why they used this universal primer. Isn't this a primer used for fungal and plant diversity? Or is this a commonly used primer for this type of sponge? Please add more explanation.  In the results, there should be a confirmation that the ITS region sequence used for genetic variation is the sequence from Terpios. For example, it is necessary to check the similarity of the ITS of the closely related Songe species. I think authors need to refer about sampling permits for all sampling sites (e.g. Great Barrier Reef). Please confirm.
Validity of the findings: Please provide the accession numbers in the Gene Bank for the COI gene sequence and the ITS gene sequence that you have obtained. Does the raw data include the ITS region sequence? There is only one GBR data in Table 1, but there are two in the row data. Why is that? In raw data,  some of the file names use Abbrevation from Table 1, and some do not. It is difficult to recognize, so please make it consistent.

Figure S1
Please exchange to a file with better resolution.Is this figure really necessary, and how does it differ from Figure 2? If so, why don't you add more explanation about this figure?
Additional comments: Minor comments
L.55, IWP region is Indo-West pacific region?
Please define it
L. 58, The results of this study indicate・・
"suggest" is more appropriate than "indicate.
L. 136, Please add references about genetic diversity in T. Hoshinota.
L. 144, Please mention the permission of great barrier reef
L.175, Please mention sequencer machine name, and more details of sequencing method. Which kit did you use? Did it performed with Sangar sequencing method?
L186, rDNA sequences of specimens obtained from the GenBank database.
Please insert the Accession No. of the rDNA sequences
L. 211-212, Only Guam is not an abbreviation. Please unify.
L. 217~ , For the ITS region, explain the sequence similarities between several different sponge taxa.
L.240, Please change "Xisha" to an abbreviation.
　　　I cannot recognize the name
L. 261, "Hap12" or "H12"?
Use the same haplotype abbreviation.
L. 266, Hap 1 is shown in Fig. 3? Please add Hap 1 in Fig. 3.
L. 290-293, Please add reference.


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: POPULATION GENETICS AND DEMOGRAPHY OF THE CORAL-KILLING CYANOBACTERIOSPONGE, TERPIOS HOSHINOTA, IN THE INDO-WEST PACIFIC
Review round: 1
Reviewer: 3

Output:
Basic reporting: It will be good to get one or two native English speakers to proofread the manuscript to improve the language.

Sufficient references. Context provided.

Minor improvements required for figures, suggestions in attached word file.

Self-contained with relevant results.
Experimental design: Original primary research.

Research question is fine and knowledge gap is identified.

Investigation performed to standard.

Minor improvements required Methods, suggestions in attached word file.
Validity of the findings: Results are novel.

Conclusions can be improved after issues are addressed.
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: POPULATION GENETICS AND DEMOGRAPHY OF THE CORAL-KILLING CYANOBACTERIOSPONGE, TERPIOS HOSHINOTA, IN THE INDO-WEST PACIFIC
Review round: 2
Reviewer: 1

Output:
Basic reporting: Sufficient, minor revisions suggested below
Experimental design: Sufficient, minor revisions suggested below
Validity of the findings: Sufficient, minor revisions suggested below
Additional comments: Sufficient, minor revisions suggested below


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: POPULATION GENETICS AND DEMOGRAPHY OF THE CORAL-KILLING CYANOBACTERIOSPONGE, TERPIOS HOSHINOTA, IN THE INDO-WEST PACIFIC
Review round: 2
Reviewer: 2

Output:
Basic reporting: Terpios habitat on coral and sometimes threaten the survival of coral reefs. Their genetic differentiation and regional differences are interesting, that are suggesting how Terpios began to spread on coral reefs. I felt a big change from the previous version. However, what makes me suspicious in this paper is that it is still difficult to determine whether the ITS2 sequence is actually derived from Terpios based on the information provided. This is because, all Terpios ITS2  sequences provided here, are homologous to the Terpios sequences in this paper, and other ITS sequence of sponges were not detected by homology search,by NCBI blastn.
Experimental design: I feel anxious about universal primers (ITS4, EF3RCHL) that are used for the identification of the ITS2 region of Terpins. This primer set is a universal primer for fungi?, and Terpios is not fungi. It might have been better to design primers based on the known sponge nucleus sequence. Anyway, there are still doubts about the method for identification of ITS2 sequence and the information of ITS2 sequence.
The author should provide the sequence information obtained by the first PCR using universal primers (ITS4, EF3RCHL), and prove that it is actually derived from Terpios by alignment analysis or by phylogenetic analysis.
Validity of the findings: 'no comment'
Additional comments: 'no comment'


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: POPULATION GENETICS AND DEMOGRAPHY OF THE CORAL-KILLING CYANOBACTERIOSPONGE, TERPIOS HOSHINOTA, IN THE INDO-WEST PACIFIC
Review round: 2
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: Objectives and conclusions are not well stated.

Objectives
1) To sample T. hoshinota from its currently known distribution in the IWP and examine the variation in its COI and nuclear ribosomal internal transcribe spacer (ITS) sequences.

All its currently known distribution in the IWP from existing literatures? If not, please list the localities sampled.

Achieved

2) To elucidate the population genetic structure and demographic history of T. hoshinota
Achieved

3) To propose hypotheses on how T. hoshinota dispersal and colonization in the IWP?
I have difficulties identifying and understand the hypothesis on T. hoshinota dispersal and colonization in the IWP in this study. Please state clearly and elaborate if there's a hypothesis.

Is “potential outbreak mechanisms of T. hoshinota may result of both long-range dispersal and local outbreaks” in the Conclusion the hypothesis that your study aims to obtain? If yes, please elaborate and distinguish how your hypothesis is different from the typical sponge dispersal and typical local outbreak.

Conclusion
Please elaborate the how the outbreak mechanism in your study is different from typical outbreak mechanisms in your Discussion. An example of a  typical outbreak is when environmental changes favour expansion of T. hoshinota population that is already established and detrimental to health of coral reefs.

Please elaborate and distinguish how your "long-range dispersal" is different from the typical sponge dispersal, and how is it relevant as your proposed hypothesis.

Please  discuss the possibility of T. hoshinota causing an outbreak “immediately” or in a  season after long-range dispersal to support your hypothesis. Are there studies or data on a population of T. hoshinota establishing on a reef after long-range dispersal and causing an outbreak immediately or in the same season or short period of time?
Additional comments: No comment


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: POPULATION GENETICS AND DEMOGRAPHY OF THE CORAL-KILLING CYANOBACTERIOSPONGE, TERPIOS HOSHINOTA, IN THE INDO-WEST PACIFIC
Review round: 3
Reviewer: 1

Output:
Basic reporting: I looked through the authors responses to my minor comments from round 2, and am fully satisfied
Experimental design: I looked through the authors responses to my minor comments from round 2, and am fully satisfied
Validity of the findings: I looked through the authors responses to my minor comments from round 2, and am fully satisfied
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: POPULATION GENETICS AND DEMOGRAPHY OF THE CORAL-KILLING CYANOBACTERIOSPONGE, TERPIOS HOSHINOTA, IN THE INDO-WEST PACIFIC
Review round: 3
Reviewer: 2

Output:
Basic reporting: The unclear point about the Terpios sequence is improved in the revised ver. There are some points where the reply comment is not reflected in the text, so I decide it is a minor revision, but it's OK that it is almost "accept".
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Before, accept, please check the following points.

 Please confirm Figure legend (I didn't find).
 The figures, those are about  "This is the result of the array alignment. This figure is included in the supplementary section"were also not included in supplementary section?
.


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: POPULATION GENETICS AND DEMOGRAPHY OF THE CORAL-KILLING CYANOBACTERIOSPONGE, TERPIOS HOSHINOTA, IN THE INDO-WEST PACIFIC
Review round: 3
Reviewer: 3

Output:
Basic reporting: I'm satisfied with the authors' revisions.
Experimental design: I'm satisfied with the authors' revisions.
Validity of the findings: I'm satisfied with the authors' revisions
Additional comments: I'm satisfied with the authors' revisions and I recommend the manuscript for publication.