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: CHARACTERIZING THE SUCKLING BEHAVIOR BY VIDEO AND 3D-ACCELEROMETRY IN HUMPBACK WHALE CALVES ON A BREEDING GROUND
Review round: 1
Reviewer: 3

Output:
Basic reporting: Overall, I think this is a well-done study and well-written manuscript. This work nicely builds on past studies about nursing/suckling behavior in humpback whales and improves our knowledge using modern technologies. It also lays the foundation to automate the detection of suckling events in biologger data. The introduction provides a solid background and establishes the need for this study. However, a few citations are needed to support some of the statements made in the introduction.  I provided comments in the "Line by line comments" section on the attached document.

The figures and tables provided are valuable and relevant to include in the manuscript. I have provided specific comments for some of them in the "Figures and Tables" section of the attached document. Overall, I would suggest making sure the captions provide enough information to stand alone from the manuscript. A few figures and tables also need additional labeling to specify which data was actually plotted/reported (mean, maximum, minimum, maximum mean, or minimum mean).

The raw data is not referenced in the main text. If you intend to provide the data with your study, additional metadata (maybe I missed it) tied to the supplemental files (definitions of the column headers and units) would be helpful.

There are a few overall minor suggestions to increase clarity and improve flow. You can choose to use them or not, but I do not think it is necessary for you to address each individual suggestion/comment in that section.  I listed some overarching comments (1-3)  and provided line-by-line suggestions at the end of the attached document under the headline "Minor suggestion."
Experimental design: This study clearly defines the research questions, the knowledge gap, and how this study has helped to close that gap. I have provided comments in the "Line by line comments" section that I think will allow others to replicate this study more accurately by asking you to include additional details in the methods section. There is an additional need for you to clarify the reporting of your results and provide a little more information as to why you chose the experimental design you used. Since this study is proof of concept for automating detection for humpback whale suckling behavior, I foresee many other studies basing their experimental design on your paper. Therefore, future studies will be improved if you leave little room for interpretation in your protocols and explain why you chose the methods you used.
Validity of the findings: Despite the small sample size, I think this is an important study to publish and introduce to the broader scientific community- for others to build upon and provide data to strengthen the models. I have provided comments in the "Line by line comments" section on the attached document that should lead to improved clarity of the discussion and ensure that the discussion and conclusions reflect and acknowledge the methods and limitations of this study.
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: CHARACTERIZING THE SUCKLING BEHAVIOR BY VIDEO AND 3D-ACCELEROMETRY IN HUMPBACK WHALE CALVES ON A BREEDING GROUND
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have addressed all the issues I pointed out before, and the latest manuscript has been clearly and well written. However, references to newer or more recent work are required since the current paper mostly references are outdated. It is more meaningful to provide newer ones. In addition, some sentences are too long (e.g., Line 107-111, and Line 528-532), which easily confuses readers. It is better to rewrite in two shorter ones.
Experimental design: The authors have added more details about the analysis process of the raw data and compared several popular machine learning methods sufficiently.
Validity of the findings: This study gave completed results and reasonable discussion, and also pointed out its limitations and future works. It’s very useful for further investigation on this kind of researches.
Additional comments: Overall, this manuscript has been well done. I have no opposing opinion for publishing this paper.


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

Input:
Title: CHARACTERIZING THE SUCKLING BEHAVIOR BY VIDEO AND 3D-ACCELEROMETRY IN HUMPBACK WHALE CALVES ON A BREEDING GROUND
Review round: 2
Reviewer: 2

Output:
Basic reporting: I believe the authors have thoroughly addressed any comments or concerns I had regarding the original manuscript. The paper reads well, and it appears that the authors have included additional text, citations, and analysis to improve the study's clarity, content, and replicability.
Experimental design: The authors clearly stated the aims of the study and the knowledge gaps they were addressing. The analysis completed within my scope of experience appears to meet high technical standards- I defer to the other reviewers to comment on the additional analysis regarding automated identification and machine learning.
Validity of the findings: The authors provided underlying data and encouraged future studies to strengthen their findings. They also addressed the impact and novelty of their study. I agree that this is an important study to further our understanding of humpback whales' suckling behavior and further our ability to use biologgers to study marine mammal behavior.
Additional comments: I believe there is a typo on line 428. It currently reads, "Additional accelerometry singles, ..." I am guessing the word is meant to be "signals". Please ignore if I am mistaken.


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: SARCOPENIA IS NOT ASSOCIATED WITH INSPIRATORY MUSCLE STRENGTH BUT WITH EXPIRATORY MUSCLE STRENGTH AMONG OLDER ADULTS REQUIRING LONG-TERM CARE/SUPPORT
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is very well written, it is clear and concise. I have made a few minor recommendations, by line number below.

The introduction shows context and is well referenced with recent evidence. The structure of the article conforms to Peer standards. The figures are clear, well labelled, and relevant. Raw data was provided and appears to be well organised.

The term “daycare” may be better written as Day Care – the correct spelling appears to differ in different countries - this may be confirmed by the Academic Editor.
Experimental design: The research question is clearly defined, and it is clearly stated how this paper fills an identified knowledge gap.
The investigation performed a high technical standard, and the methods are described with sufficient detail to replicate the study.
Validity of the findings: Replication is encouraged.
The data appear to be robust and statistically sound.
Conclusions are well stated and linked to the research question.
Additional comments: Thank you for performing this fascinating study, you have addressed a genuine knowledge gap with innovation and expertise and discovered findings that are clinically relevant. I have made a few minor recommendations below:

Abstract
The conclusion ends rather abruptly, I recommend expanding on the final sentence to indicate how your finding/s may be clinically applied. You have used 234/500 words so there is plenty of room for more detail.
Introduction
Line 60 			First sentence is a little disjointed. Please consider re-wording eg. “Respiratory sarcopenia has recently been defined as “whole- body…..”
Materials and Methods
The formatting appears to be different to the rest of the document.
Study participants
Please advise the rationale behind excluding those with respiratory conditions from your 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: SARCOPENIA IS NOT ASSOCIATED WITH INSPIRATORY MUSCLE STRENGTH BUT WITH EXPIRATORY MUSCLE STRENGTH AMONG OLDER ADULTS REQUIRING LONG-TERM CARE/SUPPORT
Review round: 1
Reviewer: 2

Output:
Basic reporting: Thank you for the opportunity to review this manuscript. Using a cross-sectional study, the authors investigated the association between respiratory parameters and sarcopenia. The topic is interesting, and the manuscript is well-written; however, there are some issues needed to be clarified.
Experimental design: 1. The authors describe the baseline characteristics (Table 1) as well as AUC curve for sarcopenia by gender (Figure 2). It would be better to show the results by gender in tables 2 and 3.
2. The authors found that maximal expiratory pressure was the only parameter that was predictive of sarcopenia. They need to explain why maximal expiratory pressure but not other parameters were associated with sarcopenia.
Validity of the findings: 1. The study was conducted among older adults who needed long-term care; thus, the findings may not be generalized to relative healthy individuals.

2. The authors aimed to identify cut-off for the obtained parameter for “the Japanese population”. I think they may need to tune down the statement about this given the small sample size (not representative) and cross-sectional design.
Additional comments: Minor concerns:
1. Given the high prevalence of sarcopenia (62.1%, 77 out of 124), I don’t think it is so meaningful to get AUCs of less than 0.7 (Figure 2).
2. Tables 2 and 3: why the authors indicate significant associations using asterisks if they already showed the exact p-values?
3. Did the authors test normal distribution for parameters such as trunk muscle mass per height squared and skeletal muscle mass index before performing correlation analysis?
4. I still don’t know how sarcopenia was defined. The authors may need to provide more details about it.


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

Input:
Title: SARCOPENIA IS NOT ASSOCIATED WITH INSPIRATORY MUSCLE STRENGTH BUT WITH EXPIRATORY MUSCLE STRENGTH AMONG OLDER ADULTS REQUIRING LONG-TERM CARE/SUPPORT
Review round: 2
Reviewer: 1

Output:
Basic reporting: Thank you for your considered responses to both reviewers. I believe the article is now close to ready for publication and my only remaining comment is regarding the first sentence. I am unsure about the term “non-systematic review” and believe that detracts from the important opening paragraph. I recommend the following first sentence,

“Respiratory sarcopenia has been recently defined as “whole-body sarcopenia, and low respiratory muscle mass followed by low respiratory muscle strength and/or deteriorated respiratory function” (Nagano et al., 2021). (Lines 62-64)”

I have no further concerns or queries.
Experimental design: No further comments
Validity of the findings: No further comments
Additional comments: Thank you for conducting this interesting 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: SARCOPENIA IS NOT ASSOCIATED WITH INSPIRATORY MUSCLE STRENGTH BUT WITH EXPIRATORY MUSCLE STRENGTH AMONG OLDER ADULTS REQUIRING LONG-TERM CARE/SUPPORT
Review round: 2
Reviewer: 2

Output:
Basic reporting: No further comment
Experimental design: No more comment
Validity of the findings: No more comment.
Additional comments: The authors have well addressed my comments. I have only one concern. It is better to clarify how each component of sarcopenia was defined. For example, low muscle strength (hand grip strength) was defined by less than 30 kg in men and less than 20 kg in women.


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: PREVALENCE OF IRON DEFICIENCY ANEMIA IN BRAZILIAN WOMEN OF CHILDBEARING AGE: A SYSTEMATIC REVIEW WITH META-ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This article reviews iron deficiency anemia (IDA). The authors acknowledge that Brazil has several programs nationwide to deal with this condition in women. The authors propose a systematic review of the prevalence of iron deficiency anemia (IDA) in Brazilian women of childbearing age, as this condition affects the development of the fetus. For this, Science, Scopus, Lilacs, SciELO and gray literature databases were used. It was concluded that IDA in women of childbearing age remains a public health problem in Brazil.
Experimental design: Although an important survey was carried out in an adequate database, there are still some doubts:
1-	the number was not clear, among the 83 works selected for meta-analyses, how many are gray literature;
2-	another issue to be considered is the authors' choice of method. Meta-analysis relies on statistics that combine the results of various scientific studies to derive a pooled estimate closest to the truth and that identify patterns or sources of disagreement between the results. However, the authors show a paradox in table
3. This study is registered as important and at the same time it is alleged that there is a serious risk of bias, a very serious inconsistency and very low quality. It is suggested that this paradox be circumvented in the statistical (quantitative) methodology so as not to harm the research based on meta-analysis.
Validity of the findings: 4-	it is not clear what the innovative result of the research is, since the lack of iron in women is already a known factor and there are measures taken by the Brazilian government to overcome it. It is also known that in less wealthy regions (in the case of this article, northern and northeastern regions of Brazil) people have a low intake of necessary nutrients for health, including iron;
5-	acronyms used in lines 148, 149 and 150 need to be specified the first time they are cited
Additional comments: It is suggested that the authors review the article and add a topic about the measures they should take so that the paradox does not occur. In this way the article will be improved to pass.


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: PREVALENCE OF IRON DEFICIENCY ANEMIA IN BRAZILIAN WOMEN OF CHILDBEARING AGE: A SYSTEMATIC REVIEW WITH META-ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: I found it a nicely written paper with recent literature. The article is written in a scientific structure.
Experimental design: It is a novel idea to test. The research question and the problem are well defined. Databases were searched extensively.
Validity of the findings: It is an impactful work and implementation of results that would benefit the community people.
Additional comments: Well done. Thanks and congratulations.


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: PREVALENCE OF IRON DEFICIENCY ANEMIA IN BRAZILIAN WOMEN OF CHILDBEARING AGE: A SYSTEMATIC REVIEW WITH META-ANALYSIS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The article is well written and with the necessary language skills. The article addresses an important public health problem regarding the health conditions of a vulnerable population segment, such as women of reproductive age. The references used are sufficient and updated to some extent. Revisions of some used references are necessary, as they are outdated. The tables were well developed and are able to represent the data found well. The supplementary files were necessary and able to resolve doubts that arose during the reading and revision. The flowchart is sufficient and self-explanatory, demonstrating the entire process of searching and selecting the articles that make up the review. The article is explanatory, but needs some revisions for its enhancement and publication.

Some comments below emerged as the reading of the work progressed and need further consideration by the authors:

C1: The systematic review itself can not determine the prevalence of IDA in brazilian women. Include the meta-analyses in abstract's background.

C2: In introduction, it is necessary to explain the difference between iron deficiency itself and iron deficiency anemia, as the authors mention both as women's health problems.

C3: On line 91, the authors mention the high prevalence of IDA found in northeastern Brazil. It is important to explain to non-brazilians the differences between Brazil's geographic regions and its particularities, specially the northeast.

C4: On line 123 the authors mention that studies that evaluated iron deficiency were included in review' formal screening, but on line 125, iron deficiency anemia was mentioned. Which one was screened?

C5: In discussion, which conditions make the South-Southeast regions provide a better quality of life that North-Northeast can not attend?

C6: Without a proper meta-regression between the pooled prevalence and the sub-groups, the authors can not claim that some prevalences were higher than others.
Experimental design: The authors well defined their object of study and carried out an extensive literature search of papers eligible for meta-analysis. However, some issues were not very clear and raise doubts about the methodological rigor of the study.

C1: Were only studies with a probabilistic sample included in the meta-analysis? It is not clear in the article. If so, it is important to emphasize as a strength of the study and the quality of evidence found. If not, I suggest that they carry out the meta-analysis only with data from investigations with probability sampling, as convenience samples can bias the results found because they do not adequately represent the contingent population of women of reproductive age with iron deficiency anemia.

C2: I mentioned it before, but it is important to emphasize that the analysis of subgroups only aggregates the results found according to interest groups, but it cannot be affirmed, without an appropriate robust meta-regression analysis, that the prevalences found in a subgroup is superior to others.
Validity of the findings: The work is unprecedented, as there is no systematic review with meta-analysis of iron deficiency anemia in women of reproductive age in Brazil. Their findings are consistent with parallel investigations and data projections from international entities. However, the low quality of the findings, possibly resulting from the inclusion of studies without random sampling, may slightly bias their findings. The conclusions are consistent and respond to what was intended to be investigated.
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: PREVALENCE OF IRON DEFICIENCY ANEMIA IN BRAZILIAN WOMEN OF CHILDBEARING AGE: A SYSTEMATIC REVIEW WITH META-ANALYSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Please, see topic number 4
Experimental design: Please, see topic number 4
Validity of the findings: Please, see topic number 4
Additional comments: After verifying the insertions in the amended text, the topics listed by this reviewer on the occasion of the first review were clarified. This gave greater consistency to the content developed by the authors. Therefore, I consider the re-review made by the authors satisfactory and recommend the publication of the second version of the article.


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: PREVALENCE OF IRON DEFICIENCY ANEMIA IN BRAZILIAN WOMEN OF CHILDBEARING AGE: A SYSTEMATIC REVIEW WITH META-ANALYSIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors made the necessary corrections, as well as informed when it was not possible to change some aspects. The work is now in a suitable format for publication, as well as adding great scientific value to the field of epidemiology of deficiency diseases in a vulnerable Brazilian segment.
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: PCATEST: TESTING THE STATISTICAL SIGNIFICANCE OF PRINCIPAL COMPONENT ANALYSIS IN R
Review round: 2
Reviewer: 1

Output:
Basic reporting: This work addresses a fundamental aspect of Principal Components Analysis (PCA) application, that unfortunately has been neglected by the general biological/ecological research community: Raw PCA is only a numerical method (and tool), not comprising statistical methods to estimate the significance of the results obtained, namely, the significant principal components (pc) extracted and the variables significantly contributing to each pc. To obtain such answers, suited statistical methods need to be coupled to PCA. The ecological research community has recursively failed on PCA application, thus presenting results and conclusions poorly grounded, to say the least. The example here presented with the re-analysis of the work by Wong and Carmona (2021) is clarifying and I praise the author for it. In the Discussion, the author gives a remarkable example of how many authors have applied incorrect PCA in articles published during 2021 in PeerJ. This example could already be shown early in the Introduction, regardless of being mentioned again in the discussion.

This problem, of enormous magnitude in ecology, nevertheless occurs also in other fields of science. Therefore, I question the author's choice of restricting its scope (writing and examples) to ecology. I suggest for the author to broaden the manuscript to other fields.

The Introduction presents the problem well. The Discussion and conclusion expand on it and present the solution very well. The proposed solution is well grounded on the results. The author did a very good job.

The English, text structure and figures comply with the journal’s requirement.

The presentation of the software, algorithm, processes and artificial data generation is vague. Reproducibility is not possible from the text alone. The article is not self-contained and, for the reader to fully understand it, in parallel he must consult the key references as well as the R script. Personally, I do not find this the best writing style and I prefer a more complete version of the manuscript. Nevertheless, I understand the intension to provide a lighter text enabling a smoother reading flow. But, in such case, the text can be improved and, at the proper places, it must be mentioned (the citations, the R script) where the respective theory/method is thoroughly detailed.

The citations are inadmissibly poor and biased, particularly in the introduction, neglecting key authors in the topic and sometimes incorrectly attributing credit to the wrong author. I also notice that the author never cites more than one reference per idea/message (set of sentences), which is utterly poor and unfair to previous authors. PCA was originally developed by Pearson (1901) and later by Hoteling (in the 1930’s). This citation is mandatory. Other authors as Ricker, Manly, Jackson or Jolliffe are classical references for PCA.
Pearson, K. (1901) On lines and planes of closest fit to systems of points in space. Philos. Mag. 2:559-572.
Manly, B.J.F. (1986) Multivariate Methods. Chapman & Hall, London, UK
Manly, B.J.F. (1991) Randomization and Monte Carlo Methods in Biology. Chapman & Hall, London, UK
Jackson, J.E. (1991) A User’s Guide to Principal Components. John Wiley & Sons, New York, USA
Jolliffe, I.T. (2002) Principal Component Analysis. Springer-Verlag, New York, USA
Jolliffe, I.T. and Cadima, G. (2016). Principal Components Analysis: a review and recent developments. Phil. Trans. Rl Soc. A. 374: 20150202.

All these would be better references for the introductory sentence in lines 27-29. They are particularly better suited than Dobriban (2020), which is not even a good citation for PCA per se but only for the application of permutation tests to PCA and FA. Following the author’s rationale, Vieira (2012) or Björklund (2019) would be as good citations for lines 27-29 as Dobriban (2020) or Abdi and Williams (2010). In fact, Vieira (2012) is a citation suited for most of the sentences in the introduction, as are many of the suggested below. Although the works by Vieira (2012), Vitale (2017), Björklund (2019) and Dobriban (2020) are the recent trend in the application of Permutation Tests to estimate the significance of results from PCA, they were not the original developers of this topic. In fact, most of their works were compilations - or built on top of – previous works. The application of Randomization methods to multivariate analysis (i.e., Permutation Tests for the estimation of significances, as well as Bootstrap and Jacknife for the estimation of confidence intervals) was started by researchers as Legendre, Peres-Neto, Jolliffe, Lebart, Jackson, Dijkseterhuis, Efron, Mehlman, ter Braak, Zwick, Velicer or Westerhuis. These citations became ‘classical’ and are mandatory to the respective sentences in the introduction. I suggest for the author to, at least, look up in the works by Vieira (2012), Vitale (2017), Björklund (2019) and Dobriban (2020) where the aforementioned references fit in his introduction.

References for the bootstrap method are not provided in the Introduction nor on the Methods, although plenty exist. These are some of the suggested above.
Experimental design: The Research question is well defined, relevant and meaningful. It is stated how the research fills an identified knowledge gap.

The research is within Scope of the journal. However, it is subjective whether it can be considered “Original primary research”. The novelty in this work is that it transposes to R software the Matlab software developed by Vieira (2012), updated with a posterior algorithm developed by Björklund (2019) and with bootstrap methods for the estimation of confidence intervals (references missing). Personally, I find this very useful to the scientific community, and worth publishing. Regarding this aspect, the author provided a very good and convincing presentation.

About “Methods described with sufficient detail and information to replicate:
Testing the method with artificial data, thus proving its validity as well as advantages, is a standard in the presentation of new numerical/statistical methods, which the author wisely followed. However, its implementation was poor:
1. How was the artificial data generated? I would prefer to see this better explained in the article. How was the algorithm for the generation of the random data? And the range of coefficients tested? What were the variances of the variables? Their associated errors (ε)? Their distributions? Their imposed effective correlations or covariances? And how? Slopes of regression lines centred on the means? Other? A few added lines suffice to satisfy this requirement.
2. The magnitude of the conditions covered by the artificially generated data seems week. In his presentation, the author should broaden the coverage of the aspects mentioned in the point 1 above. Otherwise, the author explicitly manifests that this is just an example to demonstrate the argumentation, and that its generalized prove was already done somewhere else (cite the authors who used permutations tests in PCAs applied to artificial data).

About “Rigorous investigation performed to a high technical & ethical standard”, some improvements should be done:
Relative to the PCA applied to the Ringnér (2008) data set, my personal experience is that PCAs applied to data sets with so many variables fail completely, particularly if, on the other hand, the number of observations is so small. Ringnér (2008) and the current author, have two options: (1) transpose the data matrix and do the PCA in Q-mode. (2) use the data matrix “as is” to do a PCA in R-mode, but doing it in several steps to iteratively eliminate irrelevant variables (uncorrelated with any other), thus reducing the size of the data set. The irrelevant variables bring noise that veils the true correlations. As this noise is step-wise eliminated, the true correlations emerge. A PCA as allegedly done by Ringnér (2008) is ridiculous.

The PCA applied to the Ringner (2008) data set only used 100 random simulations, which is too little. I usually use 10000. If the data set is very large, bringing computational constrains, 1000 would be admissible. 100 is inadmissible. I highlight the following aspect: with 100 replications, each represents a jump of 1% in p, meaning that p is represented at a 1% resolution. Therefore, around the α= 5% threshold, p jumps from 4% to 5% to 6%. With 1000 replications, each represents a jump of 0.1% in p. With 10000 replications, each represents a jump of 0.01% in p. Therefore, around the α=5% threshold, p jumps from 4.99% to 5% to 5.01%.
Validity of the findings: The Introduction presents the problem well. The Discussion and conclusion expand on it and present the solution, very well. The proposed solution is well grounded on the results. Replication is possible using the R script and the data is provided. Overall, the author provides a very convincing case.
Additional comments: Lines 30-31: “… the first component (PC1) accounts for most of the variance in the data …”. The use of “most” implies that pc1 accounts for more variance than all the other pcs pooled together, which is false. In an hypothetical example, accounting for 40% of the variance, pc1 may account for the largest chunk of variation, but it is certainly not the most as 60% are still unaccounted for.

Line 32: pc scores are usually called z-scores, but can also be called l-scores, depending on the use of covariance or correlation matrices and the standardization method (see Jackson, 1991). At least mention the z-score nomenclature.

Line 34: … can be used for bi- or tri-ordination plots. I personally dislike this solution as, to the unspecialized reader, it suggests some sort of correlation between successive pcs. This is exactly the opposite of what PCA does, extracting pcs subject to the restriction of being orthogonal (or orthonormal if the correlation matrix is used). (see suggested literature on PCA method).

Line 35-37: Very true. And much better alternative to Canonical Correlation Analysis, since there is nothing in nature compelling pc1 from the abiotic variables to be better correlated with pc1 of the biotic variables, pc2 from the abiotic variables to be better correlated with pc2 of the biotic variables, and so forth, as was well presented by Manly (1986) and by Jackson (1991), and well demonstrated by Cabaço et al. (2008) on the analysis of Zostera marina in Ria de Faro.

Line 41-42: This demonstration rouse way before Björklund (2019). At least by Stauffer et al (1985), Jackson (1993), Dijkseterhuis and Heiser (1995), Peres-Neto et al (2005), Vieira (2012), Jolliffe and Cadima (2016) and Vitale (2017). Hence, this sentence written “as is” and only with this citation, is false and inadmissible. The previous authors deserve the due credit.

This problem repeats throughout the manuscript. I will restrain from correcting every single incorrect citation. I leave that task for the author; who must revise extensively the citations and the criteria used to chose them.

Lines 42-45: This is a very important aspect to analyse the data from Ringnér (2008) and discuss it. But it belongs there, in the results and discussion. In the introduction, it is a bad example since the researcher has always the option to transpose the data matrix and choose between an R-mode (focus on the variables) or Q-mode PCA (focus on the observations) (see for example Lee et al. 2017). In such case, the observations act as variables and each pc weights the observations. Z-scores are estimated for each variable. The results from R-mode and Q-mode PCA applied to the same data set come hand-in-hand and can be easily transposed. Furthermore, see argumentation by Jolliffe and Cadima (2016). The fundamental aspect of these lines should be that, even if all variables are uncorrelated, just due to random error, larger and smaller pcs will inevitably arise, implying pcs higher than 1 that still only report to random associations.

Lines 72-73:  This is false in the sense that the permutation procedure was already proposed by Vieira (2012), and by others before him (see section above).

Line 73: “Vieira”

Line 99: this statistic was developed by Vieira (2012) and not by Björklund (2019).

Line 105-106: True, but still a poor and partial explanation. The fundamental aspect is that significant pcs represent factual associations between variables, whereas non-significant pcs represent only random associations.

Line 106-107: good choice. But it should be explained why: an α level is only a probability of being wright or wrong when making a decision of accepting or rejecting a null hypothesis. Hence, p<0.05 is not a warranty of rejection nor is p>0.05 a warranty of its acceptance. Statistical methods are tools helping the decision-maker, and not dogmas around 0.05 α levels. The last call should always belong to the human in charge, and the impact of making that call must always be crucial to the decision-making process.

Line 146-154: More information about the structure of the artificial data should be presented. Was the PCA done over the correlation matrix or the covariance matrix?

Line 191-192: This is the first, fundamental sentence setting the tone to the discussion. Yet, it completely misses the global picture by focusing exclusively on the topic of Life Evolution. Incorrect PCA application is spread all over sciences, including all topics covered by PeerJ.


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

Input:
Title: PCATEST: TESTING THE STATISTICAL SIGNIFICANCE OF PRINCIPAL COMPONENT ANALYSIS IN R
Review round: 2
Reviewer: 2

Output:
Basic reporting: There is a conflation of permutation tests and bootstrap throughout the article.   A permutation test involves a choice of a null hypothesis, which is unclear throughout the manuscript; the author simply refers to random noise without giving it a distribution or probability.  A bootstrap, by contrast, is a means for quantifying the uncertainty in the parameter.  The author needs to define these terms and goals much more carefully in the paper.
Experimental design: The author evaluated the bootstrapped PCA procedure and permutation PCA procedure on simulated and real datasets of varying complexity.

1.  For the simulated datasets, the authors needs to make clear what true model is from a latent factor perspective.  I think that the covariance matrix S with exchangeable correlation is equivalent to a

2.  Concluding something from one simulated dataset about the frequentist properties of a procedure is not sufficient.  The author needs to simulate datasets a certain number of times and average the estimators over the simulations.

3.  There is a sign issue with PCA in that  one can multiply all components of the PC's by negative one and still explain the same amount of variation.  This issue is not addressed.

4. It is unclear what knowledge gap the article addresses.
Validity of the findings: 1. Given the simulated data, the author needs to perform repeated simulation data generation rather than a single dataset generation.

2. R code is available to reproduce the findings.

3.  The conclusion that there needs to be better PCA analyses is an appropriate one.   However, the literature search the author was not systematic.
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: PCATEST: TESTING THE STATISTICAL SIGNIFICANCE OF PRINCIPAL COMPONENT ANALYSIS IN R
Review round: 3
Reviewer: 1

Output:
Basic reporting: The manuscript is much better now, and I congratulate the author for it. I can only wish that finally ecologists start being more careful with their applications of PCA. This manuscript can certainly give a contribution to it.
It is almost ready for publication. But I still see a few problems. Some sentences in the introduction are too long, becoming hard to follow and leading to confusion. Lines 67-71 or 80-85 are good examples. I suggest for the author to, generally speaking, try to use shorter sentences.
Some incorrect citations, persist. I will give the examples. In some cases, they are simply incorrect. In other cases, I personally would give more than one reference i.e., at least the original together with some very commonly red and cited.
Line 30: Before Cadima and Jollife (2016), the same had already been showed at least by Manly (1986), Jackson (1991) and Vieira (2012).
Line 38: The orthogonality of pc axis had already been explained at least by Manly (1986) and Jackson (1991).
Line 40-42: deserves references as Manly (1986), Jackson (1991) and Vieira (2012).
Lines 44 to 69: Manly (1986) and Vieira (2012) are actually better references than the ones used. Manly (1986) debated this earlier while Vieira (2012) debated this deeper.
Lines 55 to 57 about the SCREE Plot are repeated in lines 62 to 65. Or is there a small difference between them? A comparison against randomly generated data, in the second case? But then, separating both cases with other text in-between might not be a good idea. Vieira (2012 - Fig 2) demonstrated this SCREE Plot before Bro and Smilde (2014), having taken it from Mainly (1986) and (Jackson (1991).
Lines 58-59: Again, Manly (1986), Jackson (1991) and Vieira (2012), among many others, already debated this before Bjorklund (2019).
Lines 58-59: Besides Ringer (2008), Manly (1986), Jackson (1991) and Vieira (2012), among many others, also debated this. With a big difference that Ringer (2008) only applied to a data set whereas Manly (1986), Jackson (1991) and Vieira (2012) debated about the validity of its application, debates that were inserted in whole articles or books about the PCA method.
Lines 67-71: the author mixes in the same sentence two entirely different issues, generating a sentence that is incorrect:
i) ad-hoc tests rely on false assumptions and generally provide incorrect results leading to false conclusions. Thus, randomization tests are better suited.
ii) Among randomization tests, there are two classes: Permutation tests propose a null hypothesis and estimate significances by randomly redistributing observations breaking correlations among variables. Bootstrap and Jacknife estimate confidence intervals by randomly resampling observations without breaking correlations among variables
Consequently, the claim “Because these ad hoc procedures  …Vitale et al. (2017) suggested that permutation-based tests are better suited than other randomization approaches …” is incorrect.
Or then, it is me not quite understanding the text. The author mentions the ad hoc tests and statistical procedures. But the methods described above are generally not “statistical”.
Line 80-85: explain the concept in several, shorter sentences.
Lines 87-89: True that the metric   was proposed by Gleason & Staelin (1975) whereas  was proposed by (Vieira, 2012). However, both where proposed to be applied with permutation tests, along with other 5 metrics, by Vieira (2012) i.e., before Bjorklund (2019).

Overall, I strongly encourage the author to read carefully Vieira (2012) in order to be better informed about who did what. In such case, the author will find information that is very helpful for the problem about “axis reflection”:
Axis reflexion i.e., the permutation of signs among loadings and eigenvalues, and the implications it brings for their ranking in randomization methods, was first debated by Jackson (1995), Mehlman et al. (1995) and Peres-Neto et al. (2003 and 2005). This problem was solved by Vieira (2012) in two ways: (i) when using the “index of the loadings”, both the loadings and the eigenvalue are squared. Therefore, their original sign is irrelevant for the subsequent ranking. (ii) when using the “correlation coefficient” between pc and variable, Vieira (2012) tested ranking according to the absolute value.

Given that the present R-software is an adaptation from the Matlab software by Vieira (2012), this R-software should also be robust against axis reflection.

Lines 96-101: I may be wrong, but my feeling is that the major difference between Matlab and R is that the first is paid whereas the second is free-ware.

Line 162: “Index of the loadings”, right?

The text in lines 177 to 192, relating to Figs 1-3, should also have a table showing the eigenvalues, and loadings of each pc, and the significant ones highlighted (in bold or in color). This would help the reader understand the real example demonstrating the theory.
The above applies also to the analysis of the real data sets.

The figure from the analysis run in Q-mode should also be in the main text, and not supplementary material, and striking better that, in cases like this, Q-mode should be preferred to R-mode.

The analysis and the examples are very good. Bulls-eye! The Results and Discussion are very well written. I would not do better! My congratulations to the author.

Raw data and software are shared. The article is self-contained with relevant results and hypothesis.

Best wishes
Experimental design: Weaknesses from former submission were solved. Currently, all checks.
Validity of the findings: Weaknesses from former submission were solved. Currently, all checks.
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: PCATEST: TESTING THE STATISTICAL SIGNIFICANCE OF PRINCIPAL COMPONENT ANALYSIS IN R
Review round: 3
Reviewer: 2

Output:
Basic reporting: The author presents much improved work in the revision.  I have nothing to add.
Experimental design: The author presents much improved work in the revision.  I have nothing to add.
Validity of the findings: The author presents much improved work in the revision.  I have nothing to add.
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: MOLECULAR CHARACTERIZATION AND EXPRESSION PROFILING OF TRANSFORMER 2 AND FRUITLESS-LIKE HOMOLOGS IN THE BLACK TIGER SHRIMP, PENAEUS MONODON
Review round: 1
Reviewer: 1

Output:
Basic reporting: The MS analyses in the sequences of two genes related to sex determination in insects, tra-2 and fru, as well as their phylogenetic position and their temporal and spatial expression patterns in P. monodon.

The authors suggest that both tra-2 and fru could be involved in sex determination pathway of Penaeus monodon, one of the most traded shrimp species. Previously, several pieces of evidence have been similarly reported that tra-2 and fru may play the same role in sex determination of some crustaceans as they do in insects. However, other studies have indicated the opposite. Therefore, given that sex determination/differentiation in crustaceans is still very puzzling, new findings related to this are very interesting, especially when they come from an aquaculture species.

The MS is well written and easy to read.
Figures and tables are well presented.
A little more background/context might help non-specialist readers to better understand the difficulties in studying sex-related genes in crustaceans.
Minor comments are included in the PDF version.
Experimental design: The MS describes the sequences of an ovarian tra-2 and two fru isoforms from P. monodon.

As for phylogenetic analyses, I would say there is much room for improvement. Maximum likelihood trees and/or Bayesian trees would be preferable to neighbor-joining trees built with MEGA. Detailed information is not provided either on codon partitioning schemes or on evolutionary models. Therefore, I encourage the authors to re-run the phylogenetic analyses using more sophisticated software to obtain more robust results. In addition, more both tra-2 and fru crustacean sequences could be added to the datasets to further clarify the phylogenetic relationships of P. monodon with other crustacean/insect species, if that is one of the goals. Sequences of these genes from taxa closer to P. monodon than to E. sinensis are currently available from NCBI and could be useful. Finally, I suggest that should specify which species will be used to root the trees appropriately.

As for the RT-PCR and RT-qPCR assays, they are well done. The only drawback is that they have only used one housekeeping gene (16S rRNA), while it is strongly recommended to use more than one reference gene, and even more so if it is a mitochondrial one. However, I assume that redoing these assays is not feasible at the moment.
Validity of the findings: Novel and interesting findings are provided, as new data will be useful to further unravel sex determination in crustaceans and in particular in P. monodon.

The expression of tra-2 and fru throughout the development of P. monodon is particularly interesting, as well as their differential expression between ovary and testis.

My main concern is the validity of the phylogenetic discussion inferred from the neighbor-joining trees. Indeed, the split between P. monodon Ovatra2 and an earlier P. monodon tra2 sequence is unsupported (bootstrap=48). Therefore, these clades are not feasible. I strongly recommend re-running these analyses under ML/BI to check this. Furthermore, based on the tra-2 tree, it is proposed that the new Ovatra2 sequence in P. monodon may be involved in sexual development and that it is a paralog of the former tra2 gene. Certainly, if this is the actual phylogenetic position of Ovatra2 and tra2, further discussion of the different genomic loci would be desirable in the Discussion section.
Additional comments: The sequences of Ovatra2 and fru isoforms were obtained from an unpublished transcriptome database. I wonder why the remaining components of the Sxl-Tra/Tra2-Fru/Dsx sex determination pathways were not also included in this work, especially in the case of the sex-lethal master gene. An explanation for this would be interesting

Finally, there is evidence that this pathway (including tra-2 and fru) could be involved in sex determination in several crustacean species as in P. monodon. Further references to such work would be desirable for the sake of clarity and also to support the findings of the present MS. At the same time, there is literature arguing that tra-2 and fru have not shown any evidence of participating in sex determination of other Decapoda (see review in Chandler et al. 2017). Therefore, I suggest authors to include more references for and against tra-2 and fru as sex regulators among different decapods to illustrate the crux of sex determination in Crustacea.


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: MOLECULAR CHARACTERIZATION AND EXPRESSION PROFILING OF TRANSFORMER 2 AND FRUITLESS-LIKE HOMOLOGS IN THE BLACK TIGER SHRIMP, PENAEUS MONODON
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors report the full cDNA of three transcripts which were named PmOvtra2, Pmfru-1 and Pmfru-2. These transcripts were obtained from a transcriptome analysis that the authors claims to have performed. In this work the authors also evaluated the expression patterns of these genes and also quantified their relative abundances. Although a previous tra2 gene had been reported in this species, the authors argue that PmOvtra2 is different than the previously published. Into this context, I believe the current work gives new information about the likely participation of these genes in the sex differentiation pathways in this shrimp. However, I find some issues that need to be further addressed by the authors in order to improve the manuscript.
Introduction:
The authors point out works performed in the M. nipponense prawn but they are leaving out works done in one of the most worldwide cultivated shrimp, Penaeus vannamei. I suggest to the authors to check the following works which could give a broader perspective of sex differentiations genes that have been reported in shrimps: Lopez-Cuadros et al., 2018 (Isolation of the sex-determining gene Sex-lethal (Sxl) in Penaeus (Litopenaeus) vannamei (Boone, 1931) and characterization of its embryogenic, gametogenic, and tissue-specific expression). Galindo-Torres et al., 2019 (A natural antisense transcript of the fem-1 gene was found expressed in female gonads during the characterization, expression profile, and cellular localization of the fem-1 gene in Pacific white shrimp Penaeus vannamei). Even the authors did not mention a work performed by Robinson et al. (2014) in which the authors found that in P. monodon, the ortholog to the C. elegans fem-1 sex differentiation gene, mapped to the position where a sex-determining locus was predicted by a QTL analysis. I also think the authors need to go deeper in the chromosomal setting that shrimps seem to have (see: Li et al., 2003; Robinson et al., 2014; Staelens et al., 2008; Zhang et al., 2007; Yu et al., 2017; Perez-Enriquez, et al., 2020; Wang et al., 2021).
Materials and methods:
Although it is possible to see in the supplementary files how many biological replicas were used, the authors should mention that in the methods section. The authors did not mention how many organisms were used in each step. How many biological replicates were set for qPCR? Did the authors perform pools at the end point PCR?. Did the authors use a single organism to run end point PCR? All this part lacks of clarity.
I found the qPCR section with some issues. It is important to take into account that reference gene expression is known to vary considerably depending on the biological scenario (i.e. different tissues, maturity stage, etc.). It’s for that reason that a reference gene-stability validation is always recommended for each experimental design. It is a common practice to normalize using pre-selected reference genes, but it does not mean it is the best way. In this case, the authors are assuming that the expression of 16s rRNA is stable among all the tissues evaluated (N,Z,M,PL1,PL5,PL10,PL15 and among ovary and testis). I would recommend select at least five reference genes and evaluate their stability through GeNorm, NormFinder, bestkeeper, etc. The authors claimed they have a transcriptome of this species where they could search for potential reference genes to be validated. At the end, the authors should identify what of those five genes were the most stable in the differents scenarios they are evaluating. Moreover, they should calculate efficiency in an individual way for all of the genes, because of in practice is well known that to assume 100% efficiency is not true at all (see Vandesompele et al., 2002). On the other hand, if the authors know a reference where it has been demonstrated that the expression of r16s is found stable in all of the tissues they evaluated they should point it out.
Result:
In the recently reported P. vannamei genome (Zhang et al.,2019), there are three proteins annotated as tra2 and tra-2C. I believe these proteins should be included into the analyses performed by the authors. In fact, when I perform a blastx of the PmOvtra, it hits against P. vannamei tra.
Discussion.
Due to the pathway of sex differentiation in shrimps seem to be a mixed of genes found in D. melanogaster and C. elegans pathways of sex differentiation, I consider the authors should enlarge the discussion content. For example, including works performed in P. vannamei or even in the same P. monodon (See references pointed out in the introduction).
Other minor points:
There isn’t an accession number for any of the Pm Fruitless isoform reported, why?
Line 303: The authors use “tra” to refer a protein. Generally, a name of protein is written with the first letter capitalized or all the letters capitalized like Tra or TRA protein. Is this correct?
Line 306: The authors mention the existence of a previous work in which a tra2 gene is reported. Why didn't the authors mention this work in the introduction section?
Line 315: What does mean PM?
General comments
Line 142-143: Add the Nanodrop company name.
Terminology needs further attention. I would suggest to the authors to change the terminology used to refer to real time PCR and end point PCR. See Bustin et al., 2009 (The MIQE Guidelines:Minimum Information for Publication of Quantitative Real-Time PCR Experiments).
Why do standard deviation bars are big in figure 8? It seems that few biological replicates were used or why do the dispersions are varying considerably?. When I see the supplementary files, the Cts of these genes are not varying so much, so, why do dispersions are big?


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: MOLECULAR CHARACTERIZATION AND EXPRESSION PROFILING OF TRANSFORMER 2 AND FRUITLESS-LIKE HOMOLOGS IN THE BLACK TIGER SHRIMP, PENAEUS MONODON
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: qPCR issues.
I still find some issues that need to be addressed by the authors. The authors performed a gene stability analyses using the standard deviation as an indicator. At the end they ended up using the 16s gene as the most stable gene even though actin gene showed the lowest sd value in both, ovary-testis, and during larvae development (supplementary file 3 and 4). Why did the authors use 16s gene when it showed the higher sd value in both valuations?.  Moreover, the authors should add in the materials and method section if they performed a melting curve analysis to prove one product for each amplicon was got.  Finally, the authors should add the description of the analysis they performed to conclude 16s was the most stable gene comparing against ef1 and actin in the qPCR method section.

Result and discussion section.
In the genome of P. monodon there are 8 proteins annotated as “sex determination protein fruitless-like”, from which 4 (XP_03779110{3..6}.1) are identical and three more show different protein sequence (XP_037772638.1, XP_037799023.1, XP_037799203.1 and XP_037799203.1). These proteins are found in a different scaffold than the  fruit isoforms reported by the authors. In fact the sequence reported by the authors is found annotated as “longitudinals lacking protein”. I believe the authors should add these sequences to the phylogeny analysis or to mention why they weren’t included. The same for the Tra sequence, there are some Tra proteins annotated and some show slight differences. I believe the authors should discuss about this.

Minor points.
Line 38: Change “spiced” by spliced.
Line 57: split “Incrustace” to in crustace.
Line 76-77: Check if tra-tra2 interaction does not occur in males
Line 183: The authors say they used 5 adult females and males, but in supplementary data 3, only 4 biological samples appear.
Line 215: change “tra proteins” for Tra proteins.
Line 274:  add the name of the scaffold.
Figure 3: I suggest to the authors to improve the quality of the figure and add the genes names for clarity. For example, there are two P. vannamei proteins but without gene name (are these tra-2C?), it is hard to follow the idea. The same for figure 4.
Line 323: The author should homologue terms, here they refer to qpcr, and in other parts of the document as RT-qPCR.
In table 3, the author should add the size of the PCR product as it’s simplest to see them in a table.


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: MOLECULAR CHARACTERIZATION AND EXPRESSION PROFILING OF TRANSFORMER 2 AND FRUITLESS-LIKE HOMOLOGS IN THE BLACK TIGER SHRIMP, PENAEUS MONODON
Review round: 3
Reviewer: 1

Output:
Basic reporting: The manuscript has improved after revision. However, I found it difficult to check whether the authors followed my earlier recommendations, as the rebuttal letter available to me includes the responses to the other reviewer and not to my comments. For next time, I recommend a single letter with the responses to all reviewers in order to avoid these problems.

My main concerns remain the methodology, interpretation, and discussion of the phylogenetic analyses.
Experimental design: The authors have replaced the neighbor-joining trees of the previous version with ML trees built in RaxML.

They used MEGA to determine the best substitution model. Nevertheless, the number of models included in MEGA is very limited, so I recommend other software, such us jModelTest. In addition, although tra-2 and fru are both coding genes, whether their three partitions follow the same evolutionary model or not is not explored with specific software (e.g. PartitionFinder).

Human sequences are used as outgroups, which I consider inappropriate. Outgroups should not be too far from the study group (crustaceans). Therefore, the non-crustacean sequences could serve perfectly well as outgroups. I would eliminate human sequences as they could cause biases in the analyses.
Validity of the findings: Correction of these aspects of the phylogenetic analysis could lead to more robust results. The separation between the ovatra-2 and tra-2 clades remains unsupported (bootstrap = 74 < 90). I believe that improving the methodology of phylogenetic analyses probably could provide more support for these clades, which is vital for discussing the results.

Another issue that concerns me is that throughout section 3.2 of the Results and in the Discussion, the authors refer bootstrap as “percentage identity” and “similarity”, when the bootstrap values in a tree are not that. Bootstrap is a statistical measure of the support that the nodes/clades have in a phylogeny. I recommend re-writing these sections to avoid referring to bootstrap support of phylogenetic trees in this way because it is a serious error.
Additional comments: Here are some minor issues:
- References to the software used in the Materials and Methods section are still missing.
- Carefully review the use of italics when referring to gene/protein sequences because I notice some inconsistency.


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: MOLECULAR CHARACTERIZATION AND EXPRESSION PROFILING OF TRANSFORMER 2 AND FRUITLESS-LIKE HOMOLOGS IN THE BLACK TIGER SHRIMP, PENAEUS MONODON
Review round: 3
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: qPCR.
The authors claim that 16s is the most stable gene, but for example, in the supplement file 4, the lowest standard deviation was for actin. In the case of ovary and testis, the author should calculate this value using all the samples (ovary and testis) because they are comparing genes between these two tissues, so the reference gene should be stable in these two. Again if I calculate the sd in this case, the lowest value is assigned to the actin gene, which gives 0.97.

Minor points
line 74: Change sxl protein for Sxl protein.
Line 188: The author say they pooled around 30 individuals, is this correct? Or did they mean to say 3?


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: MOLECULAR CHARACTERIZATION AND EXPRESSION PROFILING OF TRANSFORMER 2 AND FRUITLESS-LIKE HOMOLOGS IN THE BLACK TIGER SHRIMP, PENAEUS MONODON
Review round: 4
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Thank you for amending the MS following 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: MOLECULAR CHARACTERIZATION AND EXPRESSION PROFILING OF TRANSFORMER 2 AND FRUITLESS-LIKE HOMOLOGS IN THE BLACK TIGER SHRIMP, PENAEUS MONODON
Review round: 4
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The author claims that they desired to use 16s rRNA as a reference gene even when it wasn’t the most stable gene. The authors argue that this gene might have variations in expression since some particular function during spermiogenesis among others has been reported for this gene. First of all, if the author knew this why did the authors select this gene to be tested for gene stability?.  Although the authors claim this gene might have a bias because of its function in male spermiogenesis, the SD values calculated by the authors are indicating that the gene isn’t responding with a bias in gene expression. If this claim were true, the SD value would be higher.
I didn’t find any reasonable reason why the author is using 16s rRNA. I get the impression the authors did the stability procedure just as protocol but not when the goal of identifying the most stable gene. Given the qPCR is a strong component of the manuscript, and that the qPCR lacks scientific rigor, I would reject 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: MOLECULAR CHARACTERIZATION AND EXPRESSION PROFILING OF TRANSFORMER 2 AND FRUITLESS-LIKE HOMOLOGS IN THE BLACK TIGER SHRIMP, PENAEUS MONODON
Review round: 4
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Overall, the authors have improved the manuscript following previous review comments. The current form can be accepted.

Regards


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

Input:
Title: THE RELATIONSHIPS BETWEEN TOAD BEHAVIOUR, ANTIPREDATOR DEFENCES, AND SPATIAL AND SEXUAL VARIATION IN PREDATION PRESSURE
Review round: 1
Reviewer: 1

Output:
Basic reporting: This work represents a clearly and in professionally written manuscript. The author examined how morphological, chemical, behavioral, and performance anti-predator traits are correlated and differ spatially and between sexes in a toad species. They found that body mass was negatively related to the behavioral traits examined, whereas parotoid gland area and sprint speed were not related to those behaviors. Furthermore, females showed riskier behavior than males but there was no difference in behavior between the “natural” and agrosystem habitat. Larger toads are more conspicuous to predators that are mostly gape-limited in this system, which might explain the correlation between body mass and behavior. Further, males might be more subjected to predation than females which might explain why females exhibited riskier behavior.
The author clearly is well-experienced with this system given the list of publications (some of which are by and with the author) and provides a necessary background. One figure was provided with this manuscript which I suggest to expand by adding habitat as a factor (see “Additional comments”).
Experimental design: How different traits are integrated is an important question to understand how complex phenotypes evolve. Especially in amphibians that often use different modes of defenses (i.e., behavioral, chemical, morphological, …) this question is relevant but seldomly studied. The author manages to “disentangle” the behaviors activity, exploration, and boldness by applying methods previously used in other studies examining these behaviors in amphibians. However, I believe the reviews by Réale et al. (2007) and Kelleher et al. (2018) should be referenced here, and how methodologies follow or diverge from the guidelines outlined in these reviews should be mentioned in the methods.
Réale D, Reader SM, Sol D, McDougall PT, Dingemanse NJ (2007) Integrating animal temperament within ecology and evolution. Biol Rev 82:291–318. https://doi.org/10.1111/j.1469-185X.2007.00010.x
Kelleher SR, Silla AJ, Byrne PG (2018) Animal personality and behavioral syndromes in amphibians: a review of the evidence, experimental approaches, and implications for conservation. Behav Ecol Sociobiol 72:79. https://doi.org/10.1007/s00265-018-2493-7
Validity of the findings: Given the application of a Principle Component Analysis, any correlation of the behavioral traits was removed, making the analysis statistically robust. The author makes well-stated conclusions on the basis of their results. Building up on previous studies, the author argues that male toads and larger toads are under higher predation threat which transpired in more-pronounced anti-predator traits. One constraint that I see with this work is that the exact quality of predation is unknown. Different predator species apply different senses and modes to detect prey and use different tactics to consume prey, resulting in diverging selection for anti-predator traits. In this system there is only limited knowledge about spatial and sexual differences in predator composition, limiting the conclusions that can confidently be drawn. However, I understand that this was not the main question studied here. Further, the author makes sure to address this point in the discussion.
Additional comments: I have noted some minor issues with the editing of the text that I would like to state here.
Line 50-51: I suggest changing “… how the diversity of contexts animals can be faced with could be key in …” to “… how the diversity of contexts that animals can face could be key in …” for better readability.
Line 77-78: I suggest changing to “Indeed, how animals make use of space affects …”.
Line 91: “With” instead of “of”.
Line 93: Change start of sentence to “Frequently concomitant with these substances are …”.
Line 94: Change “to” with “with”.
Line 102: “Increases” instead of “Increase”
Line 103: Use for example “However, …” instead of “Even, …”.
Line 174: Remove “in”.
Line 197: “Remainder” instead of “Remain”
Line 225: Can you please state the exact time frame that these experiments occurred within? E.g., “between 10pm and 2am”.
Line 300: Do you mean “behavioral traits measured that were correlated”?
Line 302: See above.
Line 323-324: Did you forget “was” after “PCb”? Also, I suggest stating that “PCb” was positively correlated with boldness for easier readability.
Line 333: One empty line too much in front of “Χ2”.
Figure 1: I suggest adding in the factor of habitat. Even though non-significant, habitat differences in anti-predator traits have been a major part of the research question, and thus should be presented in the figure.


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

Input:
Title: THE RELATIONSHIPS BETWEEN TOAD BEHAVIOUR, ANTIPREDATOR DEFENCES, AND SPATIAL AND SEXUAL VARIATION IN PREDATION PRESSURE
Review round: 1
Reviewer: 2

Output:
Basic reporting: This study investigates the relationship between sex, morphology, and colouration on the antipredator behaviours of natterjack toads from two contrasting habitats. The study is straightforward and well written with some minor grammatical changes needed (see general comments for my suggestions), but otherwise quite clear.

The background information on the topic is mostly sufficient, though I think it is missing some citations on amphibian (and specifically, toad) anti-predator behaviors or parotoid gland morphology. I have suggested a few relevant citations (again, see general comments), but there are more out there that could be relevant to the discussion. A discussion of whether toads are capable of self-assessment with regards to gland size / toxicity could also be helpful to the reader. Also further discussion on the sexual differences in behavior / morphology would be insightful, as the pattern appears to be different from my experience with other bufonid species (i.e. males are more dispersive and therefore bolder, females have larger toxin glands). Contrasting the findings here on E. calamita with those of other bufonids would perhaps shed light on the way that selection from different predators drives antipredator trait/behaviour evolution.

I’m happy with the way the analyses were conducted and presented at this time, so I have no changes to suggest in that regard. Some of the raw data (e.g. parotoid gland area, sprint speed, and number of squares visited during the behavioural trials) appear to be absent however, so I would request that it be added for reproducibility (either now or upon publication).
Experimental design: The experimental design is appropriate to answer the questions posed, and the data collected is sufficient to do so. The methodology is well described and could be repeated, however I would request some additional details to be more explicit (e.g. the timeline between animal capture and each experiment; again, see comments below).

From an ethical point of view, the experiment is unlikely to cause long-lasting stress for the animals, and adheres to the rules outlined by the study permits. The only issue that I can see is that the acclimation time before exploration trials (2 minutes) might be too short (I would advise longer acclimation times in future work). The toads were likely still affected by experimenter handling, and this may have influenced their behaviour during the trials.
Validity of the findings: The findings appear to be statistically sound, and well presented. They expand upon previous work in the system and link back to the original hypotheses. The study design is also well balanced between sexes and treatments. As mentioned in part 1 above, not all raw data used appears to be provided, so that is one place where I must request additional information.
Additional comments: Line 41: Change “reproduction” to reproductive

Line 44-45: Rephrase to “Indeed, selection on behaviour can hardly be regarded as directional”

Line 48-49: Rephrase to “exploratory behaviour has a consistently negative relationship”

Line 93-96: Rephrase to “Aposematic coloration (i.e. conspicuous colors and patterns that potential predators associate to unpalatability and actively avoid) can be frequently found alongside chemical deterrents (citations), thus providing their carriers with additional defense from predators (other citations).”

Line 98: Rephrase to “The relationship between body size and predation is particularly interesting.” – or something similar

Line 98 – I think this paragraph could use additional discussion of gape limited vs. unconstrained predators. This comes up later in the discussion, and is suggested to be the reason why larger toads are less bold, so I think the idea should be raised earlier in the introduction.

Line 147 – I would change “reptiles” to snakes, because the predation mode by snakes here is important for the context and both example predators listed are snakes. Unless there are also lizard predators that are relevant for E. calamita? But I am not aware of any.

Line 176 – Please include a statement of how long animals were held in captivity before the trials. I believe later on in the text it states that photographs were taken 24h post capture, and behavioural trials 24h after that? I think a clearer timeline here would be helpful and not require much additional text.

Line 190 – Parasitic lighting might be a photography term that I'm unaware of? If so, keep it! If not, I would say "In order to avoid interference from other light sources, photos were taken ... etc"

Line 218 – Again, I would explicitly state the timeline, either here or above (see previous comment)

Line 262 – Did any toads exude toxin during sprint trials? Was this information recorded? Speed here is considered in the context of an antipredator response where faster individuals are also bolder, however individuals that stand their ground against a predator and rely on toxin could also be considered “bold” as well. See:

Edelaar P, Serrano D, Carrete M, Blas J, Potti J, Tella JL (2012) Tonic immobility is a measure of boldness toward predators: an application of Bayesian structural equation modeling. Behav Ecol 23:619– 626

Hudson CM, Brown GP, Shine R. Evolutionary shifts in anti-predator responses of invasive cane toads (Rhinella marina). Behav Ecol Sociobiol. 2017;71: 134.

Line 341 – I would like to see some more amphibian specific locomotion studies here (e.g. speed to body size relationships). Perhaps the unique movement style of E. calamita differs from other amphibians/bufonids. Did you measure any other morphological traits (e.g. limb length) that could be relevant for speed?

Line 358 - Perhaps cite and discuss this MS:

Blennerhassett RA, Bell-Anderson K, Shine R, Brown GP. The cost of chemical defence: the impact of toxin depletion on growth and behaviour of cane toads (Rhinella marina). Proceedings of the Royal Society B: Biological Sciences. 2019;286: 20190867.

In the above study toads changed their activity level when glands were recently emptied, thus they may not be assessing their predation risk by their own gland size, but rather how recently they have deployed toxin / how much is available to them.

Line 383 - This is interesting given that other toad exploration / antipredator studies found no difference between sexes in cane toads and guttural toads, perhaps expand on this? See studies below.

Gruber J, Brown G, Whiting MJ, Shine R. Behavioural divergence during biological invasions: a study of cane toads (Rhinella marina) from contrasting environments in Hawai’i. R Soc Open Sci. 2018;5: 180197.

Baxter-Gilbert J, Riley JL, Measey J. Fortune favors the bold toad: urban-derived behavioral traits may provide advantages for invasive amphibian populations. Behav Ecol Sociobiol. 2021;75: 130.


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

Input:
Title: THE RELATIONSHIPS BETWEEN TOAD BEHAVIOUR, ANTIPREDATOR DEFENCES, AND SPATIAL AND SEXUAL VARIATION IN PREDATION PRESSURE
Review round: 1
Reviewer: 3

Output:
Basic reporting: I find this manuscript to be generally well written and structured, though some sentences need improved clarity of expression (listed below, I have tried to provide suggestions). There are sufficient references, and a reasonable study background has been provided to contextualize results. The manuscript figures and tables are neatly represented.

I list lines below that I would recommend improving (suggestions only):
Ln 23: change “as well” to “also”, as the former can be interpreted in multiple ways.
Ln 45: “Indeed, hardly can selection on behaviour be regarded as directional, since the fitness consequences of a given value of any behavioural trait are context-dependent.” This sentence is difficult to read, consider reversing sentence structure here for readability, or shortening the second half of the sentence.
Ln 66: change “sharks with a more exploratory behaviour” to “sharks that exhibit more exploratory behaviour”
Ln 68-71: you provide two examples here of why “the relationships between predation risk and behavioural traits may transcend plasticity,” however do not provide enough information to explain how these results don’t allow for a role for plasticity, and are different from examples above. Is it that they were done under a common garden setup? If so I would explicitly mention this.
Ln 93: “Concomitant to these substances can frequently be found aposematic colorations” sentence structure is hard to read.
 Ln 94: change “associate to” to “associate with”
Ln 113: remove “One of them was locomotion”, and add to the end of the previous sentence: “with other antipredator strategies, including locomotion.”
Ln 115: You frequently use the word “moreover”, consider changing some to “furthermore” or “additionally”.
Ln 131: consider changing “… massive, and have larger…”  to “… massive, or have larger…”  as you test some of these things independently from one another.
Ln 144: Change “under” to “during”
Ln 158: Use of the word “formation” here is ambiguous, do you mean the forest itself, the habitat structure, or something else?
Ln 207: would recommend changing “Then, after calibrating length thanks to the piece of graph paper…” to “I calibrated length using the piece of graph paper, and manually outlined…”
Ln 357: change “did not involve” to “was to associated with”
Experimental design: The experimental design is sound, and the aims and scope appropriate for this journal. The research question is well presented both within the context of the study and regarding the background information used to direct it. However, there are some aspects of the methods that need clarification or further detail for repeatability. I have tried to provide suggestions below for improved method presentation where I can.

Additional results needing representation:
- In particular, I found the ANCOVAs (how many were run and the exact predictor/response variables in each) quite hard to follow. Please clarify this in both the Methods and Results body text. Results of the ANCOVAs should be reported in tables in the main manuscript, and this will also increase clarity.
- I would recommend including the outputs of the PCAs, including eigen values for both retained and not retained PCs, in the supplementary materials. You may also want to include a visual of the PCAs.
- I would recommend making significant p-values stand out more in all relevant graphs by e.g. making them bold.

Some further notes on improving experimental reporting:
Ln 43: “boldness or exploratory” I would be explicit here as to whether these terms are interchangeable, and if not what defines them within this manuscript.
Ln 181-198: A simple diagram figure may help convey experimental setup, and would be useful for anyone wishing to replicate these methods.
Ln 196: “calibrate longitude”, do you mean “calibrate length” as you use this term on ln 207.
Ln 212: Please be explicit here what “dorsum” refers to. Does this include legs and arms? If so, how did you ensure that limb placement didn’t impact the results?
Ln 267: How was the camera affixed for sprint speed trials (height/angle)? Please mention this in Ln 218-226. Is it at all possible that the angel the camera was recording at had an impact on estimating when the toad snout crossed the white lines (larger toads would be more affected by viewing angle if the camera was low down)?
Ln 273: “I also set a dark background at one end of the raceway, which could be viewed as a shelter and encourage toads’ moving forward” Please add a reference for this statement if one is available.
Ln 301: regarding the external squares visits/visited ratio, in line 256-257, you call this “internal squared” not “external squares”
Ln 305-308: If you have already defined the PC’s greater than 1 for both PCA 1 and 2, then refer to them by their names here (i.e. PCa, PCb). Also turn this into two sentences, and make very clear how many ANCOVAs were run and for which variables, as I found this section confusing.
Ln 330-338: You do not mention the PC for PCA2 (called PC1 I think? Table 2).
Figure 1: Include in figure legend the interpretation of PCa (what do high and low values mean).
Validity of the findings: The findings presented in this manuscript are sound, and the conclusions clear and link succinctly back up to the original experimental aims.

I would like to clarify the measure of sprint speed used as part of this study, however. I am uncertain whether the speed measurement used is the best way to represent an individual toad’s sprint speed, especially because the experimental design had no repetition on the individual level. I am cautious about using the single highest speed value as I wonder if such a small distance would be bias against smaller toads, which may require many hops to cover what a large toads would do in one. I understand size correction was performed, however there is the added factor of the “encouragement” used to promote movement. How was this done, and could this have impacted results? Was the amount of encouragement needed for each toad recorded, and could this be accounted for? Alternatively, maybe find a more robust way of measuring sprint speed, e.g. an average of the top three speeds.

Finally, it would be pertinent for the discussion to include more comparisons to other amphibian behavioural and anti-predator literature, particularly when discussing parotoid gland results.

Some brief comments on particular lines:
Ln 343: change “between speed and behavioral traits” to “between speed and the behavioral traits tested as part of this study” or something similar to acknowledge that this statement doesn’t apply to all behavioral traits.
Ln 354-356: It would be good to mention somewhere in the discussion any limitations that each of these behavioural proxy measures may carry.
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: MEASUREMENT OF DISPERSION OF PM 2.5 IN THAILAND USING CONFIDENCE INTERVALS FOR THE COEFFICIENT OF VARIATION OF AN INVERSE GAUSSIAN DISTRIBUTION
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors try to study the PM2.5 time-series in Thailand using confidence intervals for the coefficient of variation of an inverse Gaussian distribution, seems to be an interesting idea but requires more work to be fruitful, so my concerns are as bellow:
Introduction:
1)	Page 2, lines 47-55: Could you please bring their results and discuss them and show the relevance of proposed techniques to the mentioned air pollution studies? To me as an environmental researcher, it is a little bit confusing and difficult to follow.
Materials and methods:
1)	Please provide the source of air quality data as a separate sub-section.
Results and discussion:
1)	Could you please explain in more detail why you chose n, µ, and the number of replicants as mentioned?
2)	Please also improve the quality of the Figures, they are hard to read.
3)	Please explain in more detail, how your results can help an environmentalist or policymakers?
4)	What are you trying to explain which can be interesting in terms of the above-mentioned statements related to air pollution focusing on PM2.5 and its environmental, social, and health impacts?
5)	The discussion is not well provided and requires to be re-written, and the final aim of the work is not clear, please bring more related studies to compare your results and show how important and/or useful your findings or proposed metho are.
Conclusion:
1)	The conclusion is a repeat of the abstract and does not provide new information, please re-write it completely!
2)	Please talk more in detail about the future works and again how they can add more value to the existing knowledge!
Experimental design: 1)	Please provide the source of air quality data as a separate sub-section.
Validity of the findings: 1)	Could you please explain in more detail why you chose n, µ, and the number of replicants as mentioned?
2)	Please also improve the quality of the Figures, they are hard to read.
3)	Please explain in more detail, how your results can help an environmentalist or policymakers?
4)	What are you trying to explain which can be interesting in terms of the above-mentioned statements related to air pollution focusing on PM2.5 and its environmental, social, and health impacts?
5)	The discussion is not well provided and requires to be re-written, and the final aim of the work is not clear, please bring more related studies to compare your results and show how important and/or useful your findings or proposed metho are.
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: MEASUREMENT OF DISPERSION OF PM 2.5 IN THAILAND USING CONFIDENCE INTERVALS FOR THE COEFFICIENT OF VARIATION OF AN INVERSE GAUSSIAN DISTRIBUTION
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: In general in terms of writing and structure, I think that the manuscript has its goal clearly stated, along with sufficient details and information in method description.

However, in term of original research, I would like to ask the authors to clarify on the difference between the current manuscript and a previous manuscript by the same authors. Namely, "Confidence Intervals for Coefficient of Variation of Inverse Gaussian Distribution" published on ICVISP 2019: Proceedings of the 3rd International Conference on Vision, Image and Signal Processing (August 2019 Article No.: 73Pages 1–6. https://doi.org/10.1145/3387168.3387254). Two of methods in the current manuscript are the same as those in the above-mentioned previously published 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: MEASUREMENT OF DISPERSION OF PM 2.5 IN THAILAND USING CONFIDENCE INTERVALS FOR THE COEFFICIENT OF VARIATION OF AN INVERSE GAUSSIAN DISTRIBUTION
Review round: 2
Reviewer: 1

Output:
Basic reporting: No further comments!
Experimental design: No
Validity of the findings: No comment
Additional comments: Thank you for addressing all 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: EVIDENCE OF EPISODIC POSITIVE SELECTION IN CORYNEBACTERIUM DIPHTHERIAE COMPLEX OF SPECIES AND ITS IMPLEMENTATIONS IN IDENTIFICATION OF DRUG AND VACCINE TARGETS
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1.	The authors should describe the rationale for and information provided by each of the pipelines in plain language for readers to better understand the input and outputs of each step.
2.	The potential roles for several genes of interest are very briefly discussed, and the putative function(s) of the proteins in the context of other species is not made clear. While references are made for each protein, the original species, and experiments from which the putative functions are derived, is not explicit.
Some examples:
Line 185-186: “This enzyme is a potent antileukemic agent.”
Lines: 257-260: The utilization of carbon sources in macrophages and citrate fermentation.
3.	In the summary results of the Abstract, the authors note that 62 different genes were detected with 45 shared across all species. Lines 176-177 appear to reference the presence of 45 genes, but this information is not expanded upon. Further discussion of the 45 genes appears to be lacking in the Results and Discussion section and should be provided.
Minor concerns:
1.	Lines 50-51: The assertion of the rise in Diphtheria attributed to C. ulcerans does not appear to be fully supported by the references provided. While Hacker et al. note that cases of Diphtheria are caused mostly by C. ulcerans in Western Europe, the most recent literature review cited (Truelove et al. 2020) notes that C. ulcerans rarely causes Diphtheria.
2.	Table 1: The predictions for virulence factors and essentiality of the proteins are derived from PBIT based upon the Materials and Methods. While this approach may be useful in the broad sense, it should be clearly indicated that these are predictions based upon PBIT and not experimentally established.
3.	Lines 197-199: The reference cited by the authors for the ABC transporter, permease protein (MntC) notes the potential for Mn and Zn import through these permease proteins, not Mg and Zn.
4.	Lines 199-201: The authors do not expand upon the significance of a potentially nonfunctional gene and how this fits within their study for drug or vaccine targets. The rationale for proposing it as a legitimate drug target is unclear (Lines 218-220).
5.	Line 247: …(Batool et al., 2020), but. - I suspect this is a typographical error.
Experimental design: 1.	Based my understanding of the methodology described, it seems that only one C. diphtheriae strain was considered (NTCT 11397) and the diversity of C. diphtheriae isolates has not been considered. If proteins from this approach are to be explored as vaccine candidates, this limited approach may not capture the targets which the authors propose to identify.
2.	The text refers to a strain NTCT 11397, which is not a strain that is within the sequenced database. I believe the authors meant NCTC 11397 – this is correct within the spreadsheets.
Validity of the findings: 1.	The Staphylococcus aureus MntC vaccine component is not the same as the MntC that was identified in their approach. The S. aureus MntC is the substrate binding lipoprotein component of the ABC transporter system, not the permease component. The mechanism for immune evasion likely stems from S. aureus MntC binding plasminogen (PMID: 25409527) – a function that is not established for the permease protein(s). The finding of MntC and all discussion related to the S. aureus MntC should be rewritten, as appropriate.
2.	Lines 209-210: While SpaE may be considered a minor pilin in C. diphtheriae NCTC13129, it is unclear if this observation holds true for other diphtheria strains or for other Corynebacteria. Furthermore, the conservation of SpaE across all species is unclear. The authors may find the information provided in studies by Trost et al. (PMID: 22505676) and Broadway et al. (PMID: 23772071) helpful.
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: EVIDENCE OF EPISODIC POSITIVE SELECTION IN CORYNEBACTERIUM DIPHTHERIAE COMPLEX OF SPECIES AND ITS IMPLEMENTATIONS IN IDENTIFICATION OF DRUG AND VACCINE TARGETS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper of Vinicius Canário Viana et al  describes a study in to the evolution of pathogenic bacterial strains belonging to the Corynebacterium diphtheriae group and identify genes that may have undergone positive selection, and they suggest that these could be useful treatment/vaccine targets

The authors use ‘diphtheria’ group throughout….this is not widely accepted in the field, I’m not opposed to it, but it should at least be qualified at first use perhaps at first use in the abstract…

the study is framed in an acceptable manner and based on a plausible hypothesis
Experimental design: I would be interested to know why Mycobacterium tuberculosis was used as an out group in the phylogenetic analysis, one could argue that it is phylogenetically distinct from the genus and perhaps a species of Corynebacterium that was not part of the ‘diphtheria’ group would be better – perhaps C. glutamicum, or C kroppenstedtii such as used as the outgroup in the analysis for positive selection.

Can the authors comment on the potential for skewing of the positive selection analysis by the use of small numbers of genomes for some taxa? Or does Posigene correct for taxon ID?
Validity of the findings: The data seem robust and there is potential novely and impact from the data, but the interpretation is sometimes not clear cut.
I felt the section of the discussion that focusses on the potential novel vaccine/therapeutic targets rather fizzles out without really identifying key candidates and rationalizing these…the authors should attempt to revise this with clearer conclusions. I was initially very excited by the title of the paper, but I’m not sure the conclusions are really born out by the way the final manuscript is written.
There are a few papers that talk about gene content in various biovars (sangal et al) and the work describing C belfanti that could be useful in framing some of the data...do any of the genes identified map to historical phenotypic differences used for biovar differentiation?
Additional comments: The whole manuscript should be revised for grammar and syntax by a proficient English language speaker to ensure the sentences reflect what the authors mean.

Line 82 – ‘fix’ should be ‘can help fix’ – positive selection doesn’t always fix the mutation – also the end of that sentence doesn’t make sense and should be reworded, I can’t help the authors as I’m not quite sure what they mean….

Line 167 ‘19 genes were identified’…as what…I am not sure what qualified these 19 genes?

182 – essential genes or proteins….as this is a bioinformatic study, they would be ‘genes encoding proteins predicted to…’

Line 221 – reference is J Timson 2016, should be corrected…


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: EVIDENCE OF EPISODIC POSITIVE SELECTION IN CORYNEBACTERIUM DIPHTHERIAE COMPLEX OF SPECIES AND ITS IMPLEMENTATIONS IN IDENTIFICATION OF DRUG AND VACCINE TARGETS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have sufficiently addressed my comments. No further comments.
Experimental design: The authors have sufficiently addressed my comments. No further comments.
Validity of the findings: The authors have sufficiently addressed my comments. No further comments.
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: INTERROGATING COVID-19 VACCINE INTENT IN THE PHILIPPINES WITH A NATIONWIDE OPEN-ACCESS ONLINE SURVEY
Review round: 1
Reviewer: 1

Output:
Basic reporting: see attached PDF file - there are comments to help make the article clearer.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: No additional comments