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: DIFFERENCES IN TAIL FEATHER GROWTH RATE IN STORM-PETRELS BREEDING IN THE NORTHERN AND SOUTHERN HEMISPHERE: A PTILOCHRONOLOGICAL APPROACH
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript “Does nutritional condition during tail moult differ in storm-petrels adopting contrasting moult-breeding strategies? A ptilochronological approach” by Ausems and colleagues, aimed to compare Growth Bar Width as a proxy for Feather Growth Rate in 4 species of storm-petrels: 2 breeding in the Northern Hemisphere with a breeding/moult overlap, and 2 breeding in the Southern Hemisphere with no breeding/moult overlap.
In general, the manuscript could benefit from a thorough check of the English grammar, for examples at lines: 77 “providing some flexibility allocation”; line 89 “when come to land”; line 196 “the values averaged per species”; line 304 “growing feathers faster lowers feather quality”.

The introduction nicely brought the subject of this study. I would make a stronger separation between the different costs of moulting and the costs of breeding (Line 59). I would also suggest the authors to use the same level of details throughout. For example, a species name is given Line 61, but not line 63.
I think the authors would benefit from reading Huntington et al. 1996 Leach’s storm-petrel. In The Birds of North America, No 233. The Academy of Natural Sciences, Philadelphia for a detailed account on the species.

Line 136. “move towards the equator” is not totally correct, as 2 of those species (LHP and WSP) are known trans-equatorial migrants (Drucker 2013, Wilson’s storm-petrel. In Neotropical Birds Online, Cornell Lab of Ornithology, Ithaca, NY; Pollet et al. 2019, Migration routes and stopover areas of Leach’s storm-petrels. Marine Ornithology 47: 55-65).

Line 415. Why not clearly state that is was among 4 species (rather than the more elusive between several species)?

Figure 1: I would include the significant correlation line for the Southern species.
Figure 3: The legend font is too small compare to the rest of the figure.
Experimental design: The methods sections would benefit from a few changes. For example, the sampling collection should be chronological. As it stands right now, the authors collected the feathers before the captured the birds.

Feather measurements: this section would require a bit more details on the methods, rather than simply citing Grubb (1989). A brief description on how the measure was done would be best.

Samples were collected in 2017 and 2018, but no mention was made on how authors dealt with pseudo-replication. Clarification is required.
Line 172, it should be “the ratio between feather mass and feather length” and not “the ratio between feather length and feather mass”.
Lines 190-195. This very long sentence would benefit from being cut into two distinct sentences. Furthermore, a more detailed description on the methods used for the literature search would be helpful.

Line 190. To better investigate nutritional condition during moult, I would suggest the authors to perform some stable isotopes analysis on the feathers. This would give them another variable to correlate with Growth Bar Width.

Is the Saino et al. 2012 reference regarding the correlation between FGR between primary and rectrices for all group of birds, or for a specific species (Barn swallow)? Does it take into account that northern storm-petrels moult their rectrices during the breeding season, but moult their primaries during migration, while barn swallow moult their rectrices and primaries during their wintering period? What are the results of the model if the feather types are included as a variable?

The aim of this study was to compare FGR between storm-petrels with different breeding/moult strategies, so I am not sure this phylogenetic analysis between FGR and FL is relevant for this study. It would be perhaps more relevant to have a comparison in FGR between species moulting while breeding and those moulting outside of the breeding season.

Supplementary table S1.  As this table pertained to phylogenetic data, it would be helpful to have the species in phylogenetic order rather than alphabetical.
Validity of the findings: no comment
Additional comments: Line 132. As you have European storm-petrels, Leach’s storm-petrels, and Wilson’s storm-petrels, I think it would be best to have black-bellied storm-petrels (compared to black-bellied, Fregatta tropica (hereafter BBSP), storm-petrels).

Line 289-290. The comment that storm-petrels feed their chick during the night is correct, but the authors failed to take into account that individuals do not come back every evening to feed their chicks. According to Huntington et al 1996, for LSP, on average, chicks are fed by 1 adult on 44% of the nights, by 2 adults on 21% of the nights, and chicks are not being fed on 35% of the nights. As such interval between feeding is 2.2 d for a 30 to 40-day old chick and 2.8 d for a 60 to 70-day old chick (when the moult is happening).

Line 324. Line 421. There is some confusion about nutritional condition and energy allocation. Stating that southern species have a better nutritional condition  (or a higher food availability as in line 421) might not be true, but it is true that they can allocate more energy to feather growth (as breeding is over).

Line 380 and Line 404. There is some discrepancies in the statement presented by the authors. Line 380: “Growth bar widths have been linked to relative nutritional condition,…. And results should be interpreted with caution” and the more assertive tone Line 404: “Our results suggest that ptilochronology may be a powerful non-invasive tool to reconstruct their relative nutritional condition…”


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: DIFFERENCES IN TAIL FEATHER GROWTH RATE IN STORM-PETRELS BREEDING IN THE NORTHERN AND SOUTHERN HEMISPHERE: A PTILOCHRONOLOGICAL APPROACH
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I am pleased to say that I find this paper much improved. I think the authors have adequately addressed the issues I raised in my previous review and because of it the paper is now much stronger. I commend the authors for their efforts in addressing these issues. Aside from this I have no further 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: DIFFERENCES IN TAIL FEATHER GROWTH RATE IN STORM-PETRELS BREEDING IN THE NORTHERN AND SOUTHERN HEMISPHERE: A PTILOCHRONOLOGICAL APPROACH
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comments
Experimental design: No comments
Validity of the findings: No comments
Additional comments: I read the new version of the manuscript by Ausems et al. after I provided comments on an earlier version of it before. I think the manuscript has improved and some of my remarks have been satisfactorily clarified. I am still not fully convinced about the strong focus provided to moult-breeding overlap as the main explanation for the observed variation in feather growth rate between North and South hemisphere storm petrels (although the title was changed the main storyline of the manuscript is the same), but authors acknowledge now in the text some of my hesitations (e.g. potential bias of the capture methods, limited knowledge of moult-breeding overlap in storm petrels). My only relevant concern is that I find a bit confusing the use of the term “nutritional condition” for the ptilochronology measurements of feather growth rate. As I mentioned before, growth bars can also reflect differences in temporal pressures so I do not find fully appropriate to use feather growth rate as a surrogate of nutritional condition, especially when the feather mass of the same feathers (as indicator of feather complexity) are ruled out from the analyses. Thus, differences in feather growth rate between storm petrel species could be explained by variation in feather complexity if the latter is negatively associated with feather growth rate between groups (see for example De la Hera et al. 2012 cited in the manuscript). In other words, feather mass can be relevant to distinguish whether feather growth rate varies because of nutritional differences between species or due to temporal constraints. This aspect also leads me to think about a more simple, and less confusing, title: “Does tail feather growth rate differ between storm-petrels breeding on the Northern and Southern hemisphere? A ptilochronological approach”.


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: REFRACTORY ASCITES—THE CONTEMPORARY VIEW ON PATHOGENESIS AND THERAPY
Review round: 1
Reviewer: 1

Output:
Basic reporting: Few grammatical mistakes noted, and a few spelling mistakes
References were provided appropriately
The review of management and pathogenesis is basic and is unchanged, no new ideas or changes were noted or highlighted
Several chapters in GI manuals explain the exact details except for some epidemiological data
The introduction is adequate
Experimental design: Medline search was performed on published articles from 2005-2018
It is unclear which data was extrapolated from the search of published papers and how it was analyzed
A series of management plans and basic physiology was discussed, no novel ideas noted
The review is written in coherent manner.
Validity of the findings: The article was well written however at no point did this reviewer note any information which was novel or new to management of this condition.
Additional comments: I believe the authors did a good job in detailing the pathophysiology of this medical condition, however the  information currently available is similar to the written paper. No novel ideas were reviewed or suggested based on the Medline search. I would suggest a meta-analysis instead to re confirm the management plans


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: REFRACTORY ASCITES—THE CONTEMPORARY VIEW ON PATHOGENESIS AND THERAPY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article draws extensively from the collected pool of literature. The material is well researched and largely correct.

A review of refractory ascites and its management is a good topic for review. The material presented in the article is relevent and would be a good resource for physicians who are not specialized in the filed of hepatology.

The introduction is appropriately concise and well structured.

The article suffers from serious errors in grammatical and sentence structuring throughout.  The authors have failed to present their findings in clear and unambiguous English. The information they have presented requires significant restructuring and grammatical revision. Virtually every paragraph suffers from errors in grammar, punctuation, or sentence structure. I would recommend the authors have the manuscript reviewed by a native English speaker for revision.
Experimental design: The survey methodology is comprehenisve and unbiased.

The research of the available material is clearly rigorous and thorough.

The methodology is clearly described.

The review is logically organized. The paragraphs and subsections are incoherent. Specifically, the section "Unusual clinical situations" Lines 150-180 is poorly organized and unclear. It also suffers from errors in grammar and sentence structuring.

The subsections under "Treatment of Refractory ascites" need to be more succinct and relevant to the topic. The sections should focus on the efficacy of each modality relative to resolution of ascites, improvement in renal perfusion, and reduction in mortality (if any). They should also include the rates of complication and any advantages and disadvantages of one modality relative to the others (if applicable).  Going into detail, for example, on the Z technique during paracentesis and  the effect of hydroxylated starch on hepatic lysosomes, is irrelevant to the topic of refractory ascites and does not require elaboration.
Validity of the findings: No comment
Additional comments: The first section from Line 33 to line 150 is relatively well organized with relatively minor errors in English. The section from lines 151 to 480 needs to be extensively re-worked for English and well as organization.  It seems that these two sections were written by different people. Regardless, the entire article needs to be reviewed by someone with better understanding of punctuation, grammar, and sentence structure.

The section under Treatment Modalities is too long and rambling. The information presented needs to be more concise and useful to the reader in terms of risk vs benefit.

The material that the authors have presented is no doubt well researched and extremely relevant to the the physician managing refractory ascites. It needs extensive revision for grammar and sentence structuring. Much of the wordage is redundant and the article becomes unfocused and wanders into unrelated topics in the Treament modalities section. I cannot recommend its publication in its current form.


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: DOWNREGULATED MIR-383-5P CONTRIBUTES TO THE PROLIFERATION AND MIGRATION OF GASTRIC CANCER CELLS AND IS ASSOCIATED WITH POOR PROGNOSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is an interesting article describing the potential association between downregulation of miR-383-5p with the proliferation, migration, prognosis and clinicopathological features of gastric cancer.

In terms of English language used, there seems to be much room for improvement despite being submitted for language editing services. Some mistakes are listed below:
- Line 23: grammar error 'were'
- Line 50: grammar error 'mortality worldwide'
- Line 52: grammar error ' GC have increased over the years, which seriously'
- Line 55: grammar error ' with multiple factors'
- Line 79: spelling error 'clinical'
- Line 88: grammar error 'were'
- Line 95: grammar error 'in humidified'
Experimental design: The experiments used are valid in supporting the hypothesis. However, there are several comments that requires attention:

- There seems to be inconsistency between the cell line nomenclature for MGC-803 versus SGC-803.
- For wound healing assay, was a cell cycle antogonist used such as mitomycin c prior to image analysis? If so, please indicate in the methodology.
Validity of the findings: - Line 145-146: It is unclear what 'After literature retrieval' means. Please elaborate.
- What is the basis of choosing 5cm as the cut off between large vs small tumour sizes? It would be beneficial to include the standards or guidelines used in terms of such classification as variations in this measurement value can greatly influence the association and conclusions made.
- Fig 1: Why is the blue imaginary line not situated at the FC 2.0 value on the x-axis?
- Table 2: Downregulated microRNAs with FC < 2.0 are included in the Table, but Line 142 of the results mentions that FC 2.0 and p<0.05 were used as the cut-off criterion. Maintaining this cut-off for miRNA Seq data will ultimately remove miR-383-5p from the list.
- Table 2 header is wrongly labelled as Table 1.
- Table 3: P-value needs to be specified e.g. is it a comparison between low vs high groups? Based on the table, it is unclear.
- Table 3: It would be beneficial if correlation regression values be included into the table to emphasize the strength of the associations.
Additional comments: Other comments are as stated below:
- It is recommended that the term 'novel' be excluded from the manuscript as this miR-383-5p has been reported several times in past literature.


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: DOWNREGULATED MIR-383-5P CONTRIBUTES TO THE PROLIFERATION AND MIGRATION OF GASTRIC CANCER CELLS AND IS ASSOCIATED WITH POOR PROGNOSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is well written and easily understood. There are minor spelling errors that requires correction. (Line 79 : cliniacl – clinical; Line 100 : confrmed – confirmed; Line 102 : appvoed – approved; Line 109 : summaried – summarized; Line 187 : Firebrose – Firebrowser)

Figures are well labeled and clear. Figure 4B needs improvement. The caption is “miR-383-5p mimics inhibited GC cells proliferation,” while the y-axis in the chart uses the word absorbance. It would be better to change it to viability to make it clearer what the chart is referring to.
Experimental design: The first part of this study focuses on bioinformatic analysis on miR-383-5p expression in gastric cancer, identifying it as a downregulated miRNA in gastric cancer tissues and cell lines. The second part of the study is a functional analysis of miR-383-5p in gastric cancer through overexpression study using miRNA mimics and control in gastric cancer cell lines, followed by bioinformatic analysis on predicted target gene.

Methodology

1. Reference number/information for approval should be included in the manuscript for patient tissue collections and experiments.

2. It is unclear what is the source of the 382 GC TCGA samples (Line 85), as based on the analysis for Figure 2A, there were only 235 GC samples with available value for miR-383-5p expression. Further clarification is needed on the sample sizes and selection criteria.

Figure 2C

1. The analysis for Figure 2C is very unclear and needs to be redone. Firstly, the caption for Figure 2C is “qRT-PCR showed that miR-383-5p was significantly down-regulated in GC … tissues.” Since the 54 GC tissue samples are normalized to normal tissues, why is the normalized expression of miR-383-5p divided by half to denote low (n=27) and high (n=27) expression, when instead, the expression that is higher than normal tissue should be considered as high expression and lower as low expression.

2. Next, how was the significant difference calculated? Was the expression of miR-383-5p in GC tissue statistically compared to normal tissue for the paired t-test? In that case, it is more appropriate and accurate to have a chart comparing the expression between GC and normal tissue. The current chart appears to list the 54 GC tissue samples from low to high expression of miR-383-5p, divide the samples into two groups (low and high expression) and compare them both, which is wrong.

Table 3

It is not clear in the methodology what factors were compared to each other to calculate significant difference. Also, the total number of samples for the variable “Differentiation Grade” does not tally to 54, unlike all the other variables.
Validity of the findings: 1. Although miR-383-5p overexpression study in GC cell lines is novel, its dysregulated expression in GC has been previously shown by Azarbarzin et al (Biochemical Genetics, 2017, v55(3), pp 244–252). In their study, Azarbarzin and colleagues identified miR-383-5p to be significantly downregulated in gastric cancer tissues without correlation to clinical characteristics. This highly relevant information was not discussed or addressed in this manuscript.

2. Since the overexpression of miR-383-5p inhibits migration and proliferation, and the predicted target proteins are associated with survival, authors should also look at the effects of the miRNA on apoptosis using appropriate assays instead of just observing viability. Apoptotic assays such as PARP assay or Caspase-3/7 detection assay need to be carried out to confirm the tumor suppressive effects of miR-383-5p. Knockdown of miR-383-5p is also required to support the findings of the overexpression assays.

3. Without target protein validation, it is not useful to discuss the implication of miR-383-5p relationship with the predicted signaling pathways. This is because as this point, the relationship is purely hypothetical and inconclusive. Ideally, luciferase assay and target protein RT-qPCR/western blot should be done to establish the target protein. An example can be seen in one of the papers cited in this manuscript (Zhao et al. 2017). Therefore, target validation is important before the findings of this manuscript can be published.

4. The conclusion in the abstract states “its target genes were involved in important signaling pathway.” As the target genes were only predicted and not confirmed by validation, it would be incorrect to conclude as such. This should be written as “its predicted target genes…” instead unless validation is carried out. Furthermore, this is different compared to the conclusion at the end of the manuscript, which does not make any reference to the target genes.
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: DOWNREGULATED MIR-383-5P CONTRIBUTES TO THE PROLIFERATION AND MIGRATION OF GASTRIC CANCER CELLS AND IS ASSOCIATED WITH POOR PROGNOSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: No further comment.
Experimental design: The authors have addressed all comments raised.
Validity of the findings: No comment.
Additional comments: The manuscript has been significantly improved, and the authors have addressed all comments raised.


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: TISSUE-SPECIFIC EVALUATION OF SUITABLE REFERENCE GENES FOR RT-QPCR IN THE POND SNAIL, LYMNAEA STAGNALIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors explore the stable reference genes for reverse transcription quantitative PCR (RT-qPCR) in the pond snail Lymnaea stagnalis.  The seven reference genes were investigated across six different tissues with RT-qPCR.  The stability of each of the genes was measured by some of the standard stability assessment algorithms.  As a result, the authors described that GAPDH and EF1α are highly stable in the tissues as well as in pooled analyses.  They also discussed those genes stability in other molluscs in the similar studies.
The main research findings of this paper will be important for the future researches in Lymnaea stagnalis and other molluscs.  I would recommend it for acceptance after the minor points listed below.

Specific comments:
1.	Line 56, 60, and 67
The authors referred RT-qPCR and in situ hybridization as the methods for investigating the gene expression.  I am not sure that the part of in situ hybridization is necessary.  The method RT-qPCR is to quantify the gene expression, and in situ hybridization is the method to identify the cells that express a particular gene.  Northern blots, “Virtual Northern” blots with amplified cDNA and RNAseq are the proper methods for the quantification and comparison of gene expression.

2. Line 59
The product in RT-qPCR in Lymnaea stagnalis (Sadamoto et al., 2010), which should also be cited in this connection here.

3. Line 110
“10 - 50 mg” should not to be the weight of CNSs from two animals.

4. Line 119, 166 and 173
The quantity of total RNA in the cDNA sample should be described.

5. Line 131, 136 and 137
This section describes about reverse transcription, and not PCR.

6.Line 228
“lowliest” is misspelled.

7.Table 2 and 3
The titles of tables are both described as “table 1”.

8. Table 3, Discussion
The authors refer to the previous result that GAPDH in Cepaea nemoralis is stable, but variable between seasons.  I cannot understand what the authors mean by these sentences.  If the authors think that GAPDH result in different species (Table 3) come from the seasonal effects, they need to develop this idea further and explain it in Discussion.


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: TISSUE-SPECIFIC EVALUATION OF SUITABLE REFERENCE GENES FOR RT-QPCR IN THE POND SNAIL, LYMNAEA STAGNALIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: Evaluation of candidate reference genes for qRT-PCR experiments in the invertebrate experimental model Lymnaea stagnalis is lacking.  It is of high importance therefore for quantification and comparisons in gene expression studies using qRT-PCR to validate the stability of candidate reference genes in different tissues and treatment groups in this species.   In this manuscript, the authors evaluated a panel of seven reference genes in different tissues (central nervous system, tentacles, lips, penis, foot and mantle) in Lymnaea including 1-alpha (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin, beta-tubulin, ubiquitin, prenylated rab acceptor protein 1, and voltage gated potassium channel (VGKC) by qRT-PCR.  To assess the stability of these genes in different tissues, the authors used the following standard stability assessment algorithms: geNorm, NormFinder, BestKeeper and RefFinder.  GAPDH and EF1 were found to be highly stable in all of the tissues examined, however VGKC was found to be the least stable gene.  Based on these results, the authors suggest that GAPDH and EF1 should be both used as reference genes for qRT-PCR studies in Lymnaea.
This is an important study because Lymnaea is a useful experimental invertebrate model used by several research groups working on learning and memory and it is necessary for qRT-PCR studies to use stable reference genes among tissues and treatments.  The manuscript is very well and clearly written.  However, there are some major issues that need to be addressed.
Major Revisions:
	The authors need to increase the N numbers as there was a huge variability in the Cq values for all the candidate reference genes examined including the GAPDH and EF1. The authors state that GAPDH and EF1a are highly stable and they should be used as reference genes for Lymnaea, however there was a high variability in their Cq values.  The highest Cq value for GAPDH was 33 and the lowest was 20.  The highest Cq value for EF1a was 30 and the lowest value was 24.
	For each candidate reference gene, the authors should optimise the different qRT-PCR conditions for each tissue individually as some Cq values were high for some tissues.  For instance, the Mean of Cq value for Lst-EF1a was 27.43 in the Foot and 27.20 in the Mantle. The mean of Cq value for Lst-GAPDH was 26.62 in the foot and 25.75 in the Mantle. Cq values of higher than 25 are too high for a reference gene.
	Results section, Figure 1:  For each candidate reference gene, graphs should be plotted for each tissue individually.  As there is variability of gene expression of the candidate reference genes among tissues, data plots and analysis should not be pooled across all tissues.  Furthermore, Individual data points should be plotted for each gene.
	Results section, Figure 2: Comprehensive rankings assigned by geNorm, NormFinder, BestKeeper and RefFinder should be performed for each individual tissue and not from pooled Cq values from all tissues.
	Conclusions on the rankings should be only drawn from Cq values from individual tissues and not from pooled Cq values from all the tissues as there is a huge variability of gene expression of the candidate reference genes among the different tissues.
Minor Revisions:
	Methods section, “Total RNA Extraction”, Line 105, “…animals were anesthetized in in 0.125%” should be changed to “…animals were anesthetized in 0.125%”.
	Methods section,” Reverse Transcription PCR section”, Line 135: “PCR reactions, 20 ng of RNA was added…” The authors must have made a typing error and they should change the 20 ng to 200 ng.
	Line 142. Nucleotides, not base pairs.
	Line 143. 57-62oC stated here for the melting temperature range but in Table 1 the temperature range is 55-60oC. Which one is correct?


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: TISSUE-SPECIFIC EVALUATION OF SUITABLE REFERENCE GENES FOR RT-QPCR IN THE POND SNAIL, LYMNAEA STAGNALIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: Line 126
The authors used Lst-tyrosine hydroxylase to check the genomic DNA contamination in the RNA samples, however the reason was not described.  The authors need to note the primer sequences and the reason to use it.
Validity of the findings: no comment
Additional comments: The authors explore the stable reference genes for reverse transcription quantitative PCR (RT-qPCR) in the pond snail Lymnaea stagnalis. The seven reference genes were investigated across six different tissues with RT-qPCR. The stability of each of the genes was measured by some of the standard stability assessment algorithms. As a result, the authors described that GAPDH and EF1α are highly stable in the tissues as well as in pooled analyses. They also discussed those genes stability in other molluscs in the similar studies.
The main research findings of this paper will be important for the future researches in Lymnaea stagnalis and other molluscs. The manuscript has been revised well. However I think that there is an improvement that should be made 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: TISSUE-SPECIFIC EVALUATION OF SUITABLE REFERENCE GENES FOR RT-QPCR IN THE POND SNAIL, LYMNAEA STAGNALIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The authors have addressed all the concerns and have revised accordingly.  Furthermore, the additional experiments performed by the authors improved the qRT-PCR results and the results are more robust as the Cq values for the candidate reference genes are lower and there is less variability. The manuscript is really well and clearly written.  Validation of the stability of candidate reference genes for qRT-PCR experiments for specific tissues in the invertebrate experimental model Lymnaea stagnalis is important and the main research findings of this paper can be used for future experiments using Lymnaea stagnalis.


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: TISSUE-SPECIFIC EVALUATION OF SUITABLE REFERENCE GENES FOR RT-QPCR IN THE POND SNAIL, LYMNAEA STAGNALIS
Review round: 3
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Lst-tyrosine hydroxylase is not the general reference gene.  The authors added the explanation that "it is a gene of interest that is frequently amplified by us as a part of a larger research project on neural development".  However, the Lst-tyrosine hydroxylase is highly expressed only in the CNS in many species, and it does not seem reasonable to use it to check the RNA quality in other tissues.  It would be better to add reference papers of the authors, or, the authors should add the explanation that this gene is expressed in many tissues in Lymnaea stagnalis.  The expression pattern of this gene in several tissues has not yet been reported in molluscan species, so it needs some explanation.  Otherwise, the amplifications in many tissues seem possibly to be due to the sample contamination.


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: TISSUE-SPECIFIC EVALUATION OF SUITABLE REFERENCE GENES FOR RT-QPCR IN THE POND SNAIL, LYMNAEA STAGNALIS
Review round: 4
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: As the authors wrote, I misunderstood the word "positive control", and now I understand the experiment to check the genomic DNA contamination.  However, I still do not understand the primer design.  The authors had better to add the explanation about the primers that they can be used for genomic DNA amplification, or that the authors usually use those primers for that purpose. The genomic DNA sample can be used to check the reliability of those primer design.  It is not necessary to say the primers can be used for RT-PCR.
The genomic DNA sequence of Lymnaea is not available now.  But, as the exon-intron structure are generally well conserved, I checked molluscan species genomic data.  The primers of LymEF1a seem to be designed in the same exon, which means the primers can amplify the genomic DNA.  Even though this is not the direct evidence, the primer design seems to be reasonable.


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: NCRNA2METS: A MANUALLY CURATED DATABASE FOR NON-CODING RNAS ASSOCIATED WITH METABOLIC SYNDROME
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is written in clear and unambigous language. However, I think that the term  "hypolipoproteinemia" mentioned as one of the five metabolic syndrome traits and used in the database is misleading, as it may refer to low levels of both Low-Density Lipoprotein cholesterol (LDL-c) and High-Density Lipoprotein cholesterol (HDL-c) which perform opposite functions. The word "hypolipoproteinemia" is not mentioned in the articles from which the associations were retrieved, nor is it commonly used in the literature as a trait associated with metabolic syndrome. Apparently, the authors meant low levels of HDL-c, so I would suggest to use the term "hypo-HDL cholesterolemia" or a broader term "dyslipoproteinemia".
Experimental design: no comment
Validity of the findings: The authors describe in detail the interface of the database. Obviously, it is well thought-out and convinient to use. Unfortunately, the website mentioned in the article is not available. I would like to explore it so that my review is complete.
Additional comments: The manuscript describes a manually curated database for noncoding RNAs associated with metabolic syndrome. This work is of great importance since metabolic syndrome has become a global threat to human health. The ncRNA2MetS database may become a valuable tool that will help researchers better study the pathogenesis of metabolic syndrome and associated diseases. In my opinion, the paper is suitable for publication in PeerJ after some revision and after the web interface is made available.

Some minor comments:

line 56: "including small non-coding RNAs (miRNAs)" As miRNA is an abbreviation for microRNA, I would suggest to rephrase to "including small non-coding RNAs, particularly microRNAs (miRNAs)"

line 106: "To meet demand" - "to meet this demand"

line 136: "DO identifier" - it is better to explain that DO refers to Desease Ontology

line 184: "the website will return all MetS-ncRNA associations meet the query" - "associations that meet the query"

line 186: "validated methods" - "validation methods"

line 219: "PARAL1" - maybe change to "Paral1"?

line 220: "The introduction to these associations in ncRNA2MetS show that" - "shows that"

line 225: "lincRNA" - "lncRNA"

line 241: "the relationship between metabolic and non-coding RNA" - "metabolic syndrome"

Figure 3: it is not intuitively clear why the ncRNAs in the central circle that are linked to multiple traits are of different color.

Figure 4: instead of "the comparison" and "a comparison" maybe use just "comparison"?
 "non-coding RNAs associated metabolic syndrome" - "associated with metabolic syndrome"


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: NCRNA2METS: A MANUALLY CURATED DATABASE FOR NON-CODING RNAS ASSOCIATED WITH METABOLIC SYNDROME
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this manuscript, the authors developed a metabolic syndrome-associated non-coding RNA database (ncRNA2MetS) to curate the associations between metabolic syndrome and non-coding RNAs, and then constructed a user-friendly website so that users can access and download all data easily. While this work might provide useful resources for the future metabolic syndrome-associated non-coding RNA study, I have some concerns:

1. In the ncRNA2MetS website, the authors should provide more related information, such as the RNA sequences, explanation and/or external links of disease ontology ID and ICD-10 classification code.

2. In order to better study the correlation between these ncRNA genes and metabolic syndrome, the authors should integrate and display more omics data of these genes, such as these genes’ multi-tissue expression data (especially in the disease-related tissues), in the database.

3. The authors should provide more online tools for users to better explore the ncRNA2MetS database, such as the NCBI online BLAST service.

4. Since this manuscript is presenting a database, it will be good to include a diagram of the data model, eg. ER model, to show some technical details about the database design.
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: NCRNA2METS: A MANUALLY CURATED DATABASE FOR NON-CODING RNAS ASSOCIATED WITH METABOLIC SYNDROME
Review round: 1
Reviewer: 3

Output:
Basic reporting: The authors developed a metabolic syndrome-associated non-coding RNA database (ncRNA2MetS) to curate the associations between metabolic syndrome and non-coding RNA. The ncRNA2MetS contains 1068 associations between five metabolic syndrome traits and 627 non-coding RNAs (543 miRNAs and 84 lncRNAs) in four species. They also developed a user-friendly website so that users can access and download all data easily. The ncRNA2MetS is a complete and high-quality data resource for exploring the role of non-coding RNA in the pathogenesis of metabolic syndrome and seeking new treatment options. The website is freely available at http://www.biomed-45 bigdata.com:50020/
Experimental design: The authors claim that there is no non-coding RNA database dedicated to metabolic syndrome. The authors need to comment if the database search engine can include other areas of disease that results from MetSyn such as gut bacteria dysbiosis, inflammatory bowel disease, cancer, etc.
Validity of the findings: The authors manually collected and curated MetS-miRNA (lncRNA) association from related articles in the PubMed database. They used ‘non-coding RNA’, ‘ncRNA’, ‘microRNA’, ‘miRNA’, ‘long non-coding RNA’, ‘lncRNA’ and each metabolic syndrome trait as search terms to search the PubMed database by Title/Abstract retrieval method. As a result, they acquired more than 3000 related articles published since 2007.
Additional comments: 1. Metabolic syndrome is a cluster of the most dangerous heart attack risk factors .... Please  define MetSyn properly.
2. miRNAs were shown to play critical roles in core processes of the insulin-related signaling pathway, carbohydrate and lipid metabolism, as well as adipocytokine signaling pathways. Up- or downregulation of certain miRNAs has been correlated with the development of insulin resistance and increased severity of T2DM. In particular, miR-7 was found to regulate pancreatic beta-cell function, differentiation and insulin secretion. Overexpression of miR-7 in mice has been associated with the development of T2DM due to impaired insulin secretion. Also, levels of miR-101, miR-375, miR-802 were significantly increased in T2DM patients while miR-143 and miR-223 levels were downregulated in obese individuals when compared to control groups. Constant exposure of pancreatic beta-cells to various metabolic stresses, including sweetener consumption, can shift the delicate balance between positive and negative regulatory miRNA, ultimately promoting pancreatic dysfunction and insulin resistance. Importantly, miRNAs can affect different bacterial species due to their capacity to enter the GI tract. This effect can partially explain the differences observed in miRNA regulatory effects.
3. The authors need to check the most recent articles on MetSyn and explain how the different conditions of disease elements may affect database profiling, additional parameters, and results.
4. What are the limitations of the database analyses?


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: NCRNA2METS: A MANUALLY CURATED DATABASE FOR NON-CODING RNAS ASSOCIATED WITH METABOLIC SYNDROME
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have taken into account the previous comments. In my opinion, no major revision is needed.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I see now that the web-site is generally available. I still cannot access it from my institution (which is strange), but it is available through my mobile Internet provider.

Some minor comments:

I have a little suggestion about the search page of the Web-interface. It would be great to make the fuzzy search more fuzzy (in the future). For example, the search for "mir 155" as microRNA type gives no results.

line 40: "specie involved" - "species involved"
line 70: "to develop database" - "to develop a database"
line 110: "various metabolic syndrome trait" - "traits"
lines 198, 199, 201: "validated methods" - "validation methods", the same for the Web-interface
line 256: "these three miRNAs plays an important role" - "play"


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: NCRNA2METS: A MANUALLY CURATED DATABASE FOR NON-CODING RNAS ASSOCIATED WITH METABOLIC SYNDROME
Review round: 2
Reviewer: 2

Output:
Basic reporting: 1. The figs are not easy to understand, and I can not find the detailed legends of their figs.
For example, in fig1, what is "other resources" ?
2. The authors need to find a native speaker to revise their writing.
Experimental design: NA
Validity of the findings: NA
Additional comments: NA


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: NCRNA2METS: A MANUALLY CURATED DATABASE FOR NON-CODING RNAS ASSOCIATED WITH METABOLIC SYNDROME
Review round: 2
Reviewer: 3

Output:
Basic reporting: acceptable
Experimental design: acceptable
Validity of the findings: acceptable
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: NCRNA2METS: A MANUALLY CURATED DATABASE FOR NON-CODING RNAS ASSOCIATED WITH METABOLIC SYNDROME
Review round: 3
Reviewer: 1

Output:
Basic reporting: no comment
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: NCRNA2METS: A MANUALLY CURATED DATABASE FOR NON-CODING RNAS ASSOCIATED WITH METABOLIC SYNDROME
Review round: 3
Reviewer: 2

Output:
Basic reporting: no comment
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: PREOPERATIVE INFLAMMATORY MARKERS OF NLR AND PLR AS INDICATORS OF POOR PROGNOSIS IN RESECTABLE HCC
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript provied the clearly inforamation of NLR and PLR in the HCC patients, the research background was shown in the paper and the literature well referenced. The authors have found that the NLR and PLR are useful prognostic factors to predict outcomes in patients with HCC who underwent live resection. The manuscript structure conforms to Peer J standards, and the authors uploaded the research raw data.
Experimental design: In China, the hepatitis infection plays a leading role in HCC occurrence and  progression, the researchers aimed to found new biomarker in the HCC patients who underwent the liver resection, in order to assisted to guide treatment and predict prognosis. The paper’s question was started with HCC clinical and was well defined.
The experimental design was rigorous, the idea of the paper was concise and the experimental results are reliable.
Validity of the findings: Inflammation palys great leading role in the pathogenesis and progression of cancer. Neutrophil count promotes tumor growth and metastasis and the mechanism may included extracellular matrix, reactive oxygen species. Platelets also can assist the tumor progression. Platelets can secrete inflammatory factors, including TGF-β and VEGF.
In this paper, the authors found that the preoperative NLR level and PLR level were closely correlated with the tumor size, TNM stage and BCLC stage, high NLR and high PLR were had the worst outcomes. The findings in this paper would assist the surgeons development the individualized management for the HCC patients.
Additional comments: This is a well-written paper containing credible results which merit publication. For the benefit of the reader, however, a number of points need clarifying and certain statements require further justification. There are given below:
1. In the lines 45, the description “hepatitis virus infection leads to liver cancer” should be descripted preciseness.

2. In the study population section, the recurrence of HCC patients who underwent liver resection were excluded or not?

3. The English language should be improved to ensure that an international audience can clearly understand your text.

4. The following study should be cited and disscued.
Preoperative Ratio of Neutrophils to Lymphocytes Predicts Postresection Survival in Selected Patients With Early or Intermediate Stage Hepatocellular Carcinoma. Medicine (Baltimore). 2016;95(5):e2722. doi: 10.1097/MD.0000000000002722.


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: PREOPERATIVE INFLAMMATORY MARKERS OF NLR AND PLR AS INDICATORS OF POOR PROGNOSIS IN RESECTABLE HCC
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors analyzed the relationship between preoperative NLR,PLR and RFS,OS from 239 HCC patients with liver resection and came to the result that NLR and PLR could be independent prognostically predictor.
This manuscript is not original since there are already similar studies analyzing the NLR and PLR predicting the prognosis of HCC (Int J Surg. 2018 Jul;55:73-80; Cell Physiol Biochem. 2017;44(3):967-981; Oncotarget. 2016 Jul 19;7(29):45283-45301) .
However, although the molecular mechanisms through which the high level of NLR and PLR are associated with poor HCC outcomes remain unknown, the manuscript discussed this issue and proposed some hypotheses.
Major:
•	It would be interesting to compare the preoperative NLR,PLR with postoperative NLR,PLR to evaluate the efficacy of surgery.
•	Please supplement more follow-up data of patients to show more detailed results and prognosis.

Minor:
•	Line 135-146: the authors combined the NLR and PLR to predict the prognosis. Please explain what is the difference between combination vs. single predictor.
•	Line 148-159 the manuscript indicates that the NLR,PLR correlates with tumor size, so can we just choose the tumor size as the predictor instead since tumor size is one of the prognostic predictor for HCC, too?  It would be better to add more discussion about the current existing prognostic predictors combining their advantages and disadvantages.


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: PREOPERATIVE INFLAMMATORY MARKERS OF NLR AND PLR AS INDICATORS OF POOR PROGNOSIS IN RESECTABLE HCC
Review round: 2
Reviewer: 1

Output:
Basic reporting: This is a revised manuscript. The authors performed a good work.
Experimental design: This is a revised manuscript. The authors performed a good work.
Validity of the findings: This is a revised manuscript. The authors performed a good work.
Additional comments: This is a revised manuscript. The authors performed a good work.


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: PREOPERATIVE INFLAMMATORY MARKERS OF NLR AND PLR AS INDICATORS OF POOR PROGNOSIS IN RESECTABLE HCC
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Please correct some minor grammar mistakes and improve English writing.


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: SUPERNUMERARY TEETH OBSERVED IN A LIVE TRUE’S BEAKED WHALE IN THE BAY OF BISCAY
Review round: 1
Reviewer: 1

Output:
Basic reporting: Be consistent throughout the ms between apical teeth and tusks. The apical teeth are now commonly referred to as “tusks” while additional teeth are called “teeth”

Be consistent in using mandible(s) instead of “jaw”.  Jaws can be upper and/or lower, while mandible is anatomically correct.

Line by line edits:
Line 28: melon and beak “aid in identification” but it is teeth placement that confirms species ID as stated in the next paragraph.

Line 44: add McLellan et at 2018 (citation included here) that provided photographic identification of M. mirus, M. europeaus and Z. cavirostris and describes species ID characters delineating each. This publication also describes overlapping distributions and abundances of these three species.

McLellan, W.A., McAlarney, R.J., Cumming, E.W., Read, A.J., Paxton, C.G.M., Bell J.T., and Pabst, D.A. 2018. Distribution and abundance of beaked whales (Family Ziphiidae) off Cape Hatteras, North Carolina, USA. Marine Mammal Science. DOI: 10.1111/mms.1250   34(4): 997-1017

Line 52: add Pont-Aven “ferry”
Line 61: to independent experts “for species identification”
Line 93: how far off was the initial sighting? An initial ID of Tursiops is probably not important here and could be dropped as beaked whale sightings were not confirmed until photographs were analyzed?
Line 120: you only personally examined osteological specimens in EU collections? There are a number of complete skulls in collections in the US, so would question this finding.
Lines 130-135: There is no possibility of misidentifying the photos included in this ms as Berardiu safter expert analysis. This section could be dropped.
Lines 148-150: Nowhere in this ms are other beaked whale species described as having been sighted in the Bay of Biscayne.
Lines 168-170: did that specimen have an alveoli associated with that tooth placement?
Experimental design: No additional comments on the experimental design.
Validity of the findings: Lines 40-44. The confirmed sightings of M. mirus is the more important take home message of this ms. If the species was not confirmed until these observations (though I did not conduct an exhaustive literature search for beaked whales sightings in the Bay of Biscayne) that is more important than supernumerary teeth. The supernumerary teeth can be an additional observation in this ms, but a redirection of the ms would also require a change to the title “Confirming True’s Beaked Whale Distribution in the Bay of Biscayne” or something to that effect.

You appear confident that the sighting contained 4 animals. Beaked whales take long extended deep dives and shorter “bounce” dives so they display a large range of distances traveled under the surface.  Following “normal” rules for lumping dolphins together as a single sighting are difficult to transfer to diving beaked whales. There is growing evidence that beaked whales are diving and rejoining other animals at the surface in a constant conveyor of animals from the bottom to the surface. It would be worth adding a stamen about the confidence you have to combine these separate surfacings into a sighting of 4 individuals.

The ms is based on two(?) images of a male True’s beaked whale with possible supernumerary teeth.  As there are a number of specimens in museum collections that you reference in Table 1, it would be a valuable addition to go into the collection records and images of those specimens to determine if very small teeth were present at necropsy/dissection. As these teeth may be embedded only in the hypodermis/epidermis there would be no record of them in the osteological specimen.
Additional comments: There is little value added between Fig 1 and Fig 5. As Fig 5 shows most of the same landmarks Fig 1 could be dropped.

Fig 3 needs to have the multiple images better lined up, numbers should be changed to letters and have a color change to be better delineated.

The pink coloration of the genital patch is almost certainly evidence of heat dumping.


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: SUPERNUMERARY TEETH OBSERVED IN A LIVE TRUE’S BEAKED WHALE IN THE BAY OF BISCAY
Review round: 1
Reviewer: 2

Output:
Basic reporting: When framing their observations/putting them in context the authors have missed important references regarding Mesoplodon sightings confirmed to species. How this can be addressed is noted in the comments to the authors.
Experimental design: No comment
Validity of the findings: The paragraph discussing the importance of platforms of opportunity is not warranted given this manuscript presents a single 68-second sighting of a species known (from strandings) to be in the northeast Atlantic.
Additional comments: As the authors note, beaked whales are a poorly known group and Mesoplodon in particular are a poorly-known yet diverse genus. True's beaked whales have been conclusively recorded as stranded specimens in the Northeast Atlantic, and there are a number of sightings of live individuals that are likely this species, but this manuscript provides photo-documentation of live individuals. The importance of that in itself is perhaps somewhat overstated (given they are known in the general area from strandings), but this manuscript also documents supernumerary teeth in a live True's beaked whale, which is worth reporting. That said, the manuscript is overly long given the content and could be shortened by 25% without losing anything important. Some of the context of the rarity of sightings of confirmed species of Mesoplodon also needs addressing (see below).

22-23 and 40-41. I understand that the authors want to emphasize how unusual their sighting is but the importance/frequency of confirmed species sightings of Mesoplodon is overstated and needs to be toned down. The statement that "most of our knowledge is from stranded specimens" is certainly true for most species of Mesoplodon, but not at all true for M. densirostris, with long-term in-depth studies of that species in both the Pacific and multiple locations in the Atlantic. The statement "there have been few sightings confirmed to species level" when referring to Mesoplodon is clearly not correct - there have been hundreds of confirmed sightings of M. densirostris (see papers from the Bahamas, Canary Islands and Hawaii), as well as numerous sightings of M. bidens, M. layardii, M. europaeus, M. peruvianus etc.

Methods

50-52. There are a lot of unnecessary details- should delete "a citizen science charity... staff and trained volunteers guiding a photography trip with Jessops Academy".

61-65. This is all in the acknowledgements and does not need to be repeated here.

76/85/93 etc. The term "pod" should be replaced with "group" throughout. Pod implies genetic relatedness (think peas in a pod), and should not be used when group would suffice.

76. "was recorded" should be "was observed"

96/97 - the ship passed the animals - saying "the animals passed the ship" implies the ship was motionless and the animals swam by.

113/114. Just note "The anal-genital patch appeared a light pink" - the rest of the information in that sentence belongs in the Discussion.

137/139. This implies that there have been no strandings in the western North Atlantic, yet Table 1 shows 7, and the NOAA stock assessment report notes four strandings in the US over a more recent 5-year period https://www.fisheries.noaa.gov/national/marine-mammal-protection/marine-mammal-stock-assessment-reports-species-stock#cetaceans---small-whales - as well as bycatch records. The authors should also see DeAngelis et al. 2018 "A description of echolocation clicks recorded in the presence of True's beaked whale" JASA, and McLellan et al. 2018 "Distribution and abundance of beaked whales off Cape Hatteras, North Carolina, USA" MMS

146-160. While it is clear that platforms of opportunity provide valuable information, given this manuscript is about a single sighting having a long paragraph extolling the value of such  platforms is a bit excessive. This paragraph could be reduced to one or two sentences.


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: LAYERED PATTERNS IN NATURE, MEDICINE, AND MATERIALS: QUANTIFYING ANISOTROPIC STRUCTURES AND CYCLICITY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article proposes a method to quantify the structure of patterns found in several natural images. The authors suggest an approach based on the binarization of 2D images. In the definition of the empirical model, the variables are quite loose; they all depends a lot on the binarization of the grayscale image. How Tm,n is measured? This issue is raised in the sensitivity of DStr section. However, the authors, at the end of the section, leave to the application to count it as positive or not. This problem of image binarization is very well treated by the image processing community and there are issues that do not depend on the application for example if the image contains noise or blur. This has to be dealt with independently of the application. The other variable in the equation also depends on that which make the DStr very sensitive. The image processing part in the Results section does not explain how this procedure is done, it refers to another article from the authors, here at least some explanation is necessary.

In line 159, reference should be presented when the authors describe different publications.

In line 239, how in Figure 4B has a maximum of 103 transects?? how this number was obtained?? By the image of the figure does not appear to have 103 transects. Also, there is an inconsistency in this figure the end lines do not meet the dot in the left figure.

In Figure 3 C the reference to the layers does not correlate with the figure A., For example, A1,1, A1,2, and A2,1 in C give in misleading idea from figure A. The caption also should be complete. This figure is also exactly the same as from another article from the same authors. I suggest changing to give another example from a different configuration.
Experimental design: The experiments were done is very few loose images. To validate the experiments, a lot more images should be used, I suggest a complete dataset and also a way to metrify the usefulness of the proposed metric. There is no comparison or clear applicability of the metric this should be more explored to argue proposal. For example, what would be the results compared to an entropic measure applied in the image?
Validity of the findings: Since the method depends a lot on the binarization of the image is very difficult to access the validity. Also, there was no comparison with other methods that measure disorder such as entropy.
Additional comments: Even though the authors describe possible uses in several different areas there was no definite example of clear use of the proposed disorder metric to gain insight in a single problem. The authors should investigate better this issue why is this metric necessary??


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: LAYERED PATTERNS IN NATURE, MEDICINE, AND MATERIALS: QUANTIFYING ANISOTROPIC STRUCTURES AND CYCLICITY
Review round: 1
Reviewer: 2

Output:
Basic reporting: This work by Igor Smolyar et al, presents a method to quantify the morphological characteristics of layered patterns and apply it to several images. It proposes three approaches: 1) Calculation of the disorder index DStr, 2) evaluation of the “layer thickness vs. layer number " graph and 3) evaluation of the" layer area vs. layer number" graph.
Although the three methods are described in the abstract, the authors do not present results for the 3rd.
It is definitely a judicious and interesting proposal for systems analysis made up of layers. The article is structured properly and the tables and figures are presented satisfactorily with only minor suggestions for changes.
It would be interesting to include the scale of the images used.
Equations can be best presented using equation editors.
The Sensitivity of DStr results, can be shown in a table. It is easier to refer.
I think it is interesting to present, for all applications, the Black & White image, used for layer evaluation.
Experimental design: no comment
Validity of the findings: The DStr sensitivity analysis results presented in the paper, shows that the method can present a high sensitivity to noise, reaching the ratio 30:1.5 (1.5% of change in the image pixels results in 30% of change in the index). This sensitivity is also highly dependent on layer structure, as can be seen when the Fish Scale and Fingerprint results are compered.   It would be interesting to evaluate whether the sensitivity depends on the absolute value of the DStr. This can be easily done with a graph showing the DStr values in X and the Sensitivity in Y for multiple images examples.
This sensitivity may also imply spurious results depending on the image conversion parameters for B & W. It would be interesting to present how the image conversion parameters can influence the DStr estimation.
The authors present the fluctuation patterns in the thicknesses of the layers, however no evaluation is made on the characterization of these fluctuations. A simple evaluation of the autocorrelation or persistence (Hurst exponent) of the series could reveal interesting characteristics of the formation mechanisms of the layers.
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: LAYERED PATTERNS IN NATURE, MEDICINE, AND MATERIALS: QUANTIFYING ANISOTROPIC STRUCTURES AND CYCLICITY
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors present a revised version of the article following the reviews comments. Even though the authors improved the description of the method, the binarization process in which the grayscale image is transformed into a binary image is not explained the authors used the reference Smolyar (2016), which it seems that uses a simple threshold method. This is of importance since the result of the threshold defines the binary image and consequently the EM and DStr.
Experimental design: no comment
Validity of the findings: I would agree with the review D that it is necessary to show clear use of the proposed metric. Stating that this is out of the scope of the article is not a good argument for that since then why is this metric important or necessary? The authors should present a more convincing argument for the necessity of the proposed metric as in the conclusion it is stated that it could be potentially used to quantify the morphological characteristics of arbitrary 2-D binary patterns. If so how?
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: GENOME-WIDE IDENTIFICATION AND EXPRESSION ANALYSIS OF THE WRKY GENES IN SUGAR BEET (BETA VULGARIS L.) UNDER ALKALINE STRESS
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comments
Experimental design: Phenotypic data lacking?
Validity of the findings: Why only five genes selected for validation?
Additional comments: Authors; Wu et al. in this study identified 58 WRKY TFs from sugarbeet genome using insilico analysis and did analyze for its chromosomal positions, gene structure, conserved motifs etc. Further, they validated a few genes using qRTPCR. Authors have generated new information which was not reported earlier. I am just wondering why the author did not record the phenotypic data of samples used for alkaline stress? why the author choose only two tissues? root and shoot why not other parts and stage data for correlations with transcripts at what stage is express more?
i have few corrections and suggestions as listed below:
1. Lines 60-63 needs to be rewritten for instance say" adversely environments'?
2. Line no. 76 "different kind of plants"? what do you mean by this
3.Line  97. what is "lower water loss"?
4. Lines 231-233 " make a new phrase
5. Line 250-252 " for validation of few genes using qRTPCR what is the basis of selection is not clear?
6. Line 338-339 " incomplete sentence?


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: GENOME-WIDE IDENTIFICATION AND EXPRESSION ANALYSIS OF THE WRKY GENES IN SUGAR BEET (BETA VULGARIS L.) UNDER ALKALINE STRESS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Wu et al., reported the identification and phylogenetic classification of WRKY genes in sugar beet and demonstrated their potential involvement in alkaline stress. The language of this manuscript is okay. However, the authors should take more efforts in improving their experimental design and elaborating their results and providing more convincing evidence on the role of WRKY genes in alkaline stress before publication. Furthermore, they need to improve the quality and resolution of all figures.
Experimental design: a.The authors should include a step of motif scan of WRKY domain in the ‘identification of WRKY genes’. Although it turned out that all genes contain WRKY motifs in their ‘conserved motif analysis’, it would be more logical if they perform that earlier. Genes without WRKY domain should be excluded from their analysis. Meanwhile, they need to explain what was used for the identification of WRKY genes, was it based on the domain sequence from Pfam (L142)?
b. The authors stated that they removed overlapping sequences manually (L190). This step raises a lot of doubts. First, how did the authors define ‘overlapping’?. Were those sequences from different genome loci? Second, it is possible that the authors removed WRKY genes that were caused by genome duplication (segmental duplication). It is not reasonable to remove those sequences. In addition, an assay for segmental duplication is highly recommended in this case since they mentioned that in the discussion (L284).
c.The authors mentioned that they named WRKY genes according to their order in the sugar beet genome sequence. However, they were ordered from a mixture of different chromosomes in Table 2. Please explain this.
d.’WRKYs are classified into 3 major groups ..based on their putative WRKY domains’. The grouping of phylogenetic trees should be determined by how they clustered rather than their domains. If the authors would like to group them by domains, what is the necessity of constructing a tree? In addition, they did not mention any result of domain scanning ahead of this. It seems that they used their later results in this analysis, which is not logical.
Validity of the findings: a.The authors said ‘similar observations were made in sesame’ (L198). First, I did not find any similarity between sugar beet and sesame. Second, I am not sure why this statement is informative to the audience.
b.The authors mentioned that WRKY genes are evenly distributed throughout the genome, which is not true (L212). In addition, the statement of ‘the enrichment of WRKY genes on chromosome 5’ is not convincing, given that there are 9 genes on chr 2 and chr2 is even shorter. Meanwhile, the scale on the side of Figure 2 is not correct, at least not proportional. Also what they marked in red does not fully match with the text. Finally, please explain why 3 genes are located on the chromosome unknown. Is that because they are located on unassembled scaffolds?
c. The authors claimed that ‘ WRKY genes in the same cluster share similar motif compositions’ (L239). However, I could not draw the same conclusion from Figure 3 since it is not organized according to their cluster. The authors should modify accordingly.
d. The authors’ statement that ‘WRKY genes within the same group share a similar structure’ is also not well-supported by their description (L248).
e. The authors did not mention why they only perform qPCR on 9 genes and how were those genes selected. Are those genes the only ones with detectable expression levels? If yes, they need to mention that. If not, they need to either provide a reason why group III was not included or perform more qPCR on the remainder of genes. And there are lots of problems in their descriptions. For example, they claimed that WRKY1,4,9,17,31 are all induced by the stress treatment. However, in some conditions, they are repressed or unchanged. I would recommend to rewrite this paragraph. They also need to explain why most genes respond to stress in an irregular pattern (going up and then down, or doing up ,down, up and then down with the increase of salt concentration). Furthermore, since this paper emphasizes on the role of WRKY in alkaline stress. The authors need to perform more analyses (e.g. prediction of gene functions using GO terms) to support their conclusion. So far, it is not convincing.
f. The authors hypothesis that ‘WRKY31 is involved in the regulation of organic acid genes’ is reckless and further evidence is needed for supporting this hypothesis.
Additional comments: a.It would be better if the authors could mention TaWRKY1 is from wheat (L93).
b.Table 1 should be supplemental.
c.L304, should be Table 2.
d.Please explain what circles and squares in Figure 1 refer to and why some genes are labeled in red. The three groups should also be labeled.
e.Figure 4, does ‘upstream and downstream’ mean UTR?


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: GENOME-WIDE IDENTIFICATION AND EXPRESSION ANALYSIS OF THE WRKY GENES IN SUGAR BEET (BETA VULGARIS L.) UNDER ALKALINE STRESS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have addressed most of my concerns. However, there are a few new points need to be clarified.
1. It seems that the clustering of WRKYs in Figure 1 into 3 groups is not based solely on phylogenetic analysis. The authors should mention where the grouping of I, II, III and IIa etc. is derived from. Presumably from Arabidopsis studies? (L325).
2. Figure S2, motif 3 is not a WRKY domain?
3. For the GO enrichment analysis, the authors should how the fold enrichment too.
4. For the 3D structure prediction, I am not sure how the result would facilitate the understanding of the functional mechanism of BvWRKY proteins.
5. L186, should be TAIR.
6. L312, the authors stated that 58 BvWRKY proteins had one or two conserved WRKYGQK domains. However, in Fig S1 it looks like there is only one domain. And if there were two, which one was used for alignment and for the phylogenetic analysis?
Experimental design: no comment
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: BIOINFORMATIC AND BIOCHEMICAL ANALYSIS OF THE KEY BINDING SITES OF THE PHEROMONE BINDING PROTEIN OF CYRTOTRACHELUS BUQUETI GUERIN-MENEVILLE (COLEOPTERA: CURCULIONIDEA)
Review round: 1
Reviewer: 1

Output:
Basic reporting: literature needs to be updated and relevant literatures are missing
Experimental design: some positive control for mutation experiment is useful, see my comments in comments fro authors
Validity of the findings: no physiology or behavioral validation
Additional comments: Yang et al., Bioinformatics analysis of the key binding sites of the pheromone binding protein of Cyrtotrachelus buqueti Guerin-Meneville (Coleoptera: Curculionidea).

Using Homology modeling and molecular docking they investigated to study how pheromone binding protein CbuqPBP1 and its ligand, dibutyl phthalate/cedar camphor interact. They have identified the binding sites; its 3D structure and they also validate the binding sites by doing site-directed mutagenesis. Which is interesting study.

The main shortcoming of the study is why they selected CbuqPBP1, and also where is the active site, in which of the 6 helixes?

We do agree that the authors did some quality check of the built 3D model.  They used PROCHECK tool for the quality check. But RSMD is not presented. And only one tool may not be enough to assess the rationality of the model. The authors can add another tool such as 3D profile. What is the quality (Stereochemistry, resolution) of the template?

Alignment and homology modeling

Conventional Protocols for Homology modeling were followed.
 template met criteria
3D structure built; quality checked
model optimized using robust web online server.

But authors did not give PDB accession number of the template and its Resolution. Given that the quality of the template is also based on its resolution. it is important to give the resolution of the initial template.

In the site directed mutagenesis we also need a positive control, to make sure other PBPs are not affected by the mutation.

What is the behavioral significance of the CbuqPBP1 and dibutyl phthalate interaction?

Is CH-π the binding site not well described it is not clear, where on the 3D structures, how its structure a little bit of description is needed.

What is the result implication we need some discussion about its significance?

Where are these amino acids on which helixes? Phe69 and His53

The objective was to study CbuqPBP1 - dibutyl phthalate interaction, but the result is talking about CbuqPBP1- cedar camphor interaction? Which one is the ligand and how it is selected?

Is cedar camphor correct name of the ligand?

The author wrote: Thus, His53 of CbuqPBP1 appears to be a key binding site residue, and Phe69 is a very important binding site for particular ligand interactions. Which particular ligand? You have to specify

Line 23. OBPS are not chemoreceptors?

Line 24 -25 give references

The English need to be revised for instance Line 25-27 not clear
Line 30-33 rephrase for instance …CH-π interaction between the benzene..., interaction is not
correct replace with interacts

Line 31 is CH-π is an active site of CbuqPBP1?

Line 35…. to which ligand?

Line 36 your objective and result are different

The introduction: shorten it by reviewing up to date knowledge of the subject matter (PBPS, your insect model and interaction between the protein and the ligand and possible implication) and the research gap you are addressing.

 For instance, poor description of Pheromone binding protein

The authors have not stated how important is Pheromone communication in Cyrtotrachelus buqueti

the economic importance of the pest better way

Line 46, what do you mean by tentacles, do you mean sensilla or antenna? Tentacles is not the olfactory organ of insects

The authors need to update themselves about the biology of the olfactory system

what do you mean receptors are widely distributed? Do you want to say they are equipped with…?
Line 50 antennal binding protein, what do you mean? You need to use recent works about OBPs as reference

Line 115-116. Why did the authors choose this coordinate for the docking? No reference or justification

Line 125 we need more description of the method for instance, add the PCR conditions for any duplicability of the experiment

The study ends at bioinformatics data alone, no validation using physiology or behavior or interpretation. Nothing has been said about the implications of the study and how it will help to manage the pest.


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

Input:
Title: BIOINFORMATIC AND BIOCHEMICAL ANALYSIS OF THE KEY BINDING SITES OF THE PHEROMONE BINDING PROTEIN OF CYRTOTRACHELUS BUQUETI GUERIN-MENEVILLE (COLEOPTERA: CURCULIONIDEA)
Review round: 1
Reviewer: 2

Output:
Basic reporting: There were many cases in the manuscript where the sentences including lines 80-82 were not properly structured, suggesting that the paper would improve if it could be edited for language and technical correctness. Every abbreviation for the Latin name of the species should be checked include lines 67,71, which should be accorded to the abbreviation rules.
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: BIOINFORMATIC AND BIOCHEMICAL ANALYSIS OF THE KEY BINDING SITES OF THE PHEROMONE BINDING PROTEIN OF CYRTOTRACHELUS BUQUETI GUERIN-MENEVILLE (COLEOPTERA: CURCULIONIDEA)
Review round: 1
Reviewer: 3

Output:
Basic reporting: In this study, the spatial structure of Cyrtotrachelus buqueti PBP1 was studied by constructing three-dimensional homoologous model, and the binding capacity of CbuqPBP1, cbuqpbp1-his53a, cbuqpbp1-phe69a mutants and Dibutyl phthalate, benzothiazole and cedar camphor were analyzed and compared by fluorescence competitive binding and molecular docking. The results showed that there were two important sites of CbuqPBP1, His53A and Phe69A.
Minor revision
(1) Line 180, CbuqPBO 1, should be CbuqPBP1
(2) Line 238-242, When the final concentration of odor substance was higher than 50 μM, the fluorescence peak did not decrease to half its value. This showed that almost no affinity was established between protein and the odor substance, and the binding constant could not be calculated (Fig. 6).
(3) In figure 4, Lane 3.4.5.6 was not marked in the diagram.
Experimental design: Major revision
(1) CbuqPBP1 is recognized as PBP protein, suggest author add the sequence alignment results with other insects PBP1 and evolutionary tree analysis.
(2) In Molecular docking results, why NribOBP3 protein was selected as model protein control? Maybe choose other insect PBP1 proteins that have resolved structures as models. The sequence similarity is probably higher than NribOBP3 protein.
Validity of the findings: Three kinds of fluorescent competitive binding substances used by CbuqPBP1 have been studied, and the site-directed mutagenesis have also been studied in PBP of other insect species, this experimental results in this paper also explain some olfactory molecular mechanism. It can also provide reference for other insect related research.
Additional comments: In this study, the spatial structure of Cyrtotrachelus buqueti PBP1 was studied by constructing three-dimensional homoologous model, and the binding capacity of CbuqPBP1, cbuqpbp1-his53a, cbuqpbp1-phe69a mutants and Dibutyl phthalate, benzothiazole and cedar camphor were analyzed and compared by fluorescence competitive binding and molecular docking. The results showed that there were two important sites of CbuqPBP1, His53A and Phe69A.
Minor revision
(1) Line 180, CbuqPBO 1, should be CbuqPBP1
(2) Line 238-242, When the final concentration of odor substance was higher than 50 μM, the fluorescence peak did not decrease to half its value. This showed that almost no affinity was established between protein and the odor substance, and the binding constant could not be calculated (Fig. 6).
(3) In figure 4, Lane 3.4.5.6 was not marked in the diagram.

Major revision
(1) CbuqPBP1 is recognized as PBP protein, suggest author add the sequence alignment results with other insects PBP1 and evolutionary tree analysis.
(2) In Molecular docking results, why NribOBP3 protein was selected as model protein control? Maybe choose other insect PBP1 proteins that have resolved structures as models. The sequence similarity is probably higher than NribOBP3 protein.


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

Input:
Title: BIOINFORMATIC AND BIOCHEMICAL ANALYSIS OF THE KEY BINDING SITES OF THE PHEROMONE BINDING PROTEIN OF CYRTOTRACHELUS BUQUETI GUERIN-MENEVILLE (COLEOPTERA: CURCULIONIDEA)
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have addressed the concern I have raised in my first review and the manuscript reads well. Deserve publication
Experimental design: addrresed
Validity of the findings: They used a different approach to prove their claim
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: BIOINFORMATIC AND BIOCHEMICAL ANALYSIS OF THE KEY BINDING SITES OF THE PHEROMONE BINDING PROTEIN OF CYRTOTRACHELUS BUQUETI GUERIN-MENEVILLE (COLEOPTERA: CURCULIONIDEA)
Review round: 2
Reviewer: 2

Output:
Basic reporting: This MS has been revised and it looks better for a publication now. However, after reading through the MS several times, I found some language problems which made me feel that some parts of the MS are weak or even a little poor in logic. For example, through the MS the authors used 11 times of “According to”, even from line 54 to 60, there are 3 times using of it in just 3 sentences, and this kind of using words made the part weird and, very poor in logic in my opinion. Besides, some other parts are similar as this one. Also, there are some grammatical errors, such as line 69 “The larva, in particular, prefer the…” should be “The Larvae...." Moreover, some words are used improperly and they are too many to be listed through the MS.
Furthermore, in the part of DISCUSSION, there are two independent paragraphs, line 258 to 263 and line 264 to 271, and could the authors integrate them into one paragraph? If not, please show the reasons.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I strongly suggested the MS should get language revise by English native speakers, and after the re-editing, the MS could be accepted.


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: OOMYCETE METABARCODING REVEALS THE PRESENCE OF LAGENIDIUM SPP. IN PHYTOTELMATA
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: The sequencing data certainly provides compelling evidence that Lagenidium spp are present in phytotelma, using two molecular methods to verify each other, and construction phylogenetic tree to complement sequencing. However, why was no attempt made to also culture any of the Lagenidium spp after sequences had been identified. Is it difficult to culture?
Validity of the findings: Could more information be provided on the relative abundance of all oomycete genera, not just Lagenidum (e.g. line 359). The read percent for Lagenidium was actually quite low for particularly P2 and P4. May be of interest to include read percent of the other oomycete genera - was the bulk of all the rest of the reads Pythium spp, and at what percent?

Line 464 - 467: Complementary approaches such as the ones presented.... Unclear what this means. This sentence implies that the role of oomycetes in phytotelma environment was evaluated using metabarcoding, but it was not. Next sentence goes on to state this. Suggest deleting or revising this sentence to indicate that environmental role was not ascertained in your study.

Line 476 - 479: It is not clear how this data is related to Fig 4, and also appears to be presenting new information in this discussion that wasn't previously shown in the results, i.e. differences in read percent of giganteum vs humanum in Sanger vs PacBio sequencing. Suggest including this information in the results first, rather than the discussion, and showing how the percentages were obtained.

Line 494 - 507: Although an interesting evolutionary discussion point, this section is not related to the original research question or to supporting results. Suggest shortening this section, or deleting it entirely.

Sequences have been deposited into GenBank, but at the time of this review, they are not yet available publically when searching the database.
Additional comments: Well written, easy to follow manuscript that describes previously unknown ecology of Lagenidium spp.


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: OOMYCETE METABARCODING REVEALS THE PRESENCE OF LAGENIDIUM SPP. IN PHYTOTELMATA
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript presents research on oomycete communities in particular Lagenidium spp. in phytotelmata. The study provides new insights on the presence of Lagenidium species and their relations with other Lagenidium species studies earlier and is worthy of publication. The paper is well written and I enjoyed reading it.
Experimental design: The MS combines Sanger and amplicon sequencing technique targeting cox1 DNA barcodes. The materials and methods are appropriate, the data analysis and presentation are adequate, and relevant discussion.
Validity of the findings: The paper is a step forward in understanding oomycetes communities in plant microenvironment with focus on Lagenidium, its possible ecological significance, and overall covers an important contribution to the literature.
Additional comments: However, there are few concerns need to be addressed before publication. First one, the transcriptomics and genomic information related to Lagenidium and links to the pathogenicity evolution highlight the importance of this study, however, is over-used throughout the manuscript diverting the focus of the MS. The results shown in MS are solely based on DNA barcode and detection via sanger or amplicon sequencing. I suggest reducing the information on genomics, transcriptomics and pathogenicity pathways throughout the MS, especially in the discussion section (Fx line 487-510). Second, the methods section mentions about nested PCR, which needs clarification. Please explain two sets of primers, overlapping region, amplicon size, thermal cycle, and the main purpose behind it. Also, giving a name to the newly designed primer will help readers.
 Another aspect is in the result section is that it is too descriptive. I suggest that you improve this section by reducing it, for instance, part of it describes methods, consider them to move in the materials and methods section. For example, line 279-results title says ‘Environmental detection of Lagenium spp’ but the actual result section starts from line 300.  Similarly, PacBio sequencing (line 322), this section is difficult to follow with lots of details which could be easily removed or move to a table (fx line 323-329).

Some specific comments
Consider adding a line describing Phytotelmata in the introduction.
Line 296-967, presence of band doesn’t mean that it is always oomycetes based on your amplicon results (sample P2)
Line 168 consider adding  ‘(NSU)’ after university
Line 365 remove the dot after sample


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: OOMYCETE METABARCODING REVEALS THE PRESENCE OF LAGENIDIUM SPP. IN PHYTOTELMATA
Review round: 1
Reviewer: 3

Output:
Basic reporting: Use of English: I don't consider myself qualified to do a thorough check, but I haven't detected any serious issues.

The background provided is enough and the literature is relevant but quite out of date, presenting only 7 references among 51 published in the last 3 years. There are more up-to-date works published based on metabarcoding approaches for oomycete identification, as the followings:
Sapkota, R. & Nicolaisen, M. Cropping history shapes fungal, oomycete and nematode communities in arable soils and affects cavity spot in carrot. Agric. Ecosyst. Environ. 257, 120–131 (2018).
Abdelfattah, A., Malacrinò, A., Wisniewski, M., Cacciola, S. O. & Schena, L. Metabarcoding: A powerful tool to investigate microbial communities and shape future plant protection strategies. Biol. Control 120, 1–10 (2018).
Ruiz Gómez, F.J., Navarro-Cerrillo, R.M., Pérez-de-Luque, A., Oβwald, W.,  Vannini, A., Morales-Rodríguez, C. Assessment of functional and structural changes of soil fungal and oomycete communities in holm oak declined dehesas through metabarcoding analysis. Sci. Rep. 9, 5315 (2019)

Raw data (FASTA files and abundance matrices) are not supplied.
Experimental design: The research is original but the experimental design does not provide enough evidence to support some of the considerations the authors stated in the discussion. The research question is quite methodological and the number of samples and the chosen controls are not enough to provide evidence of the association of >Lageindium spp. and the plants, nor even the phytotelmata. Authors analyze water DNA, and they declare there is a significant amount of invertebrate and biological debris in the water. The results do not support the statement that Lagenidium spp is able to colonize phytotelmata, nor the plant tissues.

Also, there is another issue along with the text. Authors confounded barcode with the amplified sequence on several occasions. A barcode in NGS is the short specific sequence added to a primer in order to allow multiplexing and ulterior demultiplexing of DNA libraries.
Validity of the findings: The work is whort to be published, but some conclusions stated are not supported by the results and in my opinion, the discussion should be modified.

The analysis of the abundance matrix is not carried out, nor the biodiversity indices calculus for the oomycete community.

Statistical results for the phylogenetic analysis are not provided.
Additional comments: No additional comments