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: SMALL HERBIVORES AND ABIOTIC HETEROGENEITY PROMOTE TRAIT VARIATION OF A SALTMARSH PLANT IN LOCAL COMMUNITIES
Review round: 1
Reviewer: 3

Output:
Basic reporting: Results and hypothesis need to be clarified (see General comments for the author)
Experimental design: I have some concerns regarding the Method section (see General comments for the author)
Validity of the findings: Conclusions need improvements and some methods used or results do not allow to draw certain conclusions that the authors present (see General comments for the author)
Additional comments: In this manuscript, the authors investigated whether small vertebrate herbivores affect the variability of phenotypic traits of the clonal grass Elytrigia atherica, either by direct grazing or indirectly by altering genotypic diversity or soil conditions due to its interaction with the environment. To do so, the authors used exclosures installed in plant communities of a saltmarsh in the Netherlands, in order to compare the intra-specific variability and patterns of neutral molecular markers inside and outside (open to herbivore’s grazing) the exposures. SEM models showed significant positive direct and indirect (through changes in soil abiotic conditions) relationships of herbivore pressure with plant traits variation (ITV) in early successional stages, while genetic diversity was reduced by herbivores in intermediate stages, without any significant change on ITV.

The question on how herbivory can affect the phenotypic variation and promote a change in the genetic diversity of a clonal species is interesting. The use of Path Analysis (i.e., SEM) is also appropriate to investigate indirect effects from herbivory activities. However, the methodology used seems not always the most appropriate for testing their hypothesis or at least does not allow to draw certain conclusions that the authors present. Moreover, in my opinion, some parts of the manuscript lack clarity due to inaccuracies in the writing and the way tables and figures are presented and the results and discussion need further development.

Major comments
1.- I have some concerns regarding the Method section. Firstly, the authors selected only one exclosure per successional stage so no replicates exist in this matter. This limits the possibility to present general patterns, so the conclusions must be presented considering this limitation. Second, you did used different sizes of exclosures between early (96 m2) and intermediate stages (48m2), which may be accounted for while drawing the conclusions. Third, according to L160-166, some of the leaf functional traits seem not correctly measured, as the authors did not used standard methods or only some plots were measured. This needs further explanation since you then have a biased measure and an inconsistent data collection that may affect your results.
In addition, despite I am not an expert in molecular analysis, I would assume that the genetic diversity inferences from neutral molecular markers may be affected by changes in sexual/clonal reproduction (mentioned in L85-86) rather than by herbivory itself (as examples for clonal species, see Holderegger et al. 1998; Bruun et al. 2007). This means first, that using neutral markers it would be unlikely to find that ITV is driven by the selection of these markers because they cannot control the gene expression of these traits, and second, that differential grazing selection considering plant size may be questionable, as the authors mentioned in L59-60 (or a reference is needed to corroborate this). Otherwise, plants clonal diversity might be affected by grazing, especially at early stages, but you only included one control, right?
2.- The results need a more appropriate and detailed description. Even, according to the results presented in Fig. 2 and Table 1, they are incomplete. For instance, the authors only mention height and flowering appearance as the traits affected by direct effects of herbivory in early successional stages. However, I see other significant positive effects in this case such as biomass and SLA. Each of these trait changes needs to be presented and discussed considering your predictions. Also, I would recommend to include the results of the mean values in the main text and discuss accordingly, as you commented that “Small herbivores may have more pronounced effects on plants: they not only impact trait means as studies have shown but also ITV”. This question is interesting but not discussed at all in the text (see for instance studies evaluating this but in another framework: Herrera 2017, March-Salas et al. 2021).
3.- Considering the previous points and other issues, I believe that the discussion should be toned down when considering global changes (e.g., in L249) and better defined to describe the exact conclusions that can be drawn from the analysis carried out and results. In your study, intra-specific variation could be also explained by phenotypic plasticity or genetic differentiation and this is little discussed. I also strongly recommend to compare more in deep the relevance of changes in means versus changes in trait variance (as examples, see previously mentioned studies), and discuss your findings with other closely related studies (e.g., Veneklaas et al. 2011) or others (see references).

Minor comments:
L11-12: Before mentioning that little is known about this topic, I would introduce first why this is a relevant question for plant ecologists. For instance, at this point, I cannot connect the effect of small herbivores in ITV with global changes.
L12: Change “response” by “respond”.
L16: You mean grazing outside the exclosure, right?
L17: You “measure” functional traits (also see L102).
L20: The meaning of “elevation” needs to be clarified, and please use a more specific name.
L27: I would remove “have more pronounced effects on plants: they”.
L39: Please specify in some point the differential relevance between genotypic diversity and genotypic richness in the context of your study.
L50: They are vertebrates and not “invertebrates, right?
L55-56: If this is not a question of your research, please remove.
L69: With environmental heterogeneity you refer to soil conditions (nutrients, texture, compaction…), correct? And what do you refer in L71 with microenvironmental heterogeneity?
L72-73: I would remove this sentence because you already write in different parts of the text that your question is underexplored.
L80: Use “preferring” and not “preferred”.
L82-83: This seems contradictory with what you said in L59-60.
L84: “hiding” doesn’t seems the correct term.
L85: Following grammar rules, after the point, the complete genus name should be written see also line 149).
L90: I would use “spread” instead of “growth”.
L96-98: The hypotheses are barely exposed or not clearly differentiated. I also miss here the connection of your results with changes in trait means, since this is one of the main conclusions that the authors exposed.
L112-113: I believe that this sentence is not correctly exposed. Please, rephrase.
L114-117: Despite the mentioned abundance, you cannot disentangle between the effects of hares and geese from the effects of other existing small herbivores such as rabbits, so I would keep it more open to the effect of small herbivores. Please, revise this along the entire text, especially in the results.
L121: To my knowledge, this is not an experimental system and mostly and study system. Also, you did not describe your measurements here.
L132: Say that this is Means ± SE, if this is the case.
L136: Mention when this experiment started.
L152: It was strange to me to read that you only measured in “sunny days”.
L153: I cannot follow this sentence. So please, rephrase.
L160: remove “within hours”. Also, 70 ºC seems high temperature for this purpose. I would do that at 36-42ºC.
L163: In addition to what I commented above, I could imagine than using dried samples instead of fresh material or samples with ethanol would affect the result of the DNA-sequencing.
L203: To facilitate reader’s comprehension, I would try to connect statistical analyses with the hypothesis stated in the introduction.
L221 and others: As commented before, I would use herbivory instead of hares and geese.
L221-222: Include here the p-value and also the statistic.
L222-223: I would be more neutral: “At intermediate stage, herbivores tended to affect ITV in height in grazed areas… And direct effects should be described more in deep in the next sentence.
L235: This was not the case in the effect of elevation in the variation of flower appearance.
L259: I do not think that the effect of microbial community can be related to your data/results.
L281-282: Which results indicate this response? I don’t think you can predict this with your design.
L293: You mainly summarize your results again instead of exposing the concluding remarks.
L295: Please, remove “substantial”.
L299: Maybe mention human actions is more appropriate that to relate this to global changes.
Fig. 1- I believe that the figure can be substantially improved. It could be presented as an infographic and not as separate panels. Also, as for panel D, is this an example? I would assume that this structure differs among plots. Also, what do the animals and the animals number represent? And the bar in panel B is the scale. The blue colours in D-F reduce the visualization too. Include this detail in the figure text. Panel F has the area size but not the Panel E. By the way, why not present the areas in early stages?
Fig. 2- I am missing the R2 values. Include the letter “H” in the last panel. Again, what do the animals and the animals number represent?
Fig. S2- Show significance and use letters. Maybe this figure can be moved to the main text. Also, why did you describe the results here? Cpuld them be moved to the main text?
Table S3- How do you explain that you find the same results in all the traits within each successional stage?

References
Bruun, H. H., Scheepens, J. F., & Tyler, T. (2007). An allozyme study of sexual and vegetative regeneration in Hieracium pilosella. Botany 85(1), 10-15.

Henn et al. (2018). Intraspecific trait variation and phenotypic plasticity mediate alpine plant species response to climate change. Frontiers in Plant Science 9, 1548.

Herrera (2017). The ecology of subindividual variability in plants: patterns, processes, and prospects.
Web Ecology 17(2), 51-64.

Holderegger, R., Stehlik, I., & Schneller, J. J. (1998). Estimation of the relative importance of sexual and vegetative reproduction in the clonal woodland herb Anemone nemorosa. Oecologia, 117(1-2), 105-107.

Laforest-Lapointe, Martínez-Vilalta & Retana (2014). Intraspecific variability in functional traits matters: case study of Scots pine. Oecologia 175, 1337–1348.

March-Salas, Fandos & Fitze (2021). Effects of intrinsic environmental predictability on intra-individual and intra-population variability of plant reproductive traits and eco-evolutionary consequences. Annals of Botany 127(4), 413–423.

Santana et al. (2021). Edge effects in forest patches surrounded by native grassland are also dependent on patch size and shape. Forest Ecology and Management 482, 118842.

Veneklaas, R. M., Bockelmann, A. C., Reusch, T. B., & Bakker, J. P. (2011). Effect of grazing and mowing on the clonal structure of Elytrigia atherica: a long-term study of abandoned and managed sites. Preslia 83(3), 455-470.


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: SMALL HERBIVORES AND ABIOTIC HETEROGENEITY PROMOTE TRAIT VARIATION OF A SALTMARSH PLANT IN LOCAL COMMUNITIES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors precisely solved everything that was commented on in the first version. I have no further comments at this time.
Experimental design: .
Validity of the findings: .
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: SMALL HERBIVORES AND ABIOTIC HETEROGENEITY PROMOTE TRAIT VARIATION OF A SALTMARSH PLANT IN LOCAL COMMUNITIES
Review round: 2
Reviewer: 2

Output:
Basic reporting: The revised manuscript includes substantial improvements compared with the previous version. The literature references are better presented thanks to the comments done by reviewer 1 and 2 and authors use professional English and structure. However, the hypothesis needs to be more clearly presented and considering methodological limitations, and other questions need to be attained (see detailed comments below)
Experimental design: Research question is relevant, but, as reported below, I consider that the research question and methods used still need some clarifications and a proper justification
Validity of the findings: I find several similarities with the study of Veeneklaas et al (2011) but authors used a new approach for analysing potential direct and indirect effects on this plant species and its structure. Some statistical questions need to be responded and showed in more detail to assure adequacy in the models used. Conclusions improved in the new revised version but I consider that they still need some clarifications
Additional comments: First of all, I salute the effort of the authors for implementing considerable changes in the text and to clarify my comments/questions, and of the other reviewers too. From these changes on the revised manuscript, I have some comments:
-I still consider that the hypotheses need to be more clearly formulated. The background need to be exposed in advance and not after each hypothesis, because this makes the questions difficult to be followed and masked. I strongly recommend that, in the latter part of the introduction authors should clearly present the predictions from the theoretical framework and according to your design and analysis instead of including them in the middle of the text.
-In your responses and in the main text (line 202-203), you mentioned that other studies reported that early stages are subjected to higher grazing pressure than intermediate stage, and that this was the reason of your selected areas, but any reference was added here. Also, I recommend avoiding over-interpretations as “much higher” if these affirmations are not corroborated by numbers. Similarly, this was also the case when you use “strongly impact” (e.g. line 293, 419, 420) or “substantial insights” (line 517), especially when, as you mentioned, your design is limited in certain points.
-Revise some of your conclusions. For instance, as far as I understand, the impact of ITV though plasticity is not corroborated with your study. Plasticity could have a genetic basis, being both plastic and genetic changes able to contribute to a phenotypic trend. However, inferences regarding phenotypic plasticity should be also supported by a speciﬁc set of evidentiary criteria (e.g. common garden experiment) and not only as an evidence from a null hypothesis, as seems you are doing here (see for instance Merila and Hendry 2014). Also, in line 528, I will limit it to abiotic and environmental changes instead of referring to “global changes”. So in these cases, please, tone down the message.
-I still consider that, using neutral markers the authors cannot infer a causal relationship with the phenotypic variation, so I recommend they should highlight this limitation too.
-You have mentioned in your responses and in line 222-224 that “We marked an area, ca. 6 m × 8 m, which corresponds to the smaller size of the exclosures” but it is not clear to me that you used this and not the 8 m x 12 m exclosures to analyse your data.
-I am still not convinced with the response around that “specific leaf area and leaf dry matter content was not measured in the standard way”. Please, explain the method used. In this regard, although understandable, I don´t feel the justification for the leaf dry matter is enough for a scientific work. Of course I understand that the field work frequently implies many limitations, but they should be considered to choose the design and measures to make. Considering this, I do not find a remarkable problem of using a representative subsample instead of all the samples, but it is when using unreported/untested method (which I assume if you mention “not measured in standard way”). Please, describe the method or limit this trait in your study. Also in this regard, I don´t feel “because they are easier to measure in the field” is an appropriate justification for a scientific work. The measure should be adequate to draw certain conclusions.
-You exposed in line 89-93 that large herbivores generally consume tall plants, and thus promote short plants because short plants are generally more nutritious, and it seems contradictory to me. Why large herbivores should select for taller plants rather than young and smaller plants if the larger ones are less palatable (e.g. more lignin content, lower energy assimilation, etc.)?
-In line 328, you say that “We checked residuals for each model, in all cases, models fitted the data reasonably well”. First, what “reasonable well” means? Do you refer that you visually checked that? Second, you should describe in detail the assumptions checked and the test used for that. For instance, did you used the Shapiro-Wilk normality test for checking the Normality of residuals or the Bartlett test for checking the homoscedasticity of variance? If your models did not comply these assumptions, you should apply adequate models (e.g. transformations, weighted least square regression) or use alternative ones.
-You frequently mention (e.g. 418-423, 442, …) that herbivores impact means, but you should also mention if responses/traits are negative or positively affected.
-In Line 417, I would change “can impact” by “could impact”
-In line 426-427, I don´t understand what “relative to ITV” means. Also, form the next lines, do you mean that the plant community structure may affect your results?


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: IMPACT OF CLIMATIC CONDITIONS PROJECTED AT THE WORLD CUP IN QATAR 2022 ON REPEATED MAXIMAL EFFORTS IN SOCCER PLAYERS
Review round: 1
Reviewer: 1

Output:
Basic reporting: As noted in the attachment
Experimental design: As noted in the attachment
Validity of the findings: As noted in the attachment
Additional comments: As noted in the attachment


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

Input:
Title: IMPACT OF CLIMATIC CONDITIONS PROJECTED AT THE WORLD CUP IN QATAR 2022 ON REPEATED MAXIMAL EFFORTS IN SOCCER PLAYERS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is clearly written and understandable. it is well designed and well-written, with a great introduction proposing the usefulness of the topic and a clear outline of the research question. Figures and Tables are well shown
Experimental design: The method is correctly explained. It could very easily be repeated. The interpretation was done correctly. Technically flawless study
Validity of the findings: The conclusions were made correctly in
accordance with the results. Overall, this is a nice study that could have great practical application when integrated with soccer players relating climate and their performances
Additional comments: The reviewer would like to thank the authors for their work and efforts in trying to improve sports science knowledge. The authors are commended on their efforts thus far.


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: IMPACT OF CLIMATIC CONDITIONS PROJECTED AT THE WORLD CUP IN QATAR 2022 ON REPEATED MAXIMAL EFFORTS IN SOCCER PLAYERS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Necessary explanations weere given in the file. Corrections were marked with yellow

The english language of the study should be professionally corrected.
Experimental design: Necessary explanations weere given in the file. Corrections were marked with yellow
Validity of the findings: Necessary explanations weere given in the file. Corrections were marked with yellow.
Additional comments: According to the quality of this journal, the methods section of this study is quite simple and should be improved.The reasons for the findings should be explained in the discussion section.

The authors should take into account the corrections in the file.

References should be arranged according to the journal format.

Statistical analysis section should be developed.


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: IMPACT OF CLIMATIC CONDITIONS PROJECTED AT THE WORLD CUP IN QATAR 2022 ON REPEATED MAXIMAL EFFORTS IN SOCCER PLAYERS
Review round: 2
Reviewer: 1

Output:
Basic reporting: According to the attached notes.
I'm not the best person to review English. As for the references, context among others after the evaluation of the presented version, in my view it is in agreement, as well as the rest of the topics mentioned here
Experimental design: With regard to experimental design, the manuscript, in the view of this reviewer, is in accordance with international standards for publication, presenting sufficient robustness and scientific rigor for publication.
Validity of the findings: Regarding the validity of the discoveries, these really are current, being a very important and current theme.
Additional comments: According to the attached notes, I consider the manuscript in publishing conditions


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: IMPACT OF CLIMATIC CONDITIONS PROJECTED AT THE WORLD CUP IN QATAR 2022 ON REPEATED MAXIMAL EFFORTS IN SOCCER PLAYERS
Review round: 2
Reviewer: 2

Output:
Basic reporting: First of all, the reviewer would like to thank the authors for their work and efforts in trying to improve sports science knowledge. All necessary amendments have been incorporated according to reviewer feedback.
Experimental design: An article is an interesting approach to the Impact of climatic conditions projected at the World Cup in Qatar 2022 on repeated maximal efforts in soccer players. The study is well designed and well-written, with a great introduction proposing the usefulness of the topic and a clear outline of the research question
Validity of the findings: The statistical analysis has been performed appropriately and rigorously. The manuscript was presented in an intelligible fashion and written in standard English. Overall the discussion is well-written and incorporates relevant literature.
Additional comments: Accepted.

Best 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: IMPACT OF CLIMATIC CONDITIONS PROJECTED AT THE WORLD CUP IN QATAR 2022 ON REPEATED MAXIMAL EFFORTS IN SOCCER PLAYERS
Review round: 2
Reviewer: 3

Output:
Basic reporting: The corrections were made by authors
Experimental design: The corrections were made by authors
Validity of the findings: The corrections were made by authors
Additional comments: The corrections were made by authors. They improve the manuscript. I think that the article is ready for publication.


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

Input:
Title: A TAXONOMIC OUTLINE OF THE POECILIMON AFFINIS COMPLEX (ORTHOPTERA) USING THE GEOMETRIC MORPHOMETRIC APPROACH
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is clearly written and much original.
Only a few small suggestions are reported in the attached text.
Experimental design: Original and well defined.
Validity of the findings: Findings show that the method followed by the authors is good.
Additional comments: Further, I would suggest to the authors to make a decision about some species: are they synonyms or valid species?


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

Input:
Title: A TAXONOMIC OUTLINE OF THE POECILIMON AFFINIS COMPLEX (ORTHOPTERA) USING THE GEOMETRIC MORPHOMETRIC APPROACH
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English language is clear and professional throughout the text, the Introduction and
background show the general context however I believe it is important to include the differences
between P. rumijae and P. affinis. The literature citation is appropriate for this manuscript and the
figures are relevant and well labeled and described. The structure is in accordance with PeerJ.
Experimental design: This manuscript A taxonomic outline of the Poecilimon affinis complex (Orthoptera) using the
geometric morphometric approach, represents a major contribution, especially by including a the
geometric morphometric analysis study to explore variation in the structure of the tegmen,
ovipositor, cercus and pronotum.

Below, I present some considerations about the manuscript.

1.- In the part of the methods on geometric morphometric analysis, I consider that they should be
clearer in the placement of landmarks so that these can be replicated.
2.- Why only use landmarks and not semilandmarks?
Validity of the findings: It appears that all the data are robust and statistically sound, but until it is clear how the
decision to place each landmarks was made, it will not be sufficiently clear.
Additional comments: No additional comments


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

Input:
Title: A TAXONOMIC OUTLINE OF THE POECILIMON AFFINIS COMPLEX (ORTHOPTERA) USING THE GEOMETRIC MORPHOMETRIC APPROACH
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I am not really familiar to the methods used in this study, but the authors seem to know well what they are doing. Below a few suggestions/comments with line numbers:

35 According to OSF (Cigliano et al.) the most species-rich phaneropterine genus is Anaulacomera from the Neotropics (154 valid species, Poecilimon has 145).

86 cercus > male cercus

183 complex species > species complex?

289/290 it occurs > they occur

343 than > compared to?

344 its subspecies – it cannot be a subspecies of a subspecies (P. a. affinis)

354 ff. The last sentence is not very clear.

Figure 1: Photo > Photos

Figure 3: The figures are very small. It is almost impossible to read the species names and the even smaller locality names below them without greatly zooming in. It would probably be better to enlarge both figures to full page width.

Figure 4: Same here, here, species names not as tyny as in Fig. 3 but still very small (and yellow is almost imposiible to read).

Figures 5,6: Same as in 4.


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: AN ESTIMATE OF THE AREA OF OCCUPANCY AND POPULATION SIZE OF BRACHYCEPHALUS TRIDACTYLUS (ANURA: BRACHYCEPHALIDAE) TO REASSESS ITS CONSERVATION STATUS, WITH A PROPOSAL FOR CONSERVATION MEASURES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Some methods need to be clarified. In particular, I could not follow the sampling scheme that occurred at each site (Lines 94-95).
Experimental design: As mentioned above, I did not follow all of the methods. More detail would need to be provided for them to be replicated.
Validity of the findings: My biggest concern is the estimate of total population size. This estimate is based on a density estimate from a single site, which is then extrapolated to represent all suitable habitat. The site where the density estimate was taken was not selected randomly, but was instead a site where multiple males were calling. This thus seems biased to be a higher density than average. Also, I don't think data from a single site should ever be assumed to represent all other sites. There needs to be replication.
Additional comments: The goal of this study was to identify suitable habitat for an endangered Brazilian frog, estimate densities of the frog, and from this reassess its conservation status. Prior to the study, the species was only known from a single site, and based on this work it has now been documented at seven additional sites. This is valuable information. Based on the eight known localities and habitat associations, the authors then estimate a total area of occupancy for the species. This is also valuable information, although I found the text somewhat confusing on this point (Lines 135-140). Even though this is clearly a calculation of area of occupancy, the authors are considering it extent of occurrence? Also, was the species only found in Montane Dense Ombrophilous Forest? If yes, then shouldn’t area of occupancy be limited to only this habitat type? I had trouble following the text, so I’m not sure of the answer to either of these questions.

The authors then move on to estimating population density. I found this estimate to be highly questionable to the point that I do not think it should be published. If I understand the procedure correctly, the authors found a site where multiple frogs were calling, counted the number of frogs within this area, and then extrapolated that density to the entire area of suitable habitat. Given that this site was selected because it had multiple males calling within it rather than being a randomly chosen area, it seems biased towards having a higher density than an average area of the same size. Also, this density estimate was only conducted at a single site with no replication. I object to the assumption that any individual site is representative of the entire species range. Any individual site could have a much lower or much higher density than the range-wide average. Certainly, the total population size estimated using this method seems farfetched. Do the authors honestly believe that there are 73 million individuals of the locally endemic frog?

I also do not understand how the authors concluded that the appropriate listing status is Vulnerable. They estimated an area of occupancy of ~148 km2. This would put the species in the Endangered category since area of occupancy is less than 500 km2. On the other hand, I do question whether the area of occupancy was estimated accurately. The authors surveyed 22 sites and found the frog at 7 of the sites. The survey protocol followed at each site (Lines 94-95) was impossible to follow. Thus, I have no idea how intensely each site was surveyed. If a frog was found, then clearly they are present, but if they were not found, how accurately does that represent absence? It is very difficult to prove absence, but some estimate of confidence would be very helpful. For instance, at the 7 sites where the frogs were found, were they found immediately? Were the remaining 15 sites searched for a much longer period of time and still no frogs were found? Or does the fact that the frogs use advertisement calls make it very simple to determine if they are present or not? Also, since the 22 survey sites were not picked with this species in mind (original search was for land mollusks), how likely is it that additional suitable sites outside of the current extent of occurrence were simply not searched? Looking at Figure 3, the current extent of occurrence seems to cut through a couple of the largest habitat patches, especially on the north and east sides, which I assume is where more of the mountains are located. It seems that there should at least be a discussion of how much suitable habitat likely remains unsearched, in which case maybe the species truly is Vulnerable rather than Endangered.

Line 25: Replace “In the anurans group” with “One group of anurans”.

Line 29: Delete “it”.

Line 30: Delete “threatened of extinction”.

Line 33: Change to “evaluated in loco impacts and potential impacts on the species’ population”

Line 34: Change “his” to “this”.

Lines 37-38: Change to “occurrence as 14,843.6 ha, density as one calling male per 4.05 m2, and a total”

Line 49: Change to “Anura is a priority group”

Line 59: Change “accessed” to “assessed”

Line 63: Aren’t the majority of species restricted to climatic envelopes? In other words, aren’t there some climate conditions outside of which they cannot persist? If so, then I think you must mean something more specific here about the tolerances of B. tridactylus.

Line 66: change “from” to “by”.

Line 70: Genus should be in italics.

Line 72: Change “which are associated with” to “exacerbated by”.

Line 78: Change “from” to “to”.

Line 79: Change “altitude” to “altitudinal range”.

Line 82: Change “was evidence that the species was threatened with extinction” to “led to the species being classified”.

Line 86: Change “over” to “on”.

Lines 94-95: I do not understand the sampling regime. Please explain this better.

Line 103: Not clear what you mean by “observed impacts” here.

Line 107: Insert “meters” after “five”.

Line 127: Change “amplitude” to “range”.

Lines 135-140: It is very important to differentiate between area of occupancy and extent of occurrence, as IUCN uses different thresholds for these two different quantities, but I don’t really follow how that was done.

Line 146: Delete “or, better, of one individual per m2.”

Lines 147-148: Delete “of two individuals per m2”

Line 169: Insert “an” before “advertisement”.

Line 170: Change to “we did not record the species at 15”.

Lines 172-173: If all localities are in this forest type, then why not limit the area of occupancy to only include areas with this habitat type that fall within the minimum convex polygon?

Line 173: Replace “connected through the” with “enclosed in a”.

Line 174: Change “resulting in” to “with”.

Line 176: Delete “previously”.

Line 183: The method by which total population size was calculated seems highly questionable. If I understand the method correctly, it sounds like an area with lots of males calling was first identified, and then the density of males within this area was determined. The density within this area was then extrapolated to be the same across the entire area of occupancy. However, if the area to sample was chosen because it had many calling males within it, it was presumably biased toward having a higher density than a randomly chosen region with the same area. If this method is to be used, it seems that random areas need to chosen first, and then numbers of frogs need to be counted within these random areas. Also, it seems critical that multiple sites be surveyed in this way rather than a single site. Any single site could have a non-representative density (either high or low) compared to other areas across the range.

Lines 190 and 192: Delete “limits”.

Line 197-198: How are fires going to change pasture to forest? I assume you mean the other way around.

Line 201: Replace “inland” with “interior”.

Line 204: What do you mean by “mischaracterizing”?

Line 213: It seems that this entire section should be in the Discussion rather than the Results.

Line 216: Change to “firefighting teams would serve to prevent”.

Lines 221-222: The goal of radio tracking mules is to stop palmiteiros? If yes, then this needs to be more clearly stated.

Lines 227-228: I don’t follow the logic here of brining the state closer to the people. How is this being achieved and why is it desirable?

Lines 265-267: This suggests that the minimum distance between Brachycephalus species is 4 km. Are they never sympatric?

Line 268: What do you mean by a sampling hiatus?

Line 271: Change “another 15 target points were unsuccessful” to “they were determined to be absent from another 15 target points”.

Line 272: How easily was the species detected when it was found? Were many individuals always found, or was the first individual always detected right away? In other words, how confident should we be that they were truly absent from the 15 sites where they were undetected?

Line 282: Change “sympathy” to “sympatry”.

Line 289: Change “superior to” to “higher than”.

Line 313: Insert “also” before “requires”.

Line 356: Change “IUNC” to “IUCN”.

Line 361: What does “conduct adjustment terms” mean?

Line 369: Change to “continues to suffer reduction and loss of habitat quality”.

Line 376: Why does it matter if other protected species are larger in body size?

Lines 377-379: Trying to introduce a new category of protected species in the last sentence of the paper seems strange. I don’t understand what would qualify a species as a fountain species.

Figure 3 caption: Change “state’s” to “state”.

Table 1: For Torre Embratel, the altitude range where animals were found does not fall completely within the altitude range that was searched.


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: AN ESTIMATE OF THE AREA OF OCCUPANCY AND POPULATION SIZE OF BRACHYCEPHALUS TRIDACTYLUS (ANURA: BRACHYCEPHALIDAE) TO REASSESS ITS CONSERVATION STATUS, WITH A PROPOSAL FOR CONSERVATION MEASURES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English language should be improved throughout the manuscript to ensure that an international audience can clearly understand your text. Some examples where the language could be improved include Lines 138, 268-270, 282, 298, 308 – the current phrasing makes comprehension difficult. I suggest you have a colleague who is proficient in English and familiar with the subject matter review your manuscript, or contact a professional editing service. Latinized words (e.g., in loco) must be italicized (e.g, Lines 33 and 245).

The article includes sufficient introduction and background to demonstrate how the work fits into the broader field of knowledge. Relevant prior literature is appropriately referenced.

The structure of the article conforms to an acceptable format of ‘standard sections’. Figures are relevant to the content of the article, of sufficient resolution, and appropriately described and labeled (except see Additional Comments for issue with Figure 3 caption).

The study does not follow an ecological hypothesis, but rather is a review of the conservation status of a threatened amphibian species.
Experimental design: The knowledge gap being investigated is clearly identified, and statements are made as to how the study contributes to filling that gap. The research appears to have been conducted in conformity with the prevailing ethical standards in the field.

The greatest area for improvement lies with the methods used to calculate population size and extent of occurrence (EOO), with the later having implications for the proposed IUCN category and criteria. See Additional Comments for greater detail on these issues.
Validity of the findings: All underlying data have been provided. There is very thorough discussion as to concrete conservation actions needed for the species and its habitat. Conclusions are well stated and succinct, and link directly to the objective of the study which is to reevaluate the conservation status of Brachycephalus tridactylus and propose conservation measures.
Additional comments: Lines 173–180 (Geographic and Altitudinal Distribution): It is not clear how the extent of occurrence (EOO) was calculated and whether it was done so according to IUCN guidelines. Based on the text, it seems that the authors: 1) drew a minimum convex polygon around the known localities and calculated the area of this polygon as 53,289.0 ha (532.89 km2); 2) contracted the minimum convex polygon to exclude altitudes outside of the species’ altitudinal range, as well as non-forested areas; and 3) reported the updated EOO based on the revised range as 14,843.6 ha (148.436 km2). This second EOO value seems dubious given that step 2 essentially represents a range map for the species, and the area of a minimum convex polygon around this range map (IUCN’s definition of EOO) is much closer to the value provided in step 1 (ca. 530 km2) than the final EOO value provided by the authors (148.436 km2). Either EOO value would make the species eligible for the EN category under criteria B1ab (see Conservation Status section below for further detail on this), but I still find the author’s method for deriving their final EOO to be a bit puzzling and requires clarification. Upon examination of the caption for Figure 3, I suspect that the authors may be using ‘EOO’ interchangeably with ‘species geographic range’. The light grey polygons in this figure represent the species’ presumed range, not the EOO. The EOO, according to IUCN definition, is a measure of the spread of risk calculated by a minimum convex polygon around all sites of occurrence – in this case, the solid white line is the EOO.

Lines 182–186 (Density and Population Size): Density was measured at a single site based on calling activity for 3 hours. I think that extrapolating these results to the entire range to obtain a total population size has a lot of limitations that are not discussed, such as the fact that the species is unlikely to be evenly distributed throughout the range and therefore their population size estimate may represent an overestimate.

Lines 255–260 (Conservation Status): The proposed IUCN Category and Criteria of VU B1ab(i,ii,iii) does not agree with the data provided in this manuscript. First, the EOO value is below the threshold for EN under Criterion B1. Second, he authors state that the geographic range is severely fragmented, but it is not clear whether the population is severely fragmented according to the IUCN definition (ie. “most (>50%) of its individuals are found in small and relatively isolated subpopulations”). If the population does meet the IUCN definition of a severely fragmented population, which is one of the requirements of using Criterion B, then the text should specifically state this information rather than leaving it up to the reader to speculate based on a seemingly fragmented distribution. Considering this species has an EOO of <5,000 km2, a severely fragmented population, and ongoing decline in the extent of occurrence (i), area of occupancy (ii), and area, extent and/o quality of habitat (iii), I would argue that EN B1ab(i,ii,iii) is a more appropriate listing for this species than VU.

Lines 304–306 (Conservation): I think it is important to clarify that B. tridactylus has not yet been assessed by IUCN and that the “previous classification of the species as Vulnerable” was an assessment by Bornschein, Pie & Teixeira (2019) not the IUCN Red List.


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: AN ESTIMATE OF THE AREA OF OCCUPANCY AND POPULATION SIZE OF BRACHYCEPHALUS TRIDACTYLUS (ANURA: BRACHYCEPHALIDAE) TO REASSESS ITS CONSERVATION STATUS, WITH A PROPOSAL FOR CONSERVATION MEASURES
Review round: 1
Reviewer: 3

Output:
Basic reporting: This manuscript has original and fundamental data for the evaluation and protection of a species of anuran endemic to Brazil.
The manuscript is very well written, has a review of clear terms, and has an excellent writing structure.
Bibliographic references are well up-to-date, except for those highlighted in the correction file.
All data, tables, and figures are well produced and contain important information for the complete understanding of the manuscript. I made some suggestions for improvements.
Experimental design: The manuscript presents an appropriate field methodology and data collection and analysis.
The proposal is well constructed and is essential in the level of detail presented to support a species assessment.
The authors demonstrated a great effort to obtain and analyze the data. Reflecting on important results and discussions.
Validity of the findings: This manuscript can serve as a citation for many other manuscripts dealing with species with some degree of threat.
I emphasize that the degree of detail of the information presented, with the analyzes and conclusions presented, make this manuscript an important paper for this renowned journal, in addition to serving as a model for future papers with the same focus.
Additional comments: With the details in the previous fields, I indicate that the paper should receive a major revision and few minor revisions to adapt some terms, understanding of paragraphs, and the conclusion.
Despite few and localized corrections, this manuscript must be passed on to the authors for the adequacy of this information.
This article deserves to be revised after corrections, since it brings new and important information for the protection of a species that is threatened and that, without proper subsidy, may be extinct in a short time.


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: AN ESTIMATE OF THE AREA OF OCCUPANCY AND POPULATION SIZE OF BRACHYCEPHALUS TRIDACTYLUS (ANURA: BRACHYCEPHALIDAE) TO REASSESS ITS CONSERVATION STATUS, WITH A PROPOSAL FOR CONSERVATION MEASURES
Review round: 2
Reviewer: 1

Output:
Basic reporting: This is fine.
Experimental design: There was still a lack of detail on how surveys were conducted at each individual survey site.
Validity of the findings: Despite consensus among the reviewers that improvements needed to be made in the estimate of total population size (or rather that it should be dropped) and that the extent of occurrence estimate was being mixed up with area of occupancy, the authors did not alter their original approach.
Additional comments: I was very disappointed with this revision. It seemed that the reviewers gave very clear, largely consistent suggestions, most of which were ignored. There were two main improvements. First, the authors changed the recommended status from Vulnerable to Endangered. This was good, since all three reviewers made this recommendation. Second, the distribution of the survey sites was explained much better using Figure 1. I am now confident that the authors searched at least the majority of the potential suitable habitat.

Unfortunately, many other recommendations were not followed. First, the authors persisted in providing an inaccurate estimate of total population size. Two of the reviewers pointed out that this estimate was problematic. I appreciate that the authors went to the trouble of surveying a second site. I also understand the logistical limitations to conducting surveys at completely randomized sites. However, it seems the solution should be to forego making any population estimate at all rather than making an inaccurate one. The fact that the authors found such a different density at their second survey site compared to their first one, which resulted in a greater than 10-fold change in their estimate of total population size, should have indicated to them that they are likely still far off from the true number. The authors themselves state that this species has a patchy distribution, but in their estimate calculate that the same density applies across the entire area of occupancy. Do these two statements/approaches not strike them as contradictory?

Second, while the authors have now clarified the methods used to select the survey sites, there is now even less information on how the survey was conducted at each individual site. I previously encouraged the authors to provide more details about their field methods, especially an indication of how quickly they found the species at sites where it was recorded relative to the total amount of time spent searching each site. This would provide some indication of how likely it is that the species is truly absent from sites where it was not found.

Third, the authors persisted with their previous calculation of extent of occurrence, even though all three reviewers found this to be problematic. The authors’ approach seems to be to first calculate a minimum convex polygon around all of the known localities, which is equivalent to the extent of occurrence. They then remove areas within this polygon that have unsuitable elevations or habitat types. This would lead to a calculation of area of occupancy. The authors then state that they think the area of occupancy should be even smaller, so they call their estimate of area of occupancy the extent of occurrence. If the authors aren’t confident in their estimate of area of occupancy, then they should stick with their original estimate of extent of occurrence using the minimum convex polygon.

Line 39: What does “67.0.2” mean?

Lines 41-42: Change to “We recommend changing the species’ conservation status from Vulnerable to Endangered”. Only IUCN can actual change the species’ status, so this is just a recommendation.

Line 64: Insert “usually” before “heard”.

Lines 65-66: My point from before is that you could say this about most species in the world. I think you must really want to say something more specific than this, such as the kind of climate envelope the group is restricted to.

Lines 74-75: If the small distributions are natural, then they don’t represent losses in areas of occupation or habitat quality. Maybe you mean that the small distributions in conjunction with losses in areas of occupation and quality of habitat are the main cause of threat to the species?

Line 95: Change “from” to “around”.

Line 96: Change “specialization” to “specificity”.

Line 100: Change “parts” to “quadrants”.

Line 101: Change “mountain” to “mountainous”.

Lines 113-114: Since everything before this has been described as an intentional search for B. tridactylus, this doesn’t make any sense.

Line 115: Delete “local anesthetic”.

Line 119: Change to “from its type locality”.

Lines 135-137: Exactly. This is why extrapolating density from a site where you found them to the entire range is a bad idea. You’ve just said here that there are some areas where density is zero. However, in your calculation, you are saying that all areas have the same density as your survey areas.

Lines 137-139: I am sympathetic to the reality that randomizing survey locations would not have been possible. If this means that you can’t get an accurate estimate of total population size, then don’t do one at all. Stick to providing information about geographic extent and threats. That’s the useful information anyway. An unreliable estimate of total population size isn’t helping anyone.

Lines 144-152: This reads as Intro or Discussion material. If this relates to data that you yourselves collected while in the field, then you need to be clearer about how that was done.

Lines 165-176: This seems like flawed logic. You followed the procedure for calculating area of occupancy, but then because you think it is a bad estimate for area of occupancy you call it extent of occurrence. I actually think you do have a good estimate of area of occupancy. However, if you don’t agree, then just stick to calculating extent of occurrence using the minimum convex polygon. Don’t try to refine that estimate by excluding inappropriate habitats within the polygon, since that procedure is part of calculating area of occupancy, not extent of occurrence.

Line 186: So you searched areas between 700-1140 m and found the species across almost that entire elevational range? If so, saying that the species is restricted to this elevational range seems flawed. If you didn’t search outside of this range, then you don’t have any information on whether the species occurs there or not. You clearly state that you didn’t search any sites above 1140 m, so you definitely don’t have information about whether this is the upper elevational limit for the species? How many sites between 700-714 m did you search and not find the species? It would need to be a large fraction of your total absence sites (~8 out of the 15) to be confident that this was the true lower elevational limit.

Line 204: The fact that the researchers found such different densities at the two sites they surveyed indicates that the distribution of the species is indeed patchy, especially since the lower density was found at the larger site. Doesn’t this suggest to the researchers that the larger they made the area that they surveyed, the lower a density they would find? Thus, estimating a mean density from this data and applying it across the entire range seems flawed. I’m sure that the new population estimate is much closer than the previous one, but I would think the fact that the estimate has changed more than 10-fold by the addition of just one more survey site would suggest to the researchers that the new estimate is still likely to be way off the true number.

Line 233: As I said previously, this whole section belongs in the Discussion. These are not things that you observed in the field. These are recommendations based on what you observed in the field.

Lines 247-249: Won’t the people likely be resentful of the punitive acts and thus less likely to cooperate with conservation of the state park? It seems like there are two conflicting approaches here. One could either educate the people about the importance of the environment and try to enroll them as collaborators in protection. Or, one could punish them for violations and force them to follow the conservation laws. Aren’t these two separate approaches rather than the same one?

Line 282: To meet these classifications you also need to show that extent of occurrence is below a specific cutoff. You should report the extent of occurrence that you calculated and compare it to this number.

Lines 287-289: Do you mean something more specific here, such as gaps between the distributions of sister species of Brachycephalus? Below you describe sympatry in the genus, so clearly gaps between the geographic distributions of two Brachycephalus species can actually be zero.

Line 292: Change “from” to “around”.

Lines 293-295: This is not clear.

Line 357: This is unclear.

Line 372: This is unclear.

Lines 379-380: Now reads like B. tridactylus occurs in the entire Atlantic Rainforest. Need to clarify what you really mean.

Line 391: This is unclear.

Lines 407-408: Why are these effects going to go beyond the extent of occurrence of the species? Are they going to be implemented in other areas where the species isn’t even present? Wouldn’t they benefit other species that co-occur with the focal species?

Lines 408-410: I still think it is strange to introduce this new concept in the final sentence of the paper. There is no indication of how this approach would actually work.


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

Input:
Title: AN ESTIMATE OF THE AREA OF OCCUPANCY AND POPULATION SIZE OF BRACHYCEPHALUS TRIDACTYLUS (ANURA: BRACHYCEPHALIDAE) TO REASSESS ITS CONSERVATION STATUS, WITH A PROPOSAL FOR CONSERVATION MEASURES
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: After reviewing this new version, I appreciate the effort made to address my comments, I am pleased with the responses of the authors to some of the observations I made. Having said this, I have no comments for this new version of your draft.


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: EARLY DETECTION OF CYANIDE, ORGANOPHOSPHATE AND RODENTICIDE POLLUTION BASED ON LOCOMOTOR ACTIVITY OF ZEBRAFISH LARVAE
Review round: 1
Reviewer: 1

Output:
Basic reporting: General comment: My main concern in this section is that authors propose the developed system “for utilization as part of a monitoring system and an ideal tool for environmental risk assessment”. In the introduction information of environmental relevant concentrations of all these chemicals in aquatic ecosystems, both non-directly exposed and directly exposed, is currently missing. This is basic information to understand the sensitivity of the proposed system. It is not my role to look for this info in the bibliography, but I think that the concentrations used for the authors are several orders of magnitude higher than the commonly found in the environment. I would be very surprised if rodenticides were found in aquatic ecosystems not directly exposed at concentrations higher than low ng/L… If this is the case, the proposed system is not sensitive enough for environmental applications (at least for rodenticides…)

Specific comments:

Lines 41-43: “Toxic substances, such as cyanide, organophosphates and rodenticides, have appeared in the water environment, and have even been reported to cause poisonings in humans and public safety emergencies”. I agree with the authors that: (1) it is possible to find these chemicals in water, and (2) that there are cases in humans of poisoning for most of them. But these (1) and (2) are two independent facts. In this sentence, reader can understand that levels of these compounds in water can result in human poisoning. And this is not what the references say (all of them are about suicides, homicides...). Therefore, the next concern raising is about the toxicity or safety of the normal range of concentrations of these chemicals in aquatic ecosystems, that has not been indicated in the manuscript. Authors should provide detail on the environmental levels of the eleven chemicals they have been testing in the manuscript.

Lines 60-62: “Importantly, the existing studies correlated behavioral changes of zebrafish induced by toxicant exposure with alterations in physiological indicators… “ Please, provide references.

Lines 86-88: “Compared with adult zebrafish, the behavioral changes of larvae occur at much lower toxicant concentrations, allowing earlier detection of contamination” I can't agree with this statement. It depends on the toxicant. For instance, for OP insecticides embryos or early larvae are more resistant than the adults, as adults die very quickly after respiratory arrest. As in embryos and early larvae an important part of the gas interchange takes place through the skin, they are able to increase the survival.

Lines 93-94: “In another study, the exposure to low concentrations of pesticides (1 ppb) in the aquatic environment…” In my opinion 1 ppb it is not necessary a low concentration. Many pesticides are commonly found in aquatic ecosystems in the range of low ng/L

Lines 104-105: “The aim of this work was to develop a robust system with sensitive detection capability of several toxicants in water, based on the behavioral changes of zebrafish larvae” It seems that authors intend to develop a sensitive system for detecting some chemicals in water. But first of all they should provide information on the range of concentrations of each chemicals in aquatic ecosystems, both in directly exposed locations and in non-directly exposed locations. If they are able to detect these chemicals at concentrations in the range of non-directly exposed ecosystems, then they'll have developed a sensitive system suitable for environmental monitoring. However, this hasn’t been demonstrated by the moment.
Experimental design: General comment: I have a problem with the behavioral analysis performed in this study. It has sense to analyze behavior when there is no signs of system toxicity, but it seems that only lethality was recorded. There is no indication on the general state of the larvae at the highest tested concentrations. We can see a significant decrease at the highest concentrations only because the larvae were near the dead or it was a specific effect on the nervous system?

Line 120: “The adult wildtype zebrafish (~3 months old, 2.5–3.0 cm in length)“. I must to say that I’m a bit surprised by the fact that authors have been able to obtain 3 months zebrafish with 2.5-3.0 cm. Usually it takes at least 1 year to get this size.

Lines 124-125: “The selected fish eggs were checked daily, and dead and malformed embryos…” Once fertilized, the oocyte becomes an embryo. Please, change the term “eggs” by “embryos”.

Lines 125-126: “Because the embryo receives nourishment from yolk sac, no feeding was required during the experiment”. At 28ºC most of the yolk is consumed during the first 5 days.

Lines 128-129: “At the end of the experiment, the larvae were euthanized with a saturated benzocaine solution and stored at -78º C” In the manuscript only behavioral endpoints are analyzed. Why are then stored the larvae? The true is that to have some additional endpoints (transcriptomics or metabolomics) to integrate with the behavioral endpoints should be great. The point is that if some additional analyses are going to be done with the exposed larvae, the best is to join all the info in one manuscript.

Lines 138-140: “The stock solutions of KCN, difenacoum, bromadiolone, diphcinone, coumatetralyl, flocoumafen, and fluoroacetamide were prepared in deionized H2O and stored in the dark at 4 °C” For how long the stock solutions were stored at 4ºC? Have you any evidence that there was no degradation during the stored time?

Line 141: “DMSO in the exposure solutions did not exceed 0.1%” I'm not sure if I understand correctly, but authors should explain if the final DMSO concentrations in the working solutions were different with the different concentrations of the same chemicals or among chemicals...

Line 144: In the section “Chemical exposure” should be indicated the exposure time. For me is unclear: 60 min exposure and then transferred to clean water for behavioral analysis? 60 min exposure and then behavioral analysis with the larvae still exposed? Please, clarify.

Lines 161-162: “The activity of each zebrafish larvae was analyzed during each 60 min period for three endpoints, namely, total distance traveled (mm), sinuosity (°/s) and burst count…” Please, clarify the meaning of "each 60 min period". It means the summary of the 6 periods of light (10 min each) and the 6 period of dark (10 min each)?

Lines 167-169: “The inhibitory capacity of the toxicant on the behaviour of zebrafish was assessed as the IC50, i.e. the concentration at which the cumulative distance travelled was reduced by 50% over 60-120 min compared to the control group”. I only want to congratulate the authors because they are very lucky. They have found the IC50 for 11 chemicals within the same range of concentrations (10 ppb-10 ppm) !!!

Line 170: I have seen that authors have tested normality and homocedasticity of the different distributions and they have used then the appropriate statistical test. This is a good point. However, this raise a new question: if there are some data that don’t follow a ormal distribution, why all the graphs in the Figures are based in parametric statistics (mean and SEM)? In my opinion it would be better to use boxplots with the median instead of the current bar charts.
Validity of the findings: Lines 185-186: “The larval zebrafish (6 dpf) were exposed to different concentrations of KCN (0, 0.01, 0.1, 1, 10 mg/L) for 2 h and their locomotor activity was monitored” It seems that larvae were exposed for 2h, but this is also the time covered by the behavioral analysis. I'm not sure if behavioral analysis started at the end of the 2h exposure. I don't know if larvae were also exposed during the 2 h of the behavioral analysis. All these details should be clarified.

Lines 191-194: I don't understand all the effects that authors claim to see: inhibition, excitation, avoidance... As I can't see any statistics after each comment, I suppose that there are no statistically significant differences, and therefore, there are no effects.

Lines 197-200: “Then, we assessed the behavioral impairment of zebrafish using the cumulative distances travelled by zebrafish larvae over the three cycles totaling the first 60 min (0-60 min) and over three cycles of the second 60 min (60-120 min) of exposure” I'm not extremely happy with this decision from authors. First of all, authors should justify the need to perform 6 cycles L/D. Why they are performing 6 cycles? What information they expect to obtain performing 6 cycles instead of 1 cycle? From my point of view, it is not very interesting to pooling the locomotor activity of L1/D1/L2/D2/L3/D3 and L4/D4/L5/D5/L6/D6. I don't know what exactly authors intend to obtain.

Lines 205-206: “Since 10 mg/L KCN was apparently lethal, there was no swimming detected in this exposure group” I don't understand the meaning of "10 mg/L KCN was apparently lethal" All the larvae died? Some larvae died? Not only lethality, but also any sign of system toxicity should be carefully surveyed before to start any behavioral test. On the other hand, why authors don’t include here the description of the effects on sinuosity and burst behaviors??

Lines 208 and 226: The same section title!

Lines 232 and 239: Exactly the same statistical information is provided.

Lines 287-288: “Clustering analysis of cumulative distance travelled by zebrafish larvae, a common method for assessing the neurotoxicity of toxicants to organisms” I'm not so sure that accumulative distance across 3 different L/D cycles is to common method, as it is unclear usefulness of the information that this approach provides.

Lines 292-294: “The reduction of sinuosity on zebrafish larvae indicated that the balance of movement was disrupted, and has been reported in Parkinson's-like behavior resulting from nerve injury (Bortolotto et al., 2014)” This sentence is not related with the results, but with the discussion.

Lines 299-302: “In general, the clustering pattern of concentrations was consistent at the three behavioral endpoints, i.e., consistent behavioral capacity at concentrations of 0 mg/L, 0.01 mg/L, and 0.1 mg/L, with a difference from 1 mg/L and the greatest difference from 10 mg/L” It was really necessary to perform the hierarchical clustering to get this conclusion?
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: EARLY DETECTION OF CYANIDE, ORGANOPHOSPHATE AND RODENTICIDE POLLUTION BASED ON LOCOMOTOR ACTIVITY OF ZEBRAFISH LARVAE
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1-	The manuscript entitled “Early detection of cyanide, organophosphate and rodenticide pollution based on locomotor activity of zebrafish larvae” focus a relevant topic in ecotoxicology, namely by using a behavior approach on zebrafish model to detect early responses to contaminants. Despite the high relevance and applicability of this approach, I highlight some issues related with the methodological approach, which might compromise the interpretation of results and outcomes of this manuscript.
2-	The objectives of the work are specified but the hypothesis tested is not presented.
3-	The introduction is well structured but the authors do not present the rationale behind the selection of the contaminants for this behavioral study. The authors present the mode of action in target species for the selected contaminants, but an explanation on why they were selected is missing. Why do they specifically fit the purpose of testing this behavioral assay to detect contaminants?
4-	The results presented address the main objectives of the work and are well structured and analysed.
5- Control in each figure: specify if it is relative to negative control or to solvent control. Present data on DMSO effects on behavior relatively to negative control.
Experimental design: 1-	The final concentration of DMSO used in all treatments (0.1%) is above the maximum concentration recommended for solvent use in standard guidelines, including in FET OECD 236 Guideline: “Where a solvent is used to assist in stock solution preparation, its final concentration should not exceed 100 μl/L and should be the same in all test vessels.” This corresponds to a maximum percentage of 0.01% (v/v) of solvent. As solvents are well known to cause effects in behavior, the solvents should be kept at minimal concentrations, and whenever possible, well below recommended values in order to avoid biased results and/or mixture effects from solvent + target substance.
2-	Chemical analysis is lacking in the methodological approach. The inclusion of this data is extremely relevant to fit the purpose and objective of testing the behavioral assay to detect the selected substances. In order to assure that the effects observed in the fish are elicited by a certain substance concentrations, the real concentrations tested should be properly verified to check the nominal concentrations used. The authors should add analytical chemical analysis data (stock solutions, initial solutions and) to this work to properly interpret their results and outcomes.
3-	Line 172-173 – “The adaptation period included in the analysis was an initial 10-min dark period”. This is already explained in the paragraph before (lines 155-157). However, if presented again here in statistical analysis, the authors should state if they have included in the data analysis the acclimation period or not. In fact, the acclimation period should not be included in the data analysis and this mention here is confusing.
Validity of the findings: 1-	The discussion should be more focused on interpreting and discussing the specific results obtained instead of giving a general overview about the topic. This will better fit the Introduction section. In general, the discussion lacks interpretation of the specific results obtained for the chemicals used vs. results obtained by other authors.
Additional comments: 1-	Line 115-116 - Delete: “Add your introduction here”.
2-	Lines 145-146 – It is not necessary to include along the manuscript text the authors’ credits and participation in the lab work. This information should be presented in a proper and different section of the manuscript according to journal guidelines.


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: ELECTROENCEPHALOGRAM-DERIVED PAIN INDEX FOR EVALUATING PAIN DURING LABOR
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article is written in sufficiently clear English that can be understand by vast population.
Experimental design: The experiment seek to find the correlation between Pain index (Pi) and NRS score rating for pain due to labor.
Validity of the findings: There is indeed some correlation of the Pi and NRS measured in this experiment. However, in my humble opinion the correlation is not strong and may be due to the noises recorded in the EEG, especially when the pain in question is a result of muscles contraction (which caused the recorded EMG). According to the article by An et al 2017b, the Pi is a derivative from the weighted wavelets coefficients, these coefficients may increase in value when there is a strong EMG recorded due to muscles contraction. This effect is also seen during some of the apex of labor contraction under epidural analgesia when no painful response is recorded in the NRS. The authors need to make sure that appropriate filtering of the EEG signals is applied to remove or minimize the influence of EMG. Nonetheless, I think there is some usefulness of this study as it covers a reasonable numbers of samples of about 1100 samples from 80 subjects. The author should explain the above limitation in the discussion section.
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: ELECTROENCEPHALOGRAM-DERIVED PAIN INDEX FOR EVALUATING PAIN DURING LABOR
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors address a clear goal of this paper in which they intended to come up with more reliable and quantitative measures of pain during labor. In this study, the authors claimed to develop a pain index that is derived from the frequency band EEG wavelets. In general, the flow is logical and structured accordingly. However, some improvements can be made in all of the figures and tables provided. The unit of measurement for Pi and NRS should be mentioned elsewhere in the main text and also in both exes of the graphs. No title of the table and this makes it hard for the reader to make sense of the content of the table. Presentation-wise of the table title should be improved.
Experimental design: Lack of details in EEG recording and data analysis. The authors claim that they used EEG as a device to come up with EEG frequency spectrum and develop the pain index. Lacking the details of EEG pre-processing and frequency analysis may question the validity and reliability of the findings. That information should be mentioned clearly in the method so that others can replicate it as well.

Apart from that, the authors should not miss out on the details of their EEG recording device such as sampling rate, etc. area of the electrode position, following the standard 10-20 EEG system position.
Validity of the findings: The authors need to justify of only including two stages of labor in this study. If there are other stages, should state in the literature.

In terms of sample size, the authors stated that their findings are less reliable due to the small sample size. Please state a required sample size and show the calculation for enough samples that can make their findings becoming more reliable.

In lines 269-270, the authors mentioned that they might exclude some confounding factors. This is a very ambiguous statement. Please be more specific and state what are the factors they are referring to as their limitation.
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: ELECTROENCEPHALOGRAM-DERIVED PAIN INDEX FOR EVALUATING PAIN DURING LABOR
Review round: 2
Reviewer: 1

Output:
Basic reporting: Manuscript is clear and easy to understand.
Experimental design: Experiment design is simple and easy to understand.
Validity of the findings: Results are statistically sound although not of significant scientific advancement.
Additional comments: I would like to suggest some references of similar study in which the author may refer to:

SAI, C.Y., MOKHTAR, N., YIP, H.W. et al. Objective identification of pain due to uterine contraction during the first stage of labour using continuous EEG signals and SVM. Sādhanā 44, 87 (2019).

Kumar S, Kumar A, Trikha A and Anand S. Electroencephalogram based quantitative estimation of pain for balanced anaesthesia. Measurement 59: 296–301 (2015).


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: INTEROCEPTIVE AWARENESS AND BELIEFS ABOUT HEALTH AND THE BODY AS PREDICTORS OF THE INTENSITY OF EMOTIONS EXPERIENCED AT THE BEGINNING OF THE PANDEMIC
Review round: 1
Reviewer: 1

Output:
Basic reporting: In my opinion, the introduction is way too long. I believe it could benefit from a revision to increase the focus on the important concepts only. Nevertheless, I acknowledge this is also subject to editorial decision.

To avoid misinterpretations with other definitions of interoceptive awareness, it should be specified in the abstract that the concept of interoceptive awareness adopted refers to that proposed by Mehling and colleagues in the MAIA.

Lines 128- 129: The authors wrote "However, valence is more intuitive when classifying emotional experiences, and thus in the current research, we place a strong emphasis on the valence dimension". Buy it's not cleat to me why valence. Can't we assume that the vast majority will perceive this as negative, thus with little variation along this dimension, but the level of intensity or arousal will vary in greater degree? Please clarify or revise

line 273: define emotional susceptibility

The difference between hypothesis 1 and hypothesis 2 could be clearer (line 273).

Acknowledging I am not an expert on canonical correlations, is there statistics to be reported on the associations between specific MAIA dimensions and emotions (lines 543-547)?

For clarity, examples of the beliefs questions, specifying the precise phrasing, should be given in the main text

As a suggestion, the authors could consider a way of organizing and synthesizing all the measures, analyses and main findings, for example in a summary table.

A few sentences and expressions are grammatically awkward. The manuscript could benefit from proof-reading

There is no mention to ethics approval nor informed consent
Experimental design: The analyses plan consisted in several approaches to examine the relationship between 2 variables or 2 sets of variables (i.e. canonical correlations). However, because the data set comprises, at least, 3 sets of variables (i.e. emotions, beliefs, intereoceptive beliefs) wouldn't it make sense to try to investigate how these three sets are related to each other, for example through targeted or theoretically driven mediations/moderation analyses or Structural Equation Modelling. I feel the different analyses report "a lot of the same" and it would be interesting to go beyond this level of analyses. I would like to know the authors' thoughts on this.

I am not fully convinced the emotions questions, and the way in which they were framed, are sufficiently related to the Covid-19 pandemic. If that would be the case it would have important implications for the interpretation of results. However, I might be missing some detail and the authors might be able to convince me otherwise. Please clarify.
Validity of the findings: Further description of each statistical analysis and justification for its use is needed in the methods section. Three sets of analyses are carried out but little explanation is given for why these are necessary and how they differ between them. In fact, it was very difficult for me to follow the results section.As a reader, as I was going through the long "list" of results (and many tables) I felt there were really too many analyses that are conceptually similar. This meant that, for me, that it was difficult to follow through all the analyses and extract the (most) meaningful information. I advise, not only to provide greater justification for the analyses plan but also to consider removing, or at least moving it to supplementary materials, some of the analyses (e.g. simple correlations; demographics).

The manuscript is framed as directly related to Covid-19 but I am not fully convinced the methods and discussion of results truly reflect this. In addition to my comment above on the relevance of the emotions question, it's not clear to me that enough has been done in terms of analyses/discussion to make bold claims about reactions to Covid-19. For example, how does MAIA dimensions are related to feelings related to Covid-19. Also, not much attention in discussion and analyses (e.g. canonical correlations) was given to the beliefs variables and I'd think these would be the ones mostly related to Covid-19.
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: INTEROCEPTIVE AWARENESS AND BELIEFS ABOUT HEALTH AND THE BODY AS PREDICTORS OF THE INTENSITY OF EMOTIONS EXPERIENCED AT THE BEGINNING OF THE PANDEMIC
Review round: 1
Reviewer: 2

Output:
Basic reporting: The study reported in manuscript identifies a relation among some dimensions concerning the Interoceptive awareness (Self-Regulation, Trusting and Not-Worrying dimensions) and the emotional reaction to the COVID-19 pandemic. Overall, the manuscript is well written and offers an interesting contribution to the understanding of the psychological/emotional experience of the pandemic.
I have some points here below:
I suggest the authors to reduce the introduction session, especially the paragraph from line 111 to 158, too many words are spent to talk about emotion and the literature mentioned is a bit old.
Lines 117: I would not say that emotions are reactions, emotions are goal-directed action tendencies. I invite the author to read and mention:

Moors, A., Boddez, Y., & De Houwer, J. (2017). The power of goal-directed processes in the causation of emotional and other actions. Emotion Review, 9(4), 310-318.

Moors, A., & Fischer, M. (2019). Demystifying the role of emotion in behaviour: toward a goal-directed account. Cognition and Emotion, 33(1), 94-100.

The valence and arousal are not the only dimensions in defining emotions.
In the goal-directed view, there are also: the experienced control of  the aversive stimuli, the value attributed to the goal pursued and the expectancy about the reached outcome.
See Moors, A., Fini, C., Everaert, T., Bardi, L., Bossuyt, E., Kuppens, P., & Brass, M. (2019). The role of stimulus-driven versus goal-directed processes in fight and flight tendencies measured with motor evoked potentials induced by Transcranial Magnetic Stimulation. PloS one, 14(5), e0217266.
Moreover, I do not totally agree with the all-of-none view of automaticity, invoked to separate fast and dirty automatic emotions from more complex cognitive reflective emotions.
I invite the authors to read:
Moors, A., & De Houwer, J. (2006). Automaticity: a theoretical and conceptual analysis. Psychological bulletin, 132(2), 297.
In this theoretical paper, the authors provide a deep insight of the concept of automaticity and suggest to think about the automaticity as composed by multiple features which need to be separately investigated and to be considered along a continuum in all the cognitive processes, including the emotions. Basically, they invite all the researchers to overrule the all-or-none view.
Concerning the paragraph: “Factors related to psychological attitude towards the body affecting epidemic responses”, I invite the authors to synthetize the paragraph and to talk about interoception, by adding recent literature.
See:
Iodice, P., Porciello, G., Bufalari, I., Barca, L., & Pezzulo, G. (2019). An interoceptive illusion of effort induced by false heart-rate feedback. Proceedings of the National Academy of Sciences, 116(28), 13897-13902.
Quadt, L., Critchley, H. D., & Garfinkel, S. N. (2018). The neurobiology of interoception in health and disease. Annals of the New York Academy of Sciences, 1428(1), 112-128.
Pezzulo, G., Maisto, D., Barca, L., & Van den Bergh, O. (2019). Symptom perception from a predictive processing perspective. Clinical Psychology in Europe, 1(4), 1-14.
Experimental design: The Hypothesis of the study are not clear to me and I cannot follow the rationale which has led to declare them.
By taking into consideration the mentioned literature about the relation between interoceptive awareness and emotion susceptibility, I would aspect a more sophisticated hypothesis, maybe there are only some components of the interoceptive awareness associated with the diminished  experience of negative emotion (H1).
Procedure:
The authors set as sample criteria just the age, I wonder whether it would be important asking about the use of medications which can alter the body sensations.
Validity of the findings: Results
Line 445: “Negative emotions are negatively related to perceived material status”, please reframe more clearly
Discussion:
Lines 660-662: “Interestingly, Noticing and Emotional awareness were positively related with negative emotions; however, the correlation was low.”
I invite the authors to spend some words to discuss this data which is consistent with part of the literature mentioned in the intro.
Additional comments: Check the typos


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: INTEROCEPTIVE AWARENESS AND BELIEFS ABOUT HEALTH AND THE BODY AS PREDICTORS OF THE INTENSITY OF EMOTIONS EXPERIENCED AT THE BEGINNING OF THE PANDEMIC
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I thank the authors for their effort to address my comments and appreciate the extensive revisions made. I believe the manuscript has improved considerably, especially in terms of readability. However, I still have some concerns I would like to see clarified or addressed.

I might be missing something but I still believe there is substantial conceptual repetition in the analyses performed. Path analyses does indeed seem to be a good addition to the manuscript and brought some additional depth to the study (please note that I am not an expert neither in canonical correlations nor path analyses). However, I am not sure what is the added value of reporting the simple correlations and the regressions models on top of the canonical correlations and path analyses. It is possible that I might be missing something and the authors might be able to convince me otherwise, but I’d think these are peripherical and should, at most be moved into the appendix. I am afraid I personally find the results section very difficult to digest with all the different (but conceptually similar) results. Unfortunately, this also transpires into the discussion section.
Also, if correlation analyses are to be reported these should be corrected for multiple comparisons.

Minor:
I would advice the authors to have the manuscript (including abstract) proof-read, ideally by someone in the field, as there are several awkward sentences and expressions. For example: “The COVID-19 pandemic is basically a strong stressor.” Also, I appreciate the authors effort to reduce and simplify the theoretical background in the introduction. However, in certain places the flow of information is somewhat fragmented and lacks fluidity (e.g. paragraph on stress and physiological reactions). I’d advise the authors to revise.

Hypothesis 2 seems to be missing

The description of the emotions questionnaire could be clearer. What precise question was asked? That is, were the question on felt emotions placed in the context of the pandemic or simply referred to “here and now”? These might lead to considerable different interpretations. The table added does clarify considerably some questions I had (mostly regarding how to measure the origin dimension) but a few details could still be clearer.

Please carefully review the results section as there might be several typos. For example, in line 483, should it read “negative emotions”?; in line 505, should it read “three of which”?.


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: INTEROCEPTIVE AWARENESS AND BELIEFS ABOUT HEALTH AND THE BODY AS PREDICTORS OF THE INTENSITY OF EMOTIONS EXPERIENCED AT THE BEGINNING OF THE PANDEMIC
Review round: 2
Reviewer: 2

Output:
Basic reporting: Overall, I find the manuscript improved; as required, the authors removed some less relevant pieces of introductions and added other important references from the recent literature.
I kindly encourage the authors to revise/improve the writing in order to endorse the publication of the manuscript.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: INTEROCEPTIVE AWARENESS AND BELIEFS ABOUT HEALTH AND THE BODY AS PREDICTORS OF THE INTENSITY OF EMOTIONS EXPERIENCED AT THE BEGINNING OF THE PANDEMIC
Review round: 3
Reviewer: 1

Output:
Basic reporting: Before endorsing the publication of the manuscript. I kindly invite the authors to re-phrase this paragraph:
"Attempts to understand the relationship between experiencing your body and emotions should also distinguish between the perception of sensations and their declared feeling Pezzulo
 and colleagues (2019) indicate that physiological dysfunctions are associated with self-reported
 symptoms, when an individual suffers from a disease. Authors explained the fluctuation in the
relationship between psychological dysfunctions affected by the disease and reported symptoms,
using the predictive processing perspective. The approach is based on exploration of the
 construction of the adaptive model by the brain, thus referring to predictive coding revealed in
perception and an active interference action. This conceptualisation addresses the issue of pre existing (prior) information and its interference in the construction of newly occurring
 representation, and therefore it offers an interesting perspective in understanding the role of
cognitive beliefs in experiencing a stressful situation."
It should be written more clearly, at the moment it appears quite obscure.
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: RISE, AND PRONOUNCED REGIONAL VARIATION, IN METHYLPHENIDATE, AMPHETAMINE, AND LISDEXAMFETAMINE DISTRIBUTION IN THE UNITED STATES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article is interesting and informative.
Experimental design: Methods appear sound.
Validity of the findings: Data supports findings.
Additional comments: The manuscript is interesting and informative.

Lines 93-95 "Patients prescribed stimulants had a nine-fold elevated risk of developing basal ganglia and cerebellar disorders."  This should be clarified.  Curtin et al noted in their abstract: "The association of ADHD patients prescribed psychostimulants with higher risk of diseases of the basal ganglia and cerebellum may reflect a more severe ADHD phenotype rather than a direct association between prescribed stimulant use and basal ganglia or cerebellum disorders." They noted patients with ADHD had a 2.4 fold increased risk.

In the discussion, speculation of "independent spirit of the western U.S." should be backed up with a reference or removed.  Much of the lower use in the may be driven by lack of specialty health care providers in several of the western states.

Line 274-277 should be rewritten as stimulants are one of the most effective classes of drugs in medicine with more than a 50 year history of safety and the comment may give the reader the false notion that these medications are not safe and effective when prescribed and used appropriately.


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: RISE, AND PRONOUNCED REGIONAL VARIATION, IN METHYLPHENIDATE, AMPHETAMINE, AND LISDEXAMFETAMINE DISTRIBUTION IN THE UNITED STATES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is interesting however it is currently not written for an international audience and could be improved. Some examples including referring to Schedule II stimulants in the title. This was also in the introduction with no definition or background (line 100), a definition was provided in the discussion, this should be removed and placed in the background. The abstract and article should be reviewed to make sure the context is understandable to an international audience including the title.
Whilst the information in the background is informative, the statistics and references are over 10 years old, for example, in line 71 you have the growth until 2011, however have not reported on any statistics since then. Please provide further references. There is a reference missing on line 87.
Line 100 you state “Non-medical use of these Schedule II agents is appreciable.” Please expand on what you mean by this.
In some instances, for example, line 105 you use brand names, it is important to be consistent and also state the drug name if it is necessary to use the brand name.
There are some occasions when the acronym is not provided in the manuscript for example line 144 electric health records and line 87 & 218 DSM-5, line 219 DSM-IV, line 280 DEA.
Rewrite line 263, ‘not insignificant portion’, it is unclear what you are trying to state here.
Experimental design: Whilst I get the sense the authors have conducted a significant amount of analysis, I am having difficulties following what was conducted and why. The article would be significantly improved if this section were rewritten in particularly the data analysis section.
Areas to help with clarification are providing context to ‘stimulant weight’ what is this referring to, breaking down your different studies into parts, explanation on how the daily doses were calculated, justification on why you choose 1.96 standard deviations from the mean. Expand on this new methodology by providing more tangible information.
You mention Drug Enforcement Administration Weights in the abstract however this is not discussed in the materials and methods section. This should be included in this section.
You state that you conducted linear regression analysis for the Hispanic population however you report on correlations, where is the linear regression model? I would recommend reviewing the statistics choices for better test statistics other than a correlation or justify why this was chosen. Further statistical investigation is warranted.
Validity of the findings: A clear description of your methods would help in understanding the results sections for example line 181 ‘stimulant use, by weight’.
Adding the p values, 95% confidence intervals and the tests conducted to all the figures would be beneficial for the reader. Whilst the figures are good, tables are also helpful to the reader, I would consider using some tables rather than all figures as this would be more informative to the readers and would provide a more in-depth understanding of the findings.
For Figure 4 and 5, it is unclear on the rationale of providing the figures and what the differences mean. Again, making it clearer to an international audience the differences between the different reimbursement, and clear about the Medicaid by cost and Medicaid by prescription. In some instances, you have overreached on your findings for example with the Hispanic populations, the methodology was a correlation, and then on line 236, you state the ‘low rate of stimulant use among the Hispanic communities’, did you also calculate rates?
It is good practice to identify your limitations in your study design, there is currently no limitation. Please reflect on your study design and add in the limitations to your study.
Additional comments: I commend the authors for the time they have taken in exploring this interesting topic and dataset. Whilst the manuscript is interesting and relevant, the manuscript should be tailored for an international audience with greater attention to details (for example with the use of acronyms), additionally, the method and result sections including the statistical methods should be improved upon before acceptance.