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: MIDDLE EAST RESPIRATORY SYNDROME CORONAVIRUS AND THE ONE HEALTH CONCEPT
Review round: 2
Reviewer: 1

Output:
Basic reporting: This manuscript's grammar and flow has been significantly improved. However, I believe more editing will further improve this work. A few examples:

- Capitalization should be standardized (Middle East Respiratory Syndrome Coronavirus vs Middle East respiratory syndrome coronavirus)

- Line 78: "Two of them" should be reworded

- Line 105: "the" should be removed in front of MERS-CoV and SARS-CoV

- Line 148: Citations should be consistent throughout and either numbered or named. Italics throughout should also be corrected for consistency.

Line 277: The author should either list the brucellosis species of interest or change Brucella to brucellosis

This manuscript still needs to be further organized into smaller paragraphs so it is easier to follow. For example, Section 4 contains one paragraph that is over 25 sentences long and is nearly 2 pages.
Experimental design: The survey methodology and sources cited have been significantly improved. However, there are several areas that can still be improved.

Line 98: The author should remove the sentence about a sharp decline in research unless there is data supporting it. New manuscripts are frequently being released and I’m not sure that this is a fair characterization.

Line 103: One Health was first described in 400 BC and is not a new concept

Some of the data cited is still incorrect. The author should return to the original manuscripts to verify they are cited appropriately.
Validity of the findings: The author speculates about a missing animal reservoir; however, does not include any data to support this claim and has omitted several manuscripts exploring this topic through field sampling or experimental infections.
Additional comments: This manuscript has been significantly improved; however, I feel that more improvements are required. The addition of text expanding on One Health and MERS-CoV were valuable, and I think has dramatically improved the manuscript.


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

Input:
Title: MIDDLE EAST RESPIRATORY SYNDROME CORONAVIRUS AND THE ONE HEALTH CONCEPT
Review round: 2
Reviewer: 2

Output:
Basic reporting: This manuscript review the historical background related to MERS-COV and place some target priorities for future research on this important viral diseases. The review discuss possible routes of transmission and factors affecting the virus transmission and management.
Experimental design: Study design was poorly developed and require further details; the author identified PubMed Central, Google Scholar, and Researchgate as the literature repositories; however, they never refer to the search keywords or search algorithm implemented. Some overlaps may occur between these literature databases and author should make clear how they avoided similar overlap in literature and how much unique literature he obtained from each database. The author should show a flow diagram of literature search for systematic review. Finally, i suggest author to consider using PRISMA checklist and flowchart.
Validity of the findings: The review require much details; however, it is successfully identified some research-related gaps of MERS-COV.
Additional comments: Line 30: Authors stated "other unknown reservoirs". I think that only a very poor evidence is available for bats as reservoirs (i.e. very short sequence similarity); however, MERS-Cov was reported from camels both in vivo and in vitro. Then, what other reservoirs are possibly involved?

Line 35: Replace "ex vivo" to "in vivo".

Line 42-43: Author should provide more details to support this conclusion. I would suggest author to add a reference to these information.

Line 53-59: The introduction section is poorly developed and should provide further details on MERS-Cov. Introduction presented only objectives of the review and none of historical background was presented as stated by the authors on Line 63-64.

Line 64: Author should introduce the "One Health concept".

Line 79-80: "This is a relatively short period for the emergence of a new coronavirus". How authors draw this conclusion?

Line 87: Remove ", especially from the Middle East".

Line 91-92: Cite a reference to the statement "There are many uncertain aspects of the virus evolution, pathogenesis, and transmission cycle".

Line 93-94: I don't agree with this statement. I can see a lot of progress on MERS-cov research and the virus attracted attention by FAO. I think one of the main critique to this review is the absence of any of the available FAO reports.

Line 98-102: I think author can provide more details in this context; the information mentioned herein was not sufficient to link both MERS-cov and the "One Health concept". I would suggest the authors to go more deep to the role of animals as a reservoir, particularly in case of MERS-Cov.

Line 112: Do you really mean ex vivo tissue culture or "in vivo".

Line 129: I think these evidences are limited only to positive antibody samples, so, the virus antibodies only were reported but none of the virus RNA.

Line 156: Correct "it being recognized".

Line 167: Replace "This" with "these".

Line 203: Cite a reference here for this study.

Line 216-219: The presence of antibodies doesn't necessarily reflect the role of these animals as reservoirs; however, it reflects only historical exposures.

I think author should show a flowchart for the future research priorities.

Line 331-343: Should you consider the paper published by Reeves et al. 2015. I think that the authors of this publication touched some important points related to MERS-COV geography and epidemiology.


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: MIDDLE EAST RESPIRATORY SYNDROME CORONAVIRUS AND THE ONE HEALTH CONCEPT
Review round: 2
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: I’m really appreciate the great effort of the author in both selection of topics and text. The author try to complete the story of MERS-CoV, He started with immunological aspects and MERS-CoV vaccine in his previous article and here the author tried to summarize all items related to MERS-CoV (12 subtitle) in this report. This extensive summary made the subtitles are much wide than their text and overlapping was common in the text. The lack of focusing make the message unclear.
It is well known now that the main issue in MERS-CoV is the transmission mechanism and origin. I recommend that the author focus his review on these topics, it well be more useful.
I see that the one health concept was broken into the title. The one health context is absent in the report according to CDC definition ( https://www.cdc.gov/onehealth/ and One Health Initiative http://www.onehealthinitiative.com/about.php.)
The author avoid to deals with the genetic characteristics of MERS-CoV isolated in East Africa and The Arabian Peninsula which will be the key to understand the future outbreaks.
The expected evolution of the virus and its impact on humans must be involved in the report.

L27….. you wrote “MERS-CoV is considered an ideal example of the One Health concept” How?
L 88…. The total No. of MERS-CoV human cases needs to be updated to 2434.
L 233…. Transmission of MERS-CoV from dromedary camels to humans has been proven indirectly in
               some recent reports (Azhar et al., 2014). I think that 2014 not recent, please remove “recent”


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: MIDDLE EAST RESPIRATORY SYNDROME CORONAVIRUS AND THE ONE HEALTH CONCEPT
Review round: 3
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: All comments have been addressed properly.


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: MIDDLE EAST RESPIRATORY SYNDROME CORONAVIRUS AND THE ONE HEALTH CONCEPT
Review round: 3
Reviewer: 2

Output:
Basic reporting: The introduction is focused on the aims of writing this review and the text is collective to the knowledge needed for understanding of MERS-CoV dynamic. The structure of the article is good and simple.
Experimental design: The study within the aims and scope of the journal and the cited reference are adequate and sufficient.
Validity of the findings: The article revise many of the knowledge needed for MERS-CoV and its circulation in the Middle East and world.
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: PHYLOGENETIC ANALYSIS OF FRITILLARIA CIRRHOSA D. DON AND ITS CLOSELY RELATED SPECIES BASED ON COMPLETE CHLOROPLAST GENOMES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Genus and species names should be italicized.

1.There are very many stylistic issues in the references and citaion in manuscript.
2. In manuscript, 'chloroplast, plastome, plastid' words are uncertain. Authors must revise change.
3. Line 222, plstid genomes OR cp genomes...  NOT whole plastid genomes....
4. Line 283 Single copy NOT SC
5. Authors should be check for Peerj format.
Experimental design: pass
Validity of the findings: pass
Additional comments: The manuscript by Qi and colleagues reports on the determination of chloroplast genomes from Fritillaria species with detailed analysis of phylogenetic relationships with other Fritillaria of this genus. Overall the manuscript is clearly written, although there are a few parts of the text that require modification.

Q1: Line 291-300,  Expansion and contraction of other Fritillaria chloroplast genomes are indicated that difference results are compared to this paper. Authors should be check previous paper.

Q2: In section “Phylogenetic analysis”, more than half of them can’t be searched from NCBI. Especially, the species, F. cirrhosa, which the complete chloroplast genomes have been submitted to the NCBI, but they were not considered in this paper.

Q3: Line 441-451, authors mentioned about 'complex group of F. chirrhosa'. Recently, three papers for Fritillaria chloroplast genome analysis reported phylogenic analysis using chloroplast genoems. However, this results have weak informatiom. Also, complex group should be show repetition for same specise samples. This paper is one sample used for one speceis. Thus,  discussions are carefuly consider.

Line 301-305, this paragraphe must change below to line 308.
Line 313 petA-psbL, check the size of letters
Line 323 Full name should be define before using abbreviation.
Line 395 - 397 It should be remove that sentence metioned in material and methods.
Line 413 - 423 This paragraphe should be move to part of Introduction.


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: PHYLOGENETIC ANALYSIS OF FRITILLARIA CIRRHOSA D. DON AND ITS CLOSELY RELATED SPECIES BASED ON COMPLETE CHLOROPLAST GENOMES
Review round: 1
Reviewer: 2

Output:
Basic reporting: There are many incorrect expressions in the manuscript, for example Line 69, line 100, line 286, line 362, line389-400,  line 414 and etc. We strongly suggest the author to revised throughly. Second, the latin name should alway be italic type, however "var." should be upright type. There are several places need to be revised, such as line 98,  line 105, line 191, line 215 and etc. Furthermore there are other nonstandard place that marked in the pdf file need to be carelly revised.
Experimental design: One of the major conclusion in the manuscript is "this study did not support the so-called “complex group of Fritillaria cirrhosa”, 442 suggested by Luo & Chen (1996a) as a monophyletic group", according to Fig.8, we bellieve that it lack abundant proof.
Validity of the findings: The findings in this manuscript provide a new method to to accurately identify F. cirrhosa and its closely related species.However, in our opinion,  some of the closely related species                                                                                are also traditional Chinese medicine.The authors need to state clearly what is the new findings which can expand our knowledge about  F. cirrhosa
Additional comments: The findings has significance in identifying F. cirrhosa and its closely related species, however, we believe it lacks novelty. There are many mistakes and noncosistence in the writing which need significant revisions. And the gene number in Line 222 is not accurate as there are reduplicated genes which should be excluded.


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: PHYLOGENETIC ANALYSIS OF FRITILLARIA CIRRHOSA D. DON AND ITS CLOSELY RELATED SPECIES BASED ON COMPLETE CHLOROPLAST GENOMES
Review round: 2
Reviewer: 1

Output:
Basic reporting: This paper is well changed than 1st paper.
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: PHYLOGENETIC ANALYSIS OF FRITILLARIA CIRRHOSA D. DON AND ITS CLOSELY RELATED SPECIES BASED ON COMPLETE CHLOROPLAST GENOMES
Review round: 2
Reviewer: 2

Output:
Basic reporting: 1.The title in word version is not consistent with that in PDF version.
Experimental design: 'no comment'
Validity of the findings: 'no comment'
Additional comments: line 251, rRAN or rRMA?
lin 313-314 'the single copy regions' Font sizes are inconsistent.


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 VQ GENE FAMILY IN SOYBEAN (GLYCINE MAX)
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript shows professional organization and sufficient reference and background. All the data were analyzed and interpreted appropriately.  The figures and tables are shown with good quality.
Experimental design: Methods described in the manuscript is sufficient to follow. Research question well defined, relevant and meaningful.
Validity of the findings: The impact and novelty are suitable for the publication. Data was interpreted completely and supported the conclusion.
Additional comments: Overall, the manuscript contains sufficient data to support its conclusion. The organization of the full text is scientific and professional for publication. The authors need to consider polishing the language for a better understanding of international researchers.


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 VQ GENE FAMILY IN SOYBEAN (GLYCINE MAX)
Review round: 1
Reviewer: 2

Output:
Basic reporting: This paper titled “Genome-wide identification and expression analysis of the VQ gene family in soybean (Glycine max)” reported the VQ genes in soybeans, which may inform further research. Generally, the paper is with scientific validity and adequate data on the topic. The reviewer has a few minor comments for the authors to consider.

1. Please proofread to avoid topo or grammar mistakes. For instance,
Line 52-54: "GmVQ genes were expressed in nine tissues suggested their putative function in many aspects of plant growth and development, and response to stresses in Glycine max" to "GmVQ genes that were expressed in nine tissues suggested their putative functions in many aspects of plant growth and development, and response to stresses in Glycine max"
Line 85-86: "cultivated in the A number of countries" to "cultivated in a number of countries"
2. Kindly expand the figure legends with essential information such as statistical significance and sample size if necessary.
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: GENOME-WIDE IDENTIFICATION AND EXPRESSION ANALYSIS OF THE VQ GENE FAMILY IN SOYBEAN (GLYCINE MAX)
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comments
Experimental design: No comment
Validity of the findings: No comments
Additional comments: In this manuscript, the authors carried out the genome-wide identification of the VQ motif gene family in the soybean genome Glycine max. They identified 75 VQ genes. They then classified these members, analyse their cis-elements in their promoter regions, chromosome location and gene replication as well as protein interactions with WRKY proteins. They then mainly focused on expression analysis using publicly available expression data as well as their own qRT-PCR data. They tried to analyse expression patterns at different developmental stages and expression regulation under various abiotic stresses and hormone treatments. Generally, the authors carried out some bioinformation analysis by using publicly available genome annotation databases and expression databases. Their works may provide some useful information. However, VQ genes have been identified but the authors did not mention it. Some comments are listed as below.
Genome-wide identification and evolutionary as well as expression analysis of the VQ gene family have been carried out in many many plant species. In the soybean genome, at least two reports have been published on the genome-wide identification of the VQ genes: (i) Wang, X., Zhang, H., Sun, G., Jin, Y., and Qiu, L. 2014. Identification of active VQ motif-containing genes and the expression patterns under low nitrogen treatment in soybean. Gene 543(2):237-243; (ii) Zhou Y, Yang Y, Zhou X, Chi Y, Fan B, Chen Z. 2016. Structural and Functional Characterization of the VQ Protein Family and VQ Protein Variants from Soybean. Sci Rep. 2016 6: 34663. The reference Wang et al (2014) was listed but was wrongly cited. The authors did not mention that the VQ gene family members were identified and were reported in at least these two references.
By raw comparison between the reference by Zhou et al (2016) and this study, the former identified 74 VQ genes and this study identified 75 genes. In chr2, chr4 and chr17, Zhou et al (2016) identified 2, 5 and 1 VQs but this study identified 1, 6, and 2 VQs. The authors at least need to make a comparison among published VQs and their identified VQs. Acturally, the 75 VQs can be easily available in the Phytozome website (https://phytozome.jgi.doe.gov/pz/portal.html#!results?search=0&crown=1&star=1&method=4433&searchText=vq&offset=0), which need to be further verified.
The only valuable data should be the expression data under various abiotic stresses or hormone treatments by qRT-PCR, which may be useful for someone who are interested in these VQ genes for their functional characterization.
Wang et al (2015) was cited but no reference was listed.
Wang et al (2017) was cited but two references were listed.


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 VQ GENE FAMILY IN SOYBEAN (GLYCINE MAX)
Review round: 1
Reviewer: 4

Output:
Basic reporting: See comments
Experimental design: See comments
Validity of the findings: See comments
Additional comments: In this work, the authors used bioinformatics tools to analyze the VQ proteins from Glycine max, and determined their expression level in different abiotic stress conditions.

The manuscript is difficult to follow as almost every other line in the paper has one or more English errors. Please thoroughly proof-read the manuscript and use unambiguous and professional English. For most part the methods are sufficiently detailed but at places require some clarification as noted in general comments below. Overall, the manuscript requires major revision and can only be considered again provided authors address my concerns listed below:
1)	 Introduction needs major improvement and should be rewritten as sufficient background is not provided to determine significance of the work. Previous literature characterizing the VQ protein from Glycine max has not been discussed (see for example: doi: 10.1038/srep34663). Authors should clarify how the present work adds to what is already known in the field.
2)	On lines 55-56 and on line 91, “The present study provided basic information for further analysis of the biological …”. I find this statement vague. What do authors mean by basic information here?
3)	On lines 96-99, “HMM searched VQ motif (PF05678) from the G. max protein database with values (e-value) cut-off at 1.0 (Punta et. al, 2012). The integrity of the VQ motif was determined using the online program SMART (…) with an e-value < 0.1”. It is not clear what authors want to say here. Can authors elaborate on why different e-values is being used.
4)	On line 105, “… VQ protein sequences were downloaded from phytozomes….”. It is not clear why the sequences retrieved using Pfam, as described in previous section under “Identification of VQ gene information”, were not used for phylogenetic analysis.
5)	On line 106, “…VQ genes were aligned …”. Since protein sequences were downloaded, I assume author aligned VQ proteins and not VQ genes.
6)	On line 107, “… neighbor-joining (NJ) phylogenetic tree was constructed using the ….”. Was the tree constructed using the amino acid sequences downloaded from phytozomes?
7)	On line 143, version number for the R software used should be provided.
8)	On line 183, “We identified 75 VQ genes in soybean genome…”. Authors should expand on how these 75 genes were identified? Previous studies (see for example: doi: 10.1038/srep34663) have also reported 74 VQ proteins in G. max. Authors should comment on whether the sequences identified here are similar/different from those previously characterized.
9)	On line 192, “…they were divided into 7 groups, designated …”. Authors should expand on how the 7 groups were identified.
10)	On line 195, “…we found 5 types of VQ specificity domain (Fig. 2)”. From figure 2, it is not clear what 5 types of specificity domains author is talking about. They should elaborate more on what these domains are and their possible significance. Also, what do different colors mean in Figure 2? This should be added in the figure legend.
11)	On lines 199-200, “….all the proteins excepted GmVQ22 shown motif 1, which is contained a specialty VQ domain.” Isn’t the presence of VQ motif a characteristic of VQ proteins? If so, why is this protein classified as VQ protein without this motif?
12)	In figure 3A, why is group VII split into two branches? Shouldn’t these sequences then be classified as two separate groups based on the tree presented in this figure?
13)	Authors should add to Figure 4 legends what magenta lines in the figure represent.
14)	On line 219-220, “…we found that 31 pairs of genes originated from segmental duplication, and 4 pair of genes involved in tandem duplication events.” Authors should elaborate on how they arrived at this observation.
15)	 On lines 224-225, authors should describe how they found the paralogous and orthologous pairs.
16)	On line 242, authors should describe how the 25 genes were selected for abiotioc stress response determination.
17)	On line 274-275, “….our results proved GmWRKY is similar to AtWRKY and those proteins can interacted and VQ protein”. Authors should understand that they are not proving anything here. The interactions shown are merely predictions which are not experimentally verified. Also, authors should elaborate on why they think GmWRKY is similar to AtWRKY. Authors show sequence comparison in Figure 12, but do not discuss it in the manuscript.
18)	On lines 286-288, “These proteins are highly conserved because of shorter branches and closer spacing, we assume that they have similar functions”.It is not clear what authors are trying to convey here.


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 VQ GENE FAMILY IN SOYBEAN (GLYCINE MAX)
Review round: 2
Reviewer: 1

Output:
Basic reporting: Good reporting with clarity
Experimental design: Well defined
Validity of the findings: The findings are validated and novel
Additional comments: The authors have taken care of my comments and the manuscript has improved from previous version


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 VQ GENE FAMILY IN SOYBEAN (GLYCINE MAX)
Review round: 2
Reviewer: 2

Output:
Basic reporting: NA
Experimental design: NA
Validity of the findings: NA
Additional comments: Authors have addressed all my concerns. However, the manuscript still lacks in professional English with several grammatical mistakes. I would recommend publishing the manuscript provided authors proofread and correct for mistakes in English.


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: TWISTED TALE OF THE TIGER: THE CASE OF INAPPROPRIATE DATA AND DEFICIENT SCIENCE
Review round: 1
Reviewer: 1

Output:
Basic reporting: I few references provided in the text need to be clarified:
Line 65: “…Jhala et al. (2011)…” is it a or b? Clarify please.
Lines 119-120: “…Karanth et al. (2011)…” which one a or b? Clarify please.
Lines 127-128: “…Karanth et al. (2011)…” which one a or b? Clarify please.

Some important information is missing in the supplementary material. This would also ease the work of the reviewers. Please add the information regarding abundance estimates by means of photographic capture-recapture for Pench-MP, Nagarahole, Kaziranga, Kanha and Tadoba in the suppl. material. Same comment for the encounter rate for Pench-MP, Nagarahole, Bandipur (the value provided in Fig. 1 of the present manuscript does not correspond to the value provided in the suppl. material; see also my comment Figure 1; clarify please), Kaziranga and Kanha.
Experimental design: Although "experiments" in Bandipur, Melghat and Pench-MR have lags of several years between estimating tiger densities and tiger signs and hence should not be included in the experiment/graph (e.g. Figure 1), they do furthermore not match the original source. Clarify please and correct where necessary..
Validity of the findings: The manuscript does not answer if double sampling to establish relationship between any index of abundance and actual abundance is a worthwhile effort. The argumentation would gain in strength if the theoretical model e.g. developed by Gopalaswamy et al. (2015) would be parameterized using detection probability of tiger signs instead of detection probabilities to predict the R2 statistic based on the theoretical models and if data that demonstrate that there is in fact a strong positive relationship between tiger abundance and scat index would be presented. I encourage to include these aspects in the present manuscript.
Additional comments: Major comments
Lines 125-127: The detection probability varies with the size of the sample unit (e.g. 225 km2 in Barber-Meyer et al. 2012; 166 km2 in Harihar and Pandav 2012; 188 km2 in Karanth et al. 2011a) and the definition of the sampling occasion (e.g. Barber-Meyer et al. maximum of 40 km was surveyed per grid cell with each contiguous 1-km segment considered a ‘spatial replicate’; according to Harihar and Pandav maximum survey distance as 40 km, the data were recorded along segments of 250 m as either detected or not-detected, detection histories were constructed for each cell by aggregating signs along 250 m segments at 1 km to form ‘replicates; Karanth et al. 2011b fixed the total sampling effort (distance walked) in a cell with 100%habitat at 40 km, each type of sign detection was assigned only once to each 100-m trail segment, thus yielding the standard ‘1’ (detection) or ‘0’ (nondetection) histories required for occupancy analyses. These sign detection data were aggregated at 1 km length to form ‘spatial replicates’) considered in a given survey/study. Hence when attempting to compare detection probability estimates across multiple studies it is usually necessary to make sure that the per occasion sampling effort is standardised across sampling areas. Please make sure that the detection probabilities are comparable. If not please adjust them according to the procedure described e.g. in Gopalaswamy et al. (2015).
Line 150: Scats were sampled in 2006 in Bandipur according to the information provided in the supplementary material. Correct please.
Lines 177-181: It does not make sense to calculate a coefficient of determination with only 4 points (e.g. Figure 1 in the manuscript). Ideally there should be at least 10 points. More over only three data points do not qualify and there is no reason not to include the value provided in the original source (namely 1.99/10 km) of the extreme outlier at the right corner of figure 5 in the calculation of the coefficient of determination! If this value is included the coefficient of determination will be lower (athough the sample size is still too low). Although "experiment" in Bandipur, Melghat and Pench-MR have lags of several years between estimating tiger densities and tiger signs and hence should not be included in the experiment/graph, they do furthermore not match the original source. Clarify and adjust the values where necessary (see my comment Figure 1).
The argumentation would gain in strength if it would be possible to demonstrate that there is in fact a strong positive relationship between tiger abundance and scat index. Ideally data (abundance and index collected contemporaneously and over the same spatial extent) collected according to the same design and in the same landscapes (as it is possible that scat detectability varies from one landscape to the other) showing that there is in fact a strong positive relationship between tiger sign index and tiger abundance should be presented (see also my comment "validity of the finding").

Figure 1: I see some additional discrepancies in the values presented in Figure 1. According to the information presented in the suppl. material the encounter rates (scats per 10 km) are 2.36 in Melghat (year 2005), 2.12 in Bandipur (mean encounter rate of scats; year 2006) and 2.4 in Penchar-MR (year 2004), which differ from the values presented in Figure 1. Clarify please and adjust where necessary.

Minor comments
Line 67: a space is missing before “cherry picked”
Line 70: I am not sure if “incorrect” (per se the parameter namely occupancy is correct) is the right term here. I would rather say that they used an inadequate parameter, namely occupancy, as surrogate for detection probability of tiger scats.
Line 119: What does CV mean? Coefficient of variation!? You should write the full name the first time it appears in the text.


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

Input:
Title: TWISTED TALE OF THE TIGER: THE CASE OF INAPPROPRIATE DATA AND DEFICIENT SCIENCE
Review round: 1
Reviewer: 2

Output:
Basic reporting: It is not often that one gets to review a paper such as this with all the ingredients of a scientific pot-boiler –famous names in wildlife ecology, potential statistical skulduggery, arguably a creature with the greatest mystique in human history, and national honour over its status at stake. Qureshi at al. may perhaps not have been so rhetorical over their criticism of the Gopalaswamy et al. paper had the media, ever alert for juicy stories on failure of the system, had not jumped into the fray, or if one of the co-authors had not given an interview to a prominent newspaper questioning tiger numbers and their monitoring in India. The latter’s paper may then have passed off as merely one that contributed to a staid scientific debate on animal population estimation methods. As matters stand, however, the paper has generated a lot of interest, publicity and controversy, and it would be in the interest of tiger conservation to set the record straight.

My task as a reviewer is not to do a thorough dissection of the Gopalaswamy et al. paper itself (which has gone through the process of peer-review), especially the theoretical basis of their statistical models, but to examine the claims in Qureshi et al. paper on the biases in the former which could render their conclusions infructuous. In other words, is there a strong enough case for publishing the Qureshi et al. paper in PeerJ?
Experimental design: Not relevant
Validity of the findings: Qureshi et al. make six basic observations to refute the conclusions of the Gopalaswamy et al. paper. I shall provide my assessment of whether these observations are valid or not for each of the six points.

a.Use of incorrect ecological parameters
Yes, it certainly seems that Gopalaswamy et al. have incorrectly used detection probability from tiger occupancy studies, rather than a double-blind observer design, to parametrize their theoretical model as stated by Qureshi et al.

b. Cherry picked references
Ostensibly, Gopalaswamy et al. have confined their model analyses to data from two sets of studies – Karanth and team, and Jhala and team – because these constitute the major tiger population estimation exercises from India in the past decade or more. However, Qureshi et al. have a valid point in that results of detection probability and coefficient of variation from at least two peer-reviewed papers on tiger estimation published in reputed journals have been (conveniently?) omitted by Gopalaswamy et al. thus rendering their conclusions less than robust.

c. Inappropriate and wrong data
The most telling criticism of the Gopalaswamy et al. paper is that they use invalid data (in Figure 5) on tiger count and tiger sign intensity because at half the sites their data have been collected years apart or wrongly reported (from one site). In particular, a 7-year time separation of tiger count and sign data from Bandipur would certainly make this data point invalid for deriving R2 as tiger abundance at this site could have varied considerably during this period. I do not have access to the Karanth & Kumar (2005) report from which a scat encounter rate of 1.99/10 km has been taken and reported as 3.6/10 km in Gopalaswamy et al. (2015). If this were true (and I am assuming that Qureshi et al. have been diligent in their homework and assertion), Figure 5 is indeed completely useless in deriving R2 for the tiger count-sign relationship, even if we condone the 2- or 3-year gaps in measurements at the other two sites, Pench and Melghat. Indeed, Tadoba would shift much further left along the X-axis and strengthen the value of R2 substantially. It is of course possible that Gopalaswamy et al. discovered an anomaly in the original figure for Tadoba reported in Karanth and Kumar (2005), but in this case they should have made this explicit in their paper.  However, Qureshi et al. should also not take a R2 value of 0.642 (lines 180-181) for only four valid data points too seriously. They have already made their point about the inadequacy of Figure 5 in Gopalaswamy et al. In fact, it would be interesting if they could compute R2 after merely changing the “outlier” Tadoba value to 1.99.

d. Variability in tiger capture …. mark-recapture
While I cannot make a judgment about the reasons for low precision in Karanth et al. (2004), Qureshi et al. are correct in saying that Gopalaswamy et al. should not have restricted their model to examining only data from Karanth et al. (2004).

e. Repeating non peer-reviewed literature
Yes, the statement in Gopalaswamy et al. about the improbability of a 49% increase in tiger numbers over four years has been made without a proper assessment of what the 2006 and 2010 estimates actually represent. More sites were added for the tiger population estimation in 2010 and this should have been factored in while making statements questioning the rate of increase.

f. Propaganda that is not consistent with facts
It is unfortunate that these days even scientific journals need catchy “blogs” to capture the attention of their readers. In this case, the blog in Methods in Ecology and Evolution casting doubts on tiger population trends was unjustified. Subsequent articles in the media and even journal papers on the subject have slated towards a negative view of India’s tiger assessment. Even though this is not a scientific issue, Qureshi et al. are aggrieved by the negative publicity to the outcome of a rather challenging task of estimating tiger populations on such a large scale.
Additional comments: One piece of advice for Qureshi et al. is that they should not fall into the same trap of high rhetoric. They have made their case against Gopalaswamy et al. very clearly. I suggest some minor edits to remove the rhetoric – the edited paper would still retain its scientific punch!

Suggested list of edits:
Line 18: “double sampling”
Line 19: large scale animal surveys
Line 20: replace “elegantly” with “potentially” [after all, the statistical debate is not necessarily over]
Line 21: should be “Gopalaswamy”
Line 28: Could I suggest rewriting as “selectively-picked estimates from the literature”?
Line 29: suggest replacing “suspect” with “questionable”
Line 31: suggest “statistical design of large scale animal surveys”
Line 46: suggest “basic competence of scientists”
Line 48: “a peer reviewed publication”
Line 55: suggest “of the scientific method probably more than others”
Line 59: suggest “supposedly demonstrate”
Line 64: suggest “In particular, they claim the relationship…”
Line 67: suggest “selectively picked references from the literature” [the media can report this as cherry picked!]
Lines 88-90: Should be a single sentence with a comma (occupied site, while)
Lines 96-99: Should be a single sentence with a comma (Nichols et al. 2000), where two observers)
Line 107: “the use of occupancy detection”
Line 114: again “Selectively picked references”
Line 120: Should this be Karanth 2011a?
Line 139: Gopalaswamy
Lines 179-181: Suggest rewriting this part taking into consideration my suggestions given above (C. Inappropriate and wrong data)
Line 189: Which model is being referred to? Model 1 or Model 2?
Line 200: suggest “adoption of sound and practical methods”
Line 235: The reference states Jhala et al. 2014.
Line 238: same as previous
Line 243: I am wondering the word “propaganda” can be replaced with another, equally effective word? Distortion? Distorted reports? Misleading reports?
Line 243: suggest “MEE paper by Gopalaswamy et al.”
Line 248: Gopalaswamy
Line 252: I wonder if this line is needed? PeerJ editors can decide.


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: TWISTED TALE OF THE TIGER: THE CASE OF INAPPROPRIATE DATA AND DEFICIENT SCIENCE
Review round: 1
Reviewer: 3

Output:
Basic reporting: The language used in the manuscript is too emotive. I think it needs a substantial rewrite to focus on the issues of the science in Gopalaswamy et al. exclude suggestions of deliberate falsification of data - just say the data quality undermines their results.
Experimental design: I think a table of RAI (though not a full scale review) showing examples of fits between indexes and independent abundance estimates would greatly strengthen the author's case here and wouldn't involve a lot of extra work.
Validity of the findings: This manuscript focuses on responding to G et al's critique of their earlier work. It would be improved and strengthened by first concisely dealing with references to their work but then by comparing other RAI-based findings to broadening the perspective (see above).
Additional comments: Review: Twisted tale of the tiger: the case of inappropriate data and deficient science.
Quershi et al.

Summary:
The use of indexes in animal abundance surveys clearly divides researchers – in fact, I notice a number of the authors publishing in this literature, have co-authored both for and against views on the subject! The present manuscript is a (somewhat delayed) response to Gopalaswamy et al (2015) who - a) present a simulation model to support the assertion that index-based approaches are unreliable and ineffective, and b) use the simulation to argue that the results of Jhala et al (2011) with a reported high r2 between an index of tiger sign and tiger density - is likely to be spurious.  Gopalaswamy et al critique was very personal and harsh in their criticism of Jhala et al., and given this I can understand why the present manuscript uses highly charged language. But I still I think the sections on scientific integrity and short-comings of peer review do not help the authors make an effective case. That said, I think a shortened and more focus response to G et al, is important and would help to clarify differences of view in the literature and perhaps could allay wider concerns about Indian tiger population estimates.

Main Points:

1)	The manuscript would be greatly improved if the authors removed sections on scientific integrity etc. e.g. remove first two sentences in the abstract. Lines 41-46; lines 53-55; simply refer to manuscript published in MEE, remove prestigious journal etc. Remove all phrases like “cherry picking” “highly suspect” it needs to be written as If the authors of G et al. may have been in error but not scheming to undermine J et al.
2)	A large part of G et al’s paper focuses on criticisms of RAIs. Could the authors here provide a table of examples of RAIs to give readers a better sense of typical fits to independent estimates of density. The most appropriate one I can think of which matches the case of Jhala et al 2011 is Funston et al 2010 (referred to in G et al, but not in the text strangely) – which uses a spoor count based RAI. But others use for photographic rates to provide RAIs e.g. O’Brien et al (2003); Rovero et al (2009) and Palmer et al (2018). Provide more evidence for better fits between indexes and actual density estimates. Demonstrating that indexes work in many settings, might be used to support the present manuscripts assertion that G et al’s model is based on overly pessimistic parameter values.
3)	It was really good to see the authors clarify estimates of percentage of increase in the tiger population correcting for increased survey area. However, it would be also useful to know what percentage of the total tiger population estimate is obtained from the double-counting GIS extrapolated estimate. E.g. what perc is dervided from tigers in core areas where SECR methods were used compared to the remaining population. Reading the note published by Karanth et al (2011) one could assume that all (or most) the tiger estimates were based on extrapolations from indexes. Are we talking about <20%?
4)	The authors make an interesting point about the use of occupancy statistics for G et al simulation. Could they clarify a bit further why this might not be appropriate?
5)	It may also be worth pointing out that the Karanth et al (2004) paper comparing tiger density against prey density, was based on tiger density using MMDM method and not SECR. Might this have contributed to the poorer fit between the two?


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: TWISTED TALE OF THE TIGER: THE CASE OF INAPPROPRIATE DATA AND DEFICIENT SCIENCE
Review round: 1
Reviewer: 4

Output:
Basic reporting: Detailed review provided in attachment.
Experimental design: Detailed review provided in attachment.
Validity of the findings: Detailed review provided in attachment.
Additional comments: Detailed review provided in attachment.


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: TWISTED TALE OF THE TIGER: THE CASE OF INAPPROPRIATE DATA AND DEFICIENT SCIENCE
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comments
Experimental design: adiquate
Validity of the findings: they are valid
Additional comments: The authors have addressed my comments satisfactorily.


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: TWISTED TALE OF THE TIGER: THE CASE OF INAPPROPRIATE DATA AND DEFICIENT SCIENCE
Review round: 2
Reviewer: 2

Output:
Basic reporting: Overall this revision is much improved and much clearer.
Experimental design: NA
Validity of the findings: See comments below
Additional comments: I find the revised version much improved, clearer and more concise and a more effective response to the original paper published by Gopalaswamy et al in MEE.
One point I would make is it would be great to have a paragraph from the authors, stating in broad simple terms, how they think the errors highlighted in this critique, have affected Gopalaswamy et al's results. I assume it means they are over critical of double sampling method, but it would be good to hear what the authors think in plain simple terms.. near the end of the paper.
Other than that I only have a number of minor edits to recommend (below)
L25 - abstract - show lags of (up to) several years?
L28 - change "wrong" with poor?
L76 - "tiger occupancy studies to surrogate detection probability" change to as a surrogate for..
L133 - "noticed several irregularities which invalidate these data as a scientific experiment." change to - which invalidates the use of these data to test this relationship?
L148-149 - Should you use Gopalaswamy et al 2012 in your revised figure or do you already?
L156 -" form" change to from
L174 - "experiment (IC-Karanth) to say the least are questionable" - I would say this more strongly - mentioning that this invalidates their conclusions.
L179 entire section: This section on variances in Karanth's tiger population estimates is the weakest bit to me. It seems like you are making criticising their work for the sake of it. Putting aside existing tensions, I wonder if this really contributes to this rebuttal. I think it doesn't unless you can make a clear case for why this contributes to the flaws in interpretation of G et al.
L220-1 - "for none of the national tiger status assessments (Jhala et al 2008, 2010, 2015) was tiger sign index solely used for modeling tiger abundance" rephrase - they should know that NTSAs are never based on tiger sign indexes alone..
L236-244- It is great to see that you now start to spell out the issue of what percentage of the population  is based on actual individual tigers photographed, and then how many are extrapolated through SECR, It would STILL be good to know how many are estimated from the marginal areas through your double sample method that Gopalaswamy and co are so unhappy with. What is this percentage? This would great help readers and conservationists alike understand the scope of the the uncertainty.
L248-51- "It is surprising that with all of the above mentioned inappropriateness of data that do not meet the experimental setup for testing double sampling, incorrect theoretical concepts, and selective referencing of literature the Gopalaswamy et al. (2015a) paper passed through the peer review process." It is not that surprising - most reviewers/editors normally assume the data is carefully collected and compiled. I would cut this sentence, but go on with your discussion about your view that G et al has not made a useful contribution to the debate over the use of double sampling in very large-scale surveys
L263 - "(2015a,b) due to inappropriate scientific process and data, has not contributed on" - perhaps rewrite to say, it has that G et al's results have been misleading and therefore do not make a useful contribution to the wider debate ..


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: TWISTED TALE OF THE TIGER: THE CASE OF INAPPROPRIATE DATA AND DEFICIENT SCIENCE
Review round: 2
Reviewer: 3

Output:
Basic reporting: No background or context is provided. I have suggested that the background information provided in the cover letter is moved to the main text. I have also provided ideas for the background in my previous review.

There is no hypothesis for the paper either. I have recommended that the authors ask a clear question right in the beginning.
Experimental design: This is not a primary research article. It is only a critique of an article by Gopalaswamy et al. (2015a,b). There really is no research question either.
There are many flaws in the arguments posed and several new evidences about the topic are not included (eg: Jhala et al. 2015). So there are many scientific contradictions.
There are many technical mistakes in the manuscript as well.
Validity of the findings: There are no data used in this manuscript. It is an attempted critique of Gopalaswamy et al. (2015a,b), with no real question behind that critique. The results are highly contradictory.
Additional comments: I would recommend that the authors pose their study question very carefully and clearly. And then I would recommend that they use sound statistical methods to demonstrate their case. Even if this is a critique of Gopalaswamy et al. (2015a,b) and not an original paper, they can write it as an original paper by demonstrating the whether the perceived data errors made in Gopalaswamy et al. (2015a,b) proved to be costly for the tiger sign and density relationship or not. For example, after Jhala et al. (2015) was published, which completely contradicted Jhala et al. (2011), they can show the similarity in the results between Jhala et al. (2015) and Gopalaswamy et al. (2015a,b). But they need to put in a lot of work and must not be fixed with a view that they have to verbally defend Jhala et al. (2011) and Jhala et al. (2015) even though they scientifically are at odds to one another.

-----------------------------------------------------------------------------------

INTRODUCTION

This is a review of the re-submitted version of the non-peer-reviewed manuscript titled ‘Twisted tale of the tiger: the case of inappropriate data and deficient science’ (Qureshi et al. 2018). I had earlier provided a detailed review of Qureshi et al. (2018) and it is also made available online (Gopalaswamy 2019). To avoid repeated use of pronouns and determiners, I will henceforth refer to this revised manuscript as Qureshi et al. (2018-V1). The corresponding author (Dr. Y. V. Jhala) has on behalf of the rest of the authors also written a covering letter to the PeerJ AE, with some relevant material on the topic and I will henceforth refer to this document as Jhala-Letter (2019).

Qureshi et al. (2018-V1) do not address any of the scientific contradictions and confusions discussed in Gopalaswamy (2019). The authors do not even provide scientific explanations about why they chose to ignore or accept each point raised by Gopalaswamy (2019). Consequently, all these scientific shortcomings persist in Qureshi et al. (2018-V1) and sure they do!

Qureshi et al. (2018-V1), however, have reduced the polemics that was present in Qureshi et al. (2018) as per recommendations from all the reviewers and the AE. But now, the authors have introduced a lot of new material related to the general topic about the failure of peer-review processes in Jhala-Letter (2019) instead of including all this material in Qureshi et al. (2018-V1). I have provided comments to the Jhala-Letter(2019) separately as an annotated document.

GENERAL COMMENTS

Since Qureshi et al. (2018-V1) ignore all the scientific recommendations and concerns expressed in Gopalaswamy (2019), virtually all the scientific inadequacies persist so my suggestion is for the authors to address/respond to them first.

The concerns raised and the excitement generated by Qureshi et al. (2018-V1), especially about the relationship between tiger sign indices and their respective densities, would have been scientifically relevant, for a brief period, about four years ago (i.e. between the time Gopalaswamy et al. (2015a,b) was published and the time Jhala et al. (2015) national tiger assessment report was released). Since Jhala et al. (2015) convincingly validates the findings of Gopalaswamy et al. (2015a,b), this part of the arguments developed in Qureshi et al. (2018-V1) becomes scientifically irrelevant.
To see this, I would advice the authors of Qureshi et al. (2018-V1) to study the mathematical derivations of Gopalaswamy et al. (2015a,b) carefully and then confront these derivations with the findings from Jhala et al. (2015) to see what they get before proceeding with their arguments and generating hasty conclusions.

The authors share some details via Jhala-Letter(2019) on how the journal Methods in Ecology and Evolution (a journal of the British Ecological Society) lacked the desired amount of transparency on the matter related to the publication of Gopalaswamy et al. (2015a,b). These are, of course, serious concerns about the procedures followed by such reputed journals and must be included as part of the main manuscript (Qureshi et al. 2018-V1) rather than be exclusively a letter to PeerJ. However, if they decide to include contents of Jhala-Letter(2019), they should share all the relevant facts and not a subset of them so that the readers can appreciate the flaws in the peer reviewed system, if any.  Similarly, I would recommend that they include how the journal Journal of Applied Ecology, failed to detect data issues related to the publication of Jhala et al. (2011) – details mentioned in Gopalaswamy (2019), which has now proven to be costly (Jhala et al. 2015, Gopalaswamy et al. (2015a,b)).

More generally, as mentioned in Gopalaswamy (2019), I would emphasize the need for the authors to critically assess what their question really is. Without a clear scientific question, the authors will lose track of what they are seeking to really answer. And at this point, Qureshi et al. (2018-V1) reads more as a set of disjointed and random allegations rather than a set of coherent arguments seeking a specific answer to a scientific question.

SPECIFIC COMMENTS

I only have a few of technical additions to make to Gopalaswamy (2019):

l.62-74: This explanation for double-sampling is inadequate to the cause taken up by Qureshi et al. (2018-V1) there is also a population level variance associated with the index for the tiger cases. It is not limited to the ‘bias’ they are discussing here. So this section is completely irrelevant and misleading.

l.75-92: This refers to the identity described in Royle and Nichols (2003) for the relationship between p and r. The authors provide a flawed description of this identity. So I would suggest they first read up this paper fully and then work through the mathematics before putting anything in writing and causing a lot of confusion. If the authors do not want to put this effort, I would suggest they simply delete this entire section.

l. 101-104: It will be faulty to include any double-sampling citation here (whether it is Cochran 1937, Eberhardt and Simmons 1987, Pollock et al. 2002 or even Conroy and Carroll 2009). This is because all of these statistical approaches either involve a design-based approach (eg: Cochran 1937 or Eberhardt and Simmons 1987) or a model-based thinking (eg: Pollock et al. 2002 by finding a way to estimate detection probability). Since, India’s tiger assessments (Jhala et al. (2008), Jhala et al. (2011b) or Jhala et al. (2015)) are neither formally a design- or model-based in such a context, all these citations are irrelevant to the discussion here. And Jhala et al. (2011a) is purely an index-calibration experiment.

l. 239-242: These estimates are flawed (see the statistical sections in Gopalaswamy et al. 2015a,b) and juxtapose these on Jhala et al. (2015) to see what happens.

In general, if the authors are not comfortable with all the statistics involved in these studies, I would recommend they simply delete these sections and focus only on what they know well.


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 CHARACTERIZATION OF THE HSP70 GENE FAMILY IN ALLOPOLYPLOID RAPESEED (BRASSICA NAPUS L.) COMPARED WITH ITS DIPLOID PROGENITORS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The professional and appropriate English language is used throughout. The figures are relevant, high quality and well described.The introduction session provided sufficient literature, while the list of references is not in the style of Peer J, the DOI for references should be added according to the guideline Authors.
Experimental design: The research question is well defined and meaningful, which contribute to exploration of the evolutionary adaptation of polyploidy, and methods described with sufficient detail and information to replicate.
Validity of the findings: The manuscript (MS)provides a genome-wide systematically analysis of Heat shock protein 70 (Hsp70) in allopolyploid rapeseed and its two diploid ancestors. In this study, 47 Hsp70 genes in B. napus genome, 29 members in B. rapa and 20 in B. oleracea were identified based on the published genome sequencing results, gene structure, chromosomal distribution and duplication pattern, cis-acting elements and expression pattern in four tissues of Hsp70 genes were conducted. It’s very interesting that this manuscript described the tissue-specific and C-subgenome biased expression of HSP70. These findings contribute to exploration of the evolutionary adaptation of polyploidy and will facilitate further application of BnHsp70 gene functions.
Additional comments: This is a carefully done study containing interesting results, I think that the research developed in this manuscript could be worth publishing after considering these points:
1) Although this is the first report of Hsp70 gene family in B. napa, there are some reports in the other crops as mentioned in the introduction. The authors should emphasize the novelty of the characters of Hsp70 gene family in B. napa compared with others.
2) As the manuscript described that Hsp70s is indispensable in plant development, as well as associate with plant stress resistance. However, authors didn’t describe the specific function of Hsp70s members involved in developmental processes and stress responses. I think it is very necessary to characterize the role of Hsp70s in development, drought resistance, heat tolerance and other abiotic stresses.
3) The organization of the abstract is a little vague and unclear. For example，I don’t think “many evidence”(line 38) appear in the abstract is appropriate, which should be replaced by the method used. Another, “all known 114 proteins” (line 37) should be indicated the genome source, which is easily misunderstood that they are derived from three species (B. napus, B. rapa and B. oleracea).
4）“Arabidopisis” should not be italic font in lines 70, 117, 128, 222, 226, 250, 376, 472, 482, 486.
5）“The orange boxes” should be “The green boxes” in Figure 2 legend.
6）The singular and plural forms of some words need to be modified. For example, in line 68 “have” should be “has”, line 69 “are” should be “is” and in line 330 “numbers” should be “number”.


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 CHARACTERIZATION OF THE HSP70 GENE FAMILY IN ALLOPOLYPLOID RAPESEED (BRASSICA NAPUS L.) COMPARED WITH ITS DIPLOID PROGENITORS
Review round: 1
Reviewer: 2

Output:
Basic reporting: No Comments
Experimental design: No comments
Validity of the findings: No Comments
Additional comments: Authors have conducted comprehensive analysis of Hsp70 gene family in rapeseed and its related diploid species. This analysis included detail information regarding gene structure, phylogenetic analysis,chromosomal distribution, expression pattern of HSP70 gene family and have provided important information regarding its evolution. This study also provided information regarding impact of polyploidy in the expansion of HSP 70gene family. Overall this study is of high scientific value and provides new and unique information regarding important gene family.


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 CHARACTERIZATION OF THE HSP70 GENE FAMILY IN ALLOPOLYPLOID RAPESEED (BRASSICA NAPUS L.) COMPARED WITH ITS DIPLOID PROGENITORS
Review round: 1
Reviewer: 3

Output:
Basic reporting: SPECIFIC COMMENTS:

ABSTRACT:
line 22: 47 BnHsp70 genes in B. napus. I think it is important to precise how many genes are on subgenome C or A.
line 25-26: sentence unclear: presence of orthologous (12 and 13) in which subfamiliy ?
It means also that on the 47 BnHsp70 genes only 25 are orthologous to parental genes. So, 22 are news duplicated genes in B. napus? If the case, that must be specified in the introduction. That means also that 17 B. rapa and 7 B. oleracea genes were not found in B. napus genome and lost.

INTRODUCTION:
line 52: missing space between in agriculture
At the end, the introduction needs more details :
- line 58: identification of Hsp70 genes in B. napus and parental genomes with a specific domain: which one ?
- line 71-84: What is the size of Hsp70 proteins? All the proteins have the two domains ?
- line 90-91:
duplicated in the corresponding genome : which ones ?
synteny between three genomes : which ones ? There are four genomes used : B. napus compared with A. thaliana, B. oleracea or B. rapa ?
- line 93: four tissues : indicate which one

FIGURES:
- Fig.1: in the title, put  Brassica napus in italic. Precise the cultivar (referent genome).
- Table 1 and S1: Precise the cultivar (referent genome) for B. napus, B. rapa and B. oleracea
The legends of these tables could be also supplemented briefly with the web sites used to define the characteristics.
Mw is calculated for mature proteins or with the N terminal peptide signal? Same question for pI, gravy and instability index.
- Table S2: Web site must be given.
-Fig. 2:
In the legend, for me, it is not a “phylogenetic relationship” but a dendrogramm based on similarity between nucleic sequences? Why At orthologous are not indicated? Why did you put sub-families E and F together?
 (A) “BnHsp70 paralogs were marked with red diamonds”. No red diamonds are present on the figure 2.
(B) The orange boxes indicate exons and black lines represent introns. TI is green boxes for exons !
Web site must be given.
On the figure, indicate in part A, the name of the sub-families (A to F). It will be clearest in comparison to Fig.1. What is the reason for the red lines (orthologs pairs?)?
- Fig S1: I have not found a legend. N-terminal ATPase domains, EEVD, HDEL, DVIDADFTDSK,GDAWV, SPSQ (I/V) G sequences and MEME sequences motifs must be indicated. What the significance of the boxes, amino acid in green, red?
- Table S3: Indicate in title Bn Bo and Br Hsp70 proteins.
The authors could indicate in addition in the table the membership of the subcategories for each MEME motif and also which ones are the HSP70 specific domains.
- Fig. 3: The legend has to be changed. There are some results inside.
Distribution of Hsp70 gene family members on B. rapa (A), B. oleracea (B) and B. napus (C) chromosomes. Some genes were not shown because located on unmapped chromosomes.
The chromosome name was indicated at the top of each bar. Tandem arrays of Hsp70 genes were displayed within the blue frame. The scale of all chromosomes was in millions of base (Mb).
- Fig.4: The title has to be changed. Synteny is the simultaneous presence on the same chromosome of two or more loci, regardless of their genetic binding. The notion of syntenia is increasingly used to describe the preservation of the order of genes between two related species. - Remove “Dark yellow and yellow colors represented An and Cn chomosomes of B. napus. Chromosomes of B. rapa and B. oleracea were represented by red and green colors, respectively. The different colors of lines indicated their syteny relationships in three species.” Give the significance of trait colors inside the circus.
- The Circos is complicated to read. May be it is possible to divide it in three circus: one with only Bn genes to see homeologs genes, one with Bn sub-genomeA and B. rapa genes, the last one with Bn sub-genome C and B. oleracea genes.
- Table S4: what is tPCK?
- Table S6: Absicsci acid to rewrite
- Fig.5 : It is not necessary to put three times the color scale. The quality of fig.5B is not good compare to the two others. In the legend, authors have to give the name of the two genes missing.
Experimental design: SPECIFIC COMMENTS ON MATERIALS AND METHODS:
This part is described with details and is very clear to understand methods used. Some minor points to improve this part :
- In the part “identification of Hsp70 gene members” (lines 100-125), authors should identify cultivar names for reference genomes (e.g. B. napus Darmor bzh …..). I assume those are the cultivars that are used for tissue collection (line 182).
- line 165: the reference of the mapping method (MApInspect tool) is missing.
- In the part “plant material and tissue collection” (lines 181-186), authors should precise the developmental stage of plant with BBCH scale.
- In part “analysis of Hsp70 gene expression…”, lines 190-191 : It is possible to indicate what are juvenile stem (top of the stem ?), young leaves and the age of siliques (10- DAP as flowers ?).
- line 194: I don’t understand the method to extracted the reads for each Hsp70 gene ? It was on annotated gene ontology ? If not, what were the sequences to specifically select each gene from RNA data set ?
- line 195: The method to obtain the relative gene expression levels must be more detailed.
- The plant material and RNA seq approach are published by the authors in “The Gene Structure and Expression Level Changes of the GH3 Gene Family in Brassica napus Relative to Its Diploid Ancestors. Ruihua Wang, Mengdi Li, Xiaoming Wu, Jianbo Wang. Genes, 2019, 10(1): 58. doi: 10.3390/genes10010058. This reference must be cited and the two parts could be reduced.
- Expression in root is not followed. It is because no HSp70 have been expressed in it?
Validity of the findings: SPECIFIC COMMENTS:

RESULTS:
genome_wide identification of Hsp70 genes
- line 208: “a total of 47 genes” : how many were identified from each approaches ? How many with a lack of Hsp70 specific domain? Does it mean that they are not functional? May be it could be important to indicate these genes in tables 1 and S1. No pseudogenes were identified?
- line 210: “The genes from BnHsp70-15s to BnHsp70-17s belonged to hsp110s”. Precise again that they are considered as a sub-class of Hsp70 (even if it is already mentioned in the introduction).
- Moreover, it seems to me important to compare the numbers of copies in B. napus genome and the parental ones. For example, no orthologous for AtHsp70-1 -3 -18 and -14 were found in parental genomes; or still, B. napus genome contains only one gene for At5g02500 but 6 and 5 copies were found respectively in B. rapa and B. oleracea genomes.
- No information is given about the similarity between genes or proteins? It will be interested to know if gene or protein sequences are similar or not to At orthologous or parental copies.
- For the names given for B. napus genes, it will be clearer to put inside information about the membership to the sub-genome A or C. For nomenclature, as recommended by Ostergaard and King (2008), gene names were designed according to the closet At sequence homology, taking into account A or C genome location and cluster memberships. For example: BnHsp70-2 becomes BnaC.Hsp70-2. What is the B. oleracea copy corresponding (a to f)?
- lines 217-222: The size comparaisons of Hsp70 proteins in terms of aa or kDa give the same informations. This part could be reduced.
- line 223: Authors has to mention that except BnHsp70-8s, all the proteins have negative gravity values.
- line 228: At the end of this section, authors have to compare with parental proteins as they did in lines 237-238.

Phylogenetic analyses
Why AtHsp70-1, -3, -18 and -14 were put on phylogenetic approach ? They are not detected in Bn, Bo and Br genomes?
- line 247: “subfamily E had only 5 members which were likely to be truncated”. What is the explanation? If it is the size, some others small proteins would be also designed as truncated.
- lines 253-256: “Integration analysis of the phylogenetic tree and subcellular localization prediction was similar to that described for other species, including A.thaliana, rice and pepper, which suggested that the Hsp70 genes have been broadly conserved in distinct taxa (Lin et al., 2001; Sarkar, Kundnani & Grover, 2013; Guo et al., 2016). ». For me, it is discussion.
- line 259: “All subfamilies except subfamily E included multiple sets of orthologs”. I think It could be nice to indicated the At orthogs for each sub-family.
- lines 262-265: Again, to clearly shown homeologs Bn gene, names have to be changed. For example, in sub-family E : BnHsp70-8b will become BnaC.Hsp7-8, BnHsp70-8a = BnaA.Hsp70-8. It would clearly show the links between Bn genes and parental ones (line 263 “12 Ar-An and 13 Co-Cn pairs”) and also if there are duplication in Bn compared to parental genomes or gene lost between parental genomes and Bn.

Structure of Hsp70 genes and conserved domain of Hsp70 proteins in three Brassica species
- line 271: and the end of the sentence, after “obtained”, put according sub-family membership.
- line 273: “The intron distribution of BnHsp70 genes was similar in the same alignment clusters”. What is the definition of a cluster ? If I look in the sub-family A the sub-group Hsp70-2, the number of introns changed. Authors have to be more precise.
- line 276: indication of the sub-category (A).
- line 298: Replace “Table S3 by Table 3, Fig.2C
- line 304: Replace “Motif 16 and 20 » by motifs 16 and 20

Chromosomal distribution and duplication pattern analysis of Hsp70 genes in three Brassica species
- line 323:” three of which formed a gene cluster” means that they are duplicated genes?
- The notion of synteny must be written in the text.
- lines 337 : reference for Schranz blocks is missing (Schranz et al., 2006).
- lines 339-341 : reference for LF, MF1 and MF2 subgenomes is missing.
- line 355: That mean “collinear regions”?
- line 377: mention of Table S5 is wrong.

Expression patterns of Hsp70 genes in different tissues of three Brassica species
- line 414 and line 416: “All genes except BnHsp70-6d were expressed in different tissues…”. “BnHsp70-6d lacked expression data in all samples of four tissues”. IT is not really the same idea. Authors have to change the sentences.
- line 416: “indicating most of them may play important part in the growth of these tissues”. Why the authors mentioned a role in growth tissues? It is because it was young tissues. If it is the case, it has to be mentioned.
- lines 426-428: “In addition, a similar higher expression pattern was also observed in different tissues, which was similar to Hsp70 paralogs in palaeo-polyploid soybean and pepper (Zhang et al., 2015; Guo et al., 2016).” For me, it is discussion and no result.

DISCUSSION
Genome duplications play major roles in the expansion of the BnHsp70 gene family
- lines 497-500: concerning importance of cytosolic Hsp70s. What abour the others species ? It is the same preferential retention process?

Intron gain of BnHsp70 genes and domain loss of BnHsp70 proteins
- line 518-519: “Also, previous researches have shown that variation of the number and placement of intron is a common process that has occurred during evolution”. Which references?
- line 526: Intron replace by intron.
Additional comments: This work concerning Hsp70 identification and comparison in three Brassica genomes is very well conducted. Hsp70 gene family is used to understand the origin and evolution in Brassica genomes, as well as the mechanism of polyploidy adaptability. The comparison with A. thaliana genes is also positive to understand evolution inside diploid and tetraploid genomes. The experimental work appears to be carefully done. The authors seems to be used to this kind of exercise with other paper in the same aim but focus on another gene family (GH3, doi: 10.3390/genes10010058).
The first part of introduction is focus on the polyploidization and consequences of this process. The second part gives the example of Brassica lineage. The interest of Hsp70 is developed in the third part. The authors cited others species and it would be interested to have more information that only gene numbers, for example, in terms of divergence of functions or expressions of A. thaliana, rice, soybean or pepper Hsp70s. The end of the introduction define clearly the rationale and specific hypotheses of the current experiments.
The material and methods, results and discussion are well organized. Concerning results, it would have been interesting to have the percentage of similarities of genes and proteins for each sub-class (for Bn, Bo, Br and At copies) to see if there is a strong conservation and divergence between related proteins and genes. For proteins functions, Hsp70 have two important domains and only the NBD domain is really highlighted. The secondary structure analysis is also missing. Concerning gene structures with various intron-exon numbers, the possibility of alternative splicing RNA is not pointed in result (but in discussion) with may be several expressed proteins for one gene…
The main critic of this paper is the choice to name the genes. It is logical to take reference to A. thaliana nomenclature. But, the relationship between Bn genes and parental genomes it is not enough put forward. The authors have to change the names according to nomenclature recommended by Ostergaard and King (2008). It will be easier to see, in all the paper, homologies or differences between conserved Bn genes and parental ones, the lost or duplicated genes (Table S4 for example), in expression patterns (example: line 436, BoHsp70-5b and BnHsp70-5c homologous pair with different nomenclatures). No pseudogenes were detected?
This work has to be accepted with minor revisions.


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 CHARACTERIZATION OF THE HSP70 GENE FAMILY IN ALLOPOLYPLOID RAPESEED (BRASSICA NAPUS L.) COMPARED WITH ITS DIPLOID PROGENITORS
Review round: 1
Reviewer: 4

Output:
Basic reporting: In this study, authors identified the Hsp70 gene family in allopolyploid rapeseed (Brassica napus L.), and then, compared with its diploid progenitors. It was an interesting study. I think this manuscript could be published after major revision.

Overall, the manuscript is well written. However, a better discussion part will help improve the manuscript.

The manuscript style should be revised according to the journal.

The full names of abbreviation must be given at first use, please check the whole text and revise.

The manuscript should be corrected by a native English speaker, alternatively please use one of the commercial English language editing services available.
Experimental design: well design.
Validity of the findings: Conclusions are well stated.
Additional comments: In this study, authors identified the Hsp70 gene family in allopolyploid rapeseed (Brassica napus L.), and then, compared with its diploid progenitors. It was an interesting study. I think this manuscript could be published after major revision.

Overall, the manuscript is well written. However, a better discussion part will help improve the manuscript.

The manuscript style should be revised according to the journal.

The full names of abbreviation must be given at first use, please check the whole text and revise.

The manuscript should be corrected by a native English speaker, alternatively please use one of the commercial English language editing services available.


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 CHARACTERIZATION OF THE HSP70 GENE FAMILY IN ALLOPOLYPLOID RAPESEED (BRASSICA NAPUS L.) COMPARED WITH ITS DIPLOID PROGENITORS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript has been greatly improved.
Experimental design: no more comments.
Validity of the findings: no more comments.
Additional comments: I accept most of the author's comments. The manuscript has been greatly improved and the results are very interesting. However, as we know, Hsp70s is strongly associated with plant stress resistance. in Results, I think it is very necessary to characterize the putative stress responsive functions of each Hsp70s by performaing expression analysis under diverse abiotic stresses (high temperature,high humidity and drought conditions......), or by data from public database.


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 CHARACTERIZATION OF THE HSP70 GENE FAMILY IN ALLOPOLYPLOID RAPESEED (BRASSICA NAPUS L.) COMPARED WITH ITS DIPLOID PROGENITORS
Review round: 3
Reviewer: 1

Output:
Basic reporting: Ok
Experimental design: Ok
Validity of the findings: Ok
Additional comments: I accept the 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: ANXIETY-LIKE BEHAVIOR AND WHOLE-BODY CORTISOL RESPONSES TO COMPONENTS OF ENERGY DRINKS IN ZEBRAFISH (DANIO RERIO)
Review round: 1
Reviewer: 1

Output:
Basic reporting: In the MS entitled “Anxiety-like behavior and whole-body cortisol responses to components of energy drinks in zebraﬁsh (Danio rerio)” the English language is clear, and the tables and figures are easy to understand. The topic approached in the MS is relevant because the consumption of taurine and caffeine association in energy drinks is very popular, and the synergic effects of these psychoactive are still unknown.

The MS addresses current and relevant references on the subject, however, the results found in the study do not support the main idea discussed along the MS (see next evaluation topics).

Basically, some points should be rethought:

-The introduction is extensive and approaches the isolated effects of caffeine and taurine, however, little is discussed about the effects of these drugs in association. What is known about this association? This topic should be deepened.

-Which is the prevalence of the energy drinks consumption? How these energy drinks are commonly consumed (association with alcohol)?  It can be related to which social problems?

-The hypotheses described in the end of introduction section are unnecessary.
Experimental design: The MS is original and is within the scope of the PeerJ. The research question is relevant and well defined, and the methodology used is referenced. However, the methodology applied to evaluate the effects of taurine and caffeine on anxiety-like behaviors can be mistaken or with poor reproducibility. It can be a problem to replicate this protocol in the future.

In relation to experimental design, some points should be considered:

-Was this study approved by an ethical committee? If yes, which is the approval protocol number?

-Mezzomo et al. (2016) showed that TAU exposure did not alter anxiety responses using the novel tank test. Anxiety-like responses trigger by taurine were observed only when the light dark paradigm was used. Why the authors choose to use the novel tank test to evaluate the synergic effects of taurine and caffeine?

-I suggest that the authors perform an additional experiment using the light-dark test in the same context, in a manner to increase the robustness of the findings.
Validity of the findings: The findings of the MS are inconclusive and without statistical significance to support the conclusions argued along all the MS.

Some points should be minutely evaluated:

-The main conclusion of the MS is that taurine exposure act mitigates the anxiety-producing effects of caffeine. However, the MS data not support this affirmation because do not exist statistical significance in the parameters discussed. No parameter evaluated in the novel tank was statistically altered by the association of taurine and caffeine. The MS only argue about trends, so, the results are not robust. I suggest that the experimental design be rethought and the light-dark test be done to confirm the data presented so far.

-The statistical analyse applied in the MS is incorrect. Two factors influence the results: taurine and caffeine, so the statistical analyse correct to be use is the two-way ANOVA.

-Why is the number of animals used in the experiments so dissimilar? In the novel tank test the number of fish per group was 10-38, and in cortisol analyse 19-39 fish per group. Please, explain why this number is so varied.

-Rosa et al. (2018) showed that acute caffeine exposure in the same concentration used in the present study (100 mg/L) increased the cortisol levels in zebrafish. However, the levels of cortisol were not altered in this study. How is possible justify this finding?

-Mezzomo et al. (2016) exposed the fish to 400 mg/L of taurine for 1 hour before evaluate anxiety-like behaviors. Why the authors perform taurine exposure for 15 minutes? There are previous studies that justify the use of this time of taurine exposure to evaluate anxiety behavior?

-In the study, taurine decreased the distance travelled when compared to other groups? However, this result is not approached in the discussion section. How this result can be explained?

-In the discussion section, many results that are not statistically significant are discussed. Mechanisms about the results are hypothesized and some points justified based on trends. Additional experiments are needed to support this discussion.
Additional comments: The topic of the study is original and relevant in neuroscience. The study has a clear objective, however, some methodological aspects need to be improved for the conclusions to be supported.


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: ANXIETY-LIKE BEHAVIOR AND WHOLE-BODY CORTISOL RESPONSES TO COMPONENTS OF ENERGY DRINKS IN ZEBRAFISH (DANIO RERIO)
Review round: 1
Reviewer: 2

Output:
Basic reporting: Dear Editor

This manuscript investigated anxiety-like behavior and whole-body cortisol responses to components of energy drinks in zebrafish (Danio rerio). Overall, the manuscript is well written, the methodological approach is sound, and the authors have done a nice job.
Some methodological aspects and study design should be better described to improve clarity. The major issues are listed below:
Experimental design: 1) In most studies using zebrafish the animals are fed 2-3 times a day. In this study, the animals were fed only once a day. Why this food restriction? Could this diet be involved in malnourished animals?

2) Is a week of habituation enough for the animals to get used to the new environment?

3) What is the zebrafish lineage used in the study? Different types of lineage present different behavioral profiles. The wild-type is most used being representative of population.

4) The sex effect on behavioral outcomes was not considered. There are several articles in the literature showing that males and females zebrafish may respond differently to certain situations. This could have been considered by the authors.

5) What is the density of animals per liter used in the experiment? A higher density of 5 animals per liter can alter the behavior of the animals making them more anxious.

6) Why were the animals tested on only one behavioral apparatus? Why was the light/dark teste not done?

7) I suggest that measures of zebrafish freezing behavior and measures of zebrafish motor activity be presented in graph form.

8) What are the limitations of the study?
Validity of the findings: 1) A major point is about randomization and blinding. The author did not describe that randomization was performed to allocate the "treatment" groups. What was the method used for random allocation in the treatment groups? Were experimenters blind to treatment? Were data analysts blind to treatment? These questions need to be addressed and clearly stated in the methods section.

2) How was the sample size calculated? Was it based on a pilot experiment to determine effect sizes? How were the calculations performed? being that the study presents different numbers of subjects between the groups. The n is too varied which makes it possible to type I and type II errors. Why were the experimental groups unbalanced?

3) If animals considered outliers in behavior were removed because the same animals were not removed from the cortisol analysis, knowing that these animals had behavioral problems. What was the criterion for determining the outliers?

4) Did the authors verify the potential effect of the tank? Ideally, at least two tanks should be available for each treatment.

5) Control animals spent less time in the upper zone of the tank in this study compared to other articles using the same apparatus. Would be these animals more anxious even without any intervention?

6) On the results expressed as percentage that did not explore the top of the aquarium, what measure used to realize that percentage? Number of entries or time in the upper zone? And what is the significance and advantage of this analysis in relation to the other measures that were not statistically significant? This measure was confused and not usually used in the literature. The criteria commonly used to verify a alteration in exploratory behavior would be the number of crossings between the zones and the time in the upper and bottom zones of the tank. I suggest that present the data of number of crossings and time in the bottom zone of the tank, since there was no change in the time in the upper zone. If these are significant, a alteration in exploratory behavior and type-anxious effect can be observed. If the choice is to maintain this analysis, it should be better explained and substantiated.

7) Was reported that caffeine decreased the exploratory behavior of the animal in the upper zone of the tank and that this decrease in exploratory behavior is indicative of the anxiety-like effect of caffeine. However, representative data for alteration in exploratory behavior and indication of caffeine anxiety-like (time in the bottom zone, time and duration of immobility and crossings) were not statistically significant. Can say that there was actually an effect of caffeine on these behavioral parameters? I suggest that the discussion of the results should be better addressed following the results found.

8) In Figure 1 is reported values are mean ± SEM of 10-38 fish per group. The maximum number of excluded animals reported were 2 per group, the volumes expressed in the graphs were between 10-38 animals? Why were the experimental groups unbalanced?

9) Average velocity, measured in units of distance per unit time (typically, meters per second), is the average distance traveled during some time interval. If the time is the same, the distance is proportional to average speed. Therefore, table 1 should present the same ratio between the mean speed and distance data. The authors have to revise all data because it is not correct.
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: ANXIETY-LIKE BEHAVIOR AND WHOLE-BODY CORTISOL RESPONSES TO COMPONENTS OF ENERGY DRINKS IN ZEBRAFISH (DANIO RERIO)
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors accepted the reviewers' suggestions and this improved the quality of the manuscript.
Experimental design: The authors explained the inquiries required by the reviewers and did an additional experiment to increase the robustness of the data, which improved the quality of the manuscript and the reliability of the data.
Validity of the findings: The authors explained all the questions required and altered the MS according to this. Mainly, they changed the statistical analysis to the correct form and thus the results of the manuscript became more robust. However, my only doubt is: Why did the authors not use a post-test of multiple comparisons after the application of two-way anova? The post-test could demonstrate some statistical significance not seen by the two-way anova interactions. I suggest that a post-test of multiple comparisons is still applied to the data.
Additional comments: The authors improved the quality of the manuscript by performing additional experiments, altering the statistical analysis and improving the writing of the manuscript.


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

Input:
Title: ANXIETY-LIKE BEHAVIOR AND WHOLE-BODY CORTISOL RESPONSES TO COMPONENTS OF ENERGY DRINKS IN ZEBRAFISH (DANIO RERIO)
Review round: 2
Reviewer: 2

Output:
Basic reporting: Dear Editor

After the first review of the article, the authors answered the questionings abouth some methodological aspects and study design and described with more clarity the results and discussion. Overall, the revised manuscript is significantly improved and well written. The methodological approach is suitable and sufficiently addressed each of the reviewers’ concerns.

However, some doubts that remained are listed below:
Experimental design: Mean speed, measured in units of distance per unit time (typically, meters per second), is the mean distance traveled during some time interval. If the time is the same, the distance is proportional to average speed. However, in figure 1, the data is conflicting and do not present the same ratio between mean speed and distance data.

In the Light-Dark Test some measurements can be difficult to obtain when the animal is in the black compartment. Can measures of total distance traveled (cm), mean speed (cm/s), immobility duration (s) be analyzed with Behavior Cloud motion-tracking software? I suggest quantifying the number of crossings and latency for first entry in the light zone.

In the cortisol assay the samples from Experiment 1 were allowed to dry at room temperature under a fume hood until the ether layer was fully evaporated; samples from Experiment 2 were dried with a light stream of air. Why this difference?
Validity of the findings: About de answer in Reviewer #1, comment #9. " In Experiment 1 (Novel Tank Test), several groups of undergraduate students collected the data". Were more experiments performed for the control group? Were the data grouped? Data of different experiments can't be grouped. Please, explain it in details.
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: A COMPARATIVE STUDY OF ROOT CANAL SHAPING USING PROTAPER UNIVERSAL AND PROTAPER NEXT ROTARY FILES IN PRECLINICAL DENTAL EDUCATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: •	There is unclear, ambiguous language in some sections.
•	What is/are statistical test(s) that was/ were used in this study? should be mentioned in background, methods, and results
•	What is deformation? Need to be defined
•	Figures should be included (shaping ability study)
Experimental design: The two different systems (PTU and PTN) were used by different groups of students and this hinder the power of this study, it is indirect comparison with unnecessary variables.
Validity of the findings: More information about data collection and statistical analysis is needed
Additional comments: •	There is unclear, ambiguous language in some sections.
•	What is/are statistical test(s) that was/ were used in this study? should be mentioned in background, methods, and results
•	The two different systems (PTU and PTN) were used by different groups of students and this hinder the power of this study, it is indirect comparison with unnecessary variables.
•	What is deformation? Need to be defined
•	Figures should be included (shaping ability study)


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

Input:
Title: A COMPARATIVE STUDY OF ROOT CANAL SHAPING USING PROTAPER UNIVERSAL AND PROTAPER NEXT ROTARY FILES IN PRECLINICAL DENTAL EDUCATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The study was well designed and professional english language was used (Certificate of editing and proofreading was attached). Research question well defined, relevant & meaningful. The first author has experience in this field.
Even so, the authors must be controlled the references. (for example; authors name in the text ; in a study by Alemann et al. (2017), and in the references ; Alemam AAH, Dummer PMH, Farnell DJJ. 2017, are different.)
As a result, this article is acceptable after correction of subject above.