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

Input:
Title: THE ROLE OF AMBIENT TEMPERATURE AND BODY MASS ON BODY TEMPERATURE, STANDARD METABOLIC RATE AND EVAPORATIVE WATER LOSS IN SOUTHERN AFRICAN ANURANS OF DIFFERENT HABITAT SPECIALISATION
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: I have an issue with the analysis of VPD that needs to be addressed. Please see general comments to the authors.
Validity of the findings: I have an issue with the validity of their analysis on VPD, see below.
Additional comments: Dear authors,

Thank you for the revision and the taking the time to address my comments. I appreciate the improvements, and I think the manuscript is useful and will be appreciated. But there’s one critical detail to address.

After reading the authors’ revision and inspecting their data, I think it’s imperative to make some things clear about their VPD analysis. Again, I appreciated the additional analyses in this manuscript, but the results are misleading. The authors state that VPD does not affect water loss rates, but in fact, their experimental design precludes an appropriate analysis of VPD. In the design, VPD and temperature are highly correlated. That’s unsurprising given that the authors scrubbed the airstream of water vapor. To analyze VPD, the authors binned VPDs into high and low categories within each temperature treatment. There are four issues with that: (1) there is very little variation in VPD within each temperature treatment leading to arbitrary binning of VPDs into categories, (2) VPDs in the high category are lower than VPDs in the low categories in different temperatures (and vice versa), (3) high or low VPDs are not present in some of the temperature treatments, and (4) an interaction term between VPD and each temperature treatment (since they are factors in the analysis) needs to be incorporated into the model to get an idea of VPDs effect within each temperature treatment. Given the first 3 issues, I would not recommend following the 4th recommendation.

That being said, there is a way to evaluate the relative role of temperature vs VPD, even though they are highly correlated in the authors’ dataset. I would recommend using VPD and air temperature (Tcham) as covariates in separate analyses in nlme and compete them to see which variable has the highest R2. When I did that analysis, VPD was a better predictor than temperature in 2/3 of the species (code below). For me, the statistical evidence and physical expectation of VPD’s effect on water loss rates suggests that the authors should be using VPD as a covariate, and not use temperature at all.

I have a feeling that the authors will have an aversion to that analysis because the predominant way of thinking about physiology is through the lens of temperature. So I suggest another option for the authors: remove VPD from the analysis, add a supplemental figure or analysis on VPD alone (as I provided), and indicate in the manuscript that the authors did not design the experiment to test for VPD but it’s possible it may be driving the effect of temperature. It could simply be a couple sentences in the results and discussion.

Minor comments

What are the units of VPD? And please provide a citation for the equations that you used to calculate it.

line 26: typo “and on”

line 33: change “and” to “or”

#R code
library(dplyr)
library(nlme)
library(MuMIn)

peerj <- read.csv("~/Desktop/peerj.csv")

#make subsets of data for each species
ami <- select(filter(peerj, Species == "Amietia"),c(Species,ID,Sex,Fri,Tcham,Mass_F,Mass_Log,VH2O,MVH2O,VPD,VPD_C))
scl <- select(filter(peerj, Species == "Sclerophrys"),c(Species,ID,Sex,Fri,Tcham,Mass_F,Mass_Log,VH2O,MVH2O,VPD,VPD_C))
xen <- select(filter(peerj, Species == “Xenopus"),c(Species,ID,Sex,Fri,Tcham,Mass_F,Mass_Log,VH2O,MVH2O,VPD,VPD_C))

#compare r.squared’s for each species with VPD and temperature
ami_mod <- lme(VH2O ~ VPD, random=(~1|ID), data=ami)
scl_mod <- lme(VH2O ~ VPD, random=(~1|ID), data=scl)
xen_mod <- lme(VH2O ~ VPD, random=(~1|ID), data=xen)

#results
r.squaredGLMM(ami_mod)
      R2m       R2c
0.3126473 0.4313033

r.squaredGLMM(scl_mod)
      R2m       R2c
0.2231434 0.2231434

r.squaredGLMM(xen_mod)
      R2m       R2c
0.4368583 0.4368583

ami_mod <- lme(VH2O ~ Tcham, random=(~1|ID), data=ami)
scl_mod <- lme(VH2O ~ Tcham, random=(~1|ID), data=scl)
xen_mod <- lme(VH2O ~ Tcham, random=(~1|ID), data=xen)

#results
r.squaredGLMM(ami_mod)
      R2m       R2c
0.2647765 0.3553159

r.squaredGLMM(scl_mod)
     R2m      R2c
0.214727 0.214727

r.squaredGLMM(xen_mod)
      R2m       R2c
0.4501313 0.4501313


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

Input:
Title: THE ROLE OF AMBIENT TEMPERATURE AND BODY MASS ON BODY TEMPERATURE, STANDARD METABOLIC RATE AND EVAPORATIVE WATER LOSS IN SOUTHERN AFRICAN ANURANS OF DIFFERENT HABITAT SPECIALISATION
Review round: 2
Reviewer: 2

Output:
Basic reporting: This revised manuscript is much improved over the previous version. It is more readable than before and seems that authors dealt with almost all the methodological issues raised before by the reviewers. However, the writing still needs some attention to clarity, mainly in the Discussion section. With some further work, I find that it will be a good contribution to the knowledge of the area.
Experimental design: Ok with almost all replies/clarifications from the authors. But see my new comments below at “Authors replies and my new comments” and also at "Line-by-line".
Validity of the findings: I could not follow the rationale of not including interactions among variables in the models. Please also deal with the line-by-line comments below to clarify the new issues raised at the present version of the MS.
Additional comments: Line-by-line:

Line 26: “…the role of Ta and on Tb, whole-animal EWL and whole-animal SMR…” to keep this sequence in which the results are presented below and also in other sections. Also keep it throughout the text in order to facilitate to the readers.

Line 32: “Tb” instead of “body temperature”.

Lines 66-67: Gillooly et al. 2001 as citation for “low mass-specific rates of …water loss”?? If not, please provide a satisfactory reference for water loss issue.

Line 69: ecology is a vague term here. Following the references cited in this passage, it should be “habitat use” or something in this line.

Line 73: typo? “,” instead of “;”

Line 79: “other groups”. Which ones? Maybe something in these lines: “compared to other groups, such as terrestrial and aquatic ones”.

Lines 82-83: increases passive heat loss? Or decreases? Increasing skin resistance and consequently reducing EWL would reduce heat loss, so that overheating is a problem for such animals at high temperatures.

Line 84: "skin resistance to EWL" instead of "skin water resistance". To keep it similar with usual terms in the literature of the area.

Lines 84-85: “due to changes in blood delivery to the skin following its lipids composition.” Blood delivery of what? Maybe reword this passage in order to clarify it.

Line 100: “affects Tb, EWL and SMR” instead of “affects SMR, EWL and Tb”.

Line 101: Delete: "We selected species depending on their apparent dependency on water".  Thus, the new version: "…African anuran species: the principally-aquatic African..."

Lines 104-106: I still cannot get your point here (and I commented on this in the last revision): "We tested how the variation in Ta affects (...) Next, we determine whether these species differ in their responses to Ta fluctuations."
What is the difference between these questions? It is not clear to the readers and authors should be more specific. In addition, if the term "determine" is kept after the modification, change it to another similar and more appropriate word.

Lines 106-107: “We expect that differences in species’ response are associated with differences in ecological specialisation.” I suggest that you follow the sequence that you presented your physiological variables: Tb, EWL, SMR. So: “…specialisation. We expected Tb increasing with Ta, but also Tb to be influenced by Mb, such that larger individuals would have lower Tb, specifically at higher Ta. We also expect aquatic species to show higher EWL…”.

Lines 107-110: It is a bit inconsistent once you describe in your methods (lines 121-122) that the aquatic species (X. laevis) frequently disperse overland. So why would you expect it to show higher EWL compared the semi-aquatic species? In other words, why conditions that may lead to reduced potential for evolutionary adaptation (of water balance traits?) are buffered in aquatic environments of X. laevis and not of A. delalandii?
The problem here is the way that you described it in the text, so that it makes this inconsistence evident when aligning it with your result expectations.

Line 256: I am not sure if I fully understood your data analyses. Please provide the rationale of not including interactions among variables in the models.

Line 271: Change to: “We found that changes in Tb were not related to Mb…”

Line 275-276: “sex” instead of “males and females”. Keep the terms similarly throughout the section.

Line 277: “had a significant effect of…”. What does it mean? Would it be(?): “whole-animal SMR increased with Ta”.

Line 281: “We found that changes in Ta had a significant effect on Tb”. Would not be better to describe the direction of this effect instead of just reporting that there was an effect (that the readers may be still not familiar with)? Whenever possible, provide this kind of information (direction) to the readers.

Line 353: “…aquatic urodeles (Olalla-Tárraga & Rodríguez, 2007) and in anurans (Gouveia et al., 2019)”.
https://www.amnat.org/an/newpapers/JanGouveia.html; https://dx.doi.org/10.1086/700833.

Line 356: when I go to see the Figure 2 to check it, this not apply for Sclerophys (15C and 25C are lower than 5, 10, 20C for example). This should be well explained in the text here.

Line 361: change “determine” to “investigate”.

Line 365-366: Line 365-366: The definition of Rc is not the “ability” to reduce rates of water loss. This reads awkward (and should be modified or excluded) since Rc is a value of mass (water) transference through a physical barrier, which is calculated from EWL rates.

Line 367: Maybe should be: "although high Rc may not be beneficial to large-bodied frogs for thermoregulation (due to possibility of overheating at high Ta's)". Since some of the readers may not be familiar with the issue.

Line 368-369: "However, in this study we did not find any significant effect of Mb, VPD and sex on rates of whole-animal EWL in the three-species considered".
First, it seems not presented in logical sequence since you were discussing Mb and Rc effects in the sentences above. So, VPD and sex suddenly appeared in a mixed way at this point of the discussion. I suggest you modify it. Second, "Mb... did influence whole-animal EWL" (Lines 301-302), and so this needs clarification.

Lines 369-371: I could not understand this sentence. This should be rewritten for clarity. “Furthermore, there seems to be a difference in the onset of evaporative cooling with high Rc or atypical frog species only employing EWL at high ambient temperatures compared low and moderate Rc or typical frogs”.

Lines 372-375: Again. I could not follow the rationale of your sequence in this sentence since in the last two sentences you were discussing about relationships among EWL, Rc and thermoregulation. Then, you conclude the paragraph with "and in this study we found that Tb is influenced by Mb in both S. capensis and A. delalandii". Clarify why you included Mb at this point of the text or change it accordingly.

Lines 376-378: Although you "expected that S. capensis would be capable of maintaining Tb close to Ta (based on its terrestrial life style), better than the other two largely aquatic species", it does not appear in any other part of the text. Thus, you could include it at the final of the Introduction section to previously communicate your expectations to the readers.

Lines 383-386: I suggest avoiding hyperbolical expression as "largely follows" since your dataset contains a limited range of species and habitat uses in comparison to Young et al 2005. Also, in the specific case of R. marina, I do not understand why you are not discussing the role of Mb component at this part of the text, since that is why this species is considered “atypical” (large body-size decreases the rates of EWL). Thus, this paragraph needs some work to connect variables of the problems you are discussing as well your own findings.

Lines 388-394: Very good discussion and connection of ideas. So, it is an additional motivation to improve the text above.

Lines 396-409: That is the only paragraph discussing SMR since the other one at this same sub-section (Lines 410-423) seems not a part of it. Thus, SMR section needs some further work to align with other sub-sections above (Tb and EWL).

Lines 405-406: It was what I would expect for both SMR and EWL since this species presents the largest body size in comparison to the other ones. For this kind of comparison (which led to the conclusion of the paragraph), I think it was more appropriate to compare specific values of EWL and SMR (V of H2O or O2 per gram or cm2 of animal per time). I am not sure if you can freely conclude this last idea with absolute values.

Lines 410-423: This in the context of habitat use and not of SMR, thus should be shorten (since it repeats some points later as ecology, habitat specialisation) to be incorporated to the conclusion section.

Lines 413-416: I commented on this in the last revision that this one of the very interesting finding of your study.
Line 423: This sentence closes the paragraph being very unspecific in my point of view. Ecology of what? Habitat use? Habit? Complete it according to your theoretical variables.

Line 426: Change to : "investigated such that it was, in most but not all cases, positively correlated with..”

Lines 429-431: Mb (allometry) is important when analysing all physiological processes. So I could not follow what was the point that you highlight here since Mb must be controlled before analysing changes in physiological traits. The same apply to the Lines 434-437. Please modify the text accordingly or highlight better what was your point in these paragraphs when discussing the role of Mb.

Line 431: “(Olalla-Tárraga & Rodriguez, 2007; Gouveia et al, 2019)”

Figures 2 and 3. Is it correct for 30C and 35? In my pdf version it seems they are overlapped (EWL and SMR data). Also, it seems that the terrestrial species show higher rates of EWL than the aquatic ones at 20C and 30C. Why?

Authors replies and my new comments.

Authors: Incorrect. The reviewer may have missed this point as it was outlined in line 217-220. See also lines line 178 – 180.
My new comment: Actually, it was not clear in the last version and I can see that you modified the text to achieve clarity.

Authors: We are not sure what the reviewer identifies as the problem here. It is standard procedure in respirometry to adjust chamber size to animal size, and subsequently adjust flow rates to obtain acceptable signals of gasses measured. The animal should be able to rest comfortably in the chamber and turn around, but not walk and jump around. We adjusted flow rate with chamber size aiming to maintain similar humidity and oxygen deficits in the chambers.
My new comment: Ok, but are you sure that the same apply for EWL measurements? That is why I raised this issue in the last round asking for EWL and resistance comparisons but I could not find it in the new version.

Authors: The percentage body mass loss in the 2 hr experimental period was (mean ±SE) 3. 2 1± 1.66 (Xenopus laevis), 3.91 ±3.84 % (Sclerophrys capensis and 5.2 ±2.85 % (Amietia delalandii). We have to assume the “other factor” referred to here, concerned VPD? Please see our response further above where we check that VPD did not confound our analyses as far as we could tell.
My new comment: I suggest you include all these percentages in the methods section.


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

Input:
Title: THE ROLE OF AMBIENT TEMPERATURE AND BODY MASS ON BODY TEMPERATURE, STANDARD METABOLIC RATE AND EVAPORATIVE WATER LOSS IN SOUTHERN AFRICAN ANURANS OF DIFFERENT HABITAT SPECIALISATION
Review round: 3
Reviewer: 1

Output:
Basic reporting: I have no further comments. The article is acceptable.
Experimental design: I have no further comments. The article is acceptable.
Validity of the findings: I have no further comments. The article is acceptable.
Additional comments: I have no further comments. The article is acceptable.


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

Input:
Title: THE ROLE OF AMBIENT TEMPERATURE AND BODY MASS ON BODY TEMPERATURE, STANDARD METABOLIC RATE AND EVAPORATIVE WATER LOSS IN SOUTHERN AFRICAN ANURANS OF DIFFERENT HABITAT SPECIALISATION
Review round: 3
Reviewer: 2

Output:
Basic reporting: Suggested improvements: what we learned from your findings that we did not know before? It is somewhat hard to find a take-home message, mainly from the Mb conclusions.
Experimental design: Measurements of EWL should be taken in experimental chambers of the same size for all the focal species since air turnover within the test container is different in different container sizes. The authors argued that it was not relevant since they did not compared rates of EWL among species. However, as it can be read, the manuscript discussion I centered in comparing species (of different habitat specialisation). The authors should also acknowledge more clearly the potential confounding effects of differential feeding states[A] (before their experiments) and dehydration (during their trials). The authors argued that they did not observe loss in mass that was great enough to suggest we had an issue with dehydration. However, does it mean “great enough”? A statistical test or reference should be provided to confirm this assumption since it is known that more dehydrated anurans decrease their rates of EWL[B], Tb[C], performance [D] and so forth [E].

A-Witters,L.R., Sievert,L., 2001.Feeding causes thermophily in the woodhouse´s toad (Bufo woodhousii). J.Therm.Biol.26,205–208.

B-Rodolfo C. O. Anderson, Rafael P. Bovo, Carlos E. Eismann, Amauri A. Menegario, and Denis V. Andrade, "Not Good, but Not All Bad: Dehydration Effects on Body Fluids, Organ Masses, and Water Flux through the Skin of Rhinella schneideri (Amphibia, Bufonidae)," Physiological and Biochemical Zoology 90, no. 3 (May/June 2017): 313-320.

C-Tracy C. R., Christian K. A., O'Connor M. P., & Tracy C. R. (1993). Behavioral thermoregulation by Bufo americanus: The importance of the hydric environment. Herpetologica, 49, 375–382.

D-Preest M. R., & Pough F. H. (2003). Effects of body temperature and hydration state on organismal performance of toads, Bufo americanus . Physiological and Biochemical Zoology, 76, 229–239.

E- Tracy, C. R., K. A. Christian, G. Betts, and C. R. Tracy. 2008. Body temperature and resistance to evaporative water loss in tropical Australian frogs. Comparative Biochemistry and Physiology A 150:102–108.
Validity of the findings: No more comments.
Additional comments: Abstract

Lines 21-23: It is hard to follow the rationale. This sentence should be rewritten.
What kind of process related to pronounced sexual dimorphism in anurans are influenced?  Why more general factors like sex and body mass (Mb) would be related to a very specific factor like vapour pressure deficit (VPD)? Does not make sense for most of the readers.
VPD seems an operational variable and should be replaced by its theoretical variable, i.e. what VPD mean in practical terms.

If Abstract section has limit of words, the description of the three species with common names and etc. could be shorten. The most important is to highlight the habitat use of the three focal species.

You found that: 1-Tb, whole-animal EWL and whole-animal SMR increased with increasing Ta; 2-Tb increased with Mb, except in the more aquatic species; 3-whole-animal EWL was not affected by VPD, Mb or sex; 4-Whole-animal SMR increased with Mb in the more terrestrial species. From these findings, the conclusion was: “Although we found that Ta is very important in influencing key physiological processes” (that is ok), "the generality of Mb as an important factor should be taken in the context of species habitat specialisation."
What does you specifically mean? What does your work add to the knowledge gap? What is(are) the take home message(s) in practical terms? How could this link to the three physiological traits tested in the hypotheses? Be more specific and connect it with your findings in order to communicate the conclusion of your research better than it is. Even reading some of the authors’ replies, it is still hard to find a simple message from Mb effects on the physiological traits tested: “Lastly, looking specifically at the influence of Mb in our results, we are of the view that these species-specific Mb trends suggest complex Mb dependency that will not be easily solved statistically by lumping all, that is having mass instead of species will not be feasible.” Therefore, this is the job of the writers and not of the readers in highlighting “the generality of Mb as an important factor” that closes the Abstract and it seems one of the main conclusions of the manuscript.

Introduction

I commented on this in another round of review, however I could not find any predictions on expected effects of Ta on Tb at the final of the Introduction section. Although it is expected that both Ta and Tb would change in the same direction, this may not be true at certain conditions as, for instance, at high Ta’s when Tb decreases due to loss of water by evaporation through skin.

Line 104: positive?negative? relationship between water loss and activity patters.

Methods

Check for misspelling of “Respirometry“ throughout the text.

Anuran species were fed with different diet. Please explain why you used this treatment since different acclimation conditions may affect physiological traits in a differential way. Also, explain why you are confident that individuals did not get food differentially since there were more than one per plastic boxes at least for 2 species (there is no information on the third, Xenopus laevis). Different captivity conditions ("treatments") may imply in differences when analyzing physiological responses.
Author reply: Although this may be true, we tried to feed species the diet that they would encounter in nature. In addition to feeding individuals in groups as highlighted by the reviewer, after each experimental run, frogs were placed in individual holding cages and were fed before they were returned to the larger plastic terraria (holding cages), see lines 218- 223.
My reply: It is extraordinary to learn that in nature Xenopus feed on ox-heart. Authors were evasive in their reply to my comment and thus I suggest they acknowledge the potential of this effect on the physiological measurements on the Methods section. Also acknowledge your method for feeding animals not individually may have implied in different conditions of fasting since some individuals could have fed and other not.

Why did you used 2 hours for EWL measurements for all individuals independent of their body size? Do you have evidence that this time period did not caused dehydration, and then you include another factor other than temperature influencing the rates of EWL?
Authors reply: The percentage body mass loss in the 2 hr experimental period was (mean ±SE) 3. 2 1± 1.66 (Xenopus laevis), 3.91 ±3.84 % (Sclerophrys capensis and 5.2 ±2.85 % (Amietia delalandii). We have to assume the “other factor” referred to here, concerned VPD? Please see our response further above where we check that VPD did not confound our analyses as far as we could tell.
My reply: Basically, if some individuals or species become more dehydrated than others, this fact could imply in differential changes in Tb, EWL, and SMR, since the effect of dehydration would be a confounding factor and, thus, should be acknowledge in the text.

Thermoregulation and Tb relationships should not be discussed without referring their interplay with hydro-regulation. Even if you mention it in some lines below at EWL sub-section, the influence of body mass on Tb and metabolism cannot be decoupled from hydration state. If so, please provide rational explanation.
Authors reply: We always hydrated our frogs between experiments runs as indicated in the manuscript (see line 151-154) and even after experiments. During experiments, lasting two hours, we did not observe loss in mass that was great enough to suggest we had an issue with dehydration.
My reply: I meant that thermoregulation and Tb relationships should not be discussed without referring their interplay with hydro-regulation. It was not about the methods / experimental design.
By the way, what does you mean with “great enough”? A statistical test or reference should be provided to confirm this assumption.

Discussion:

Line 365: I have already commented on this: “Species with high rates of Rc…”
Rc is not a rate and I explained why in a revision before.

Lines 366-367: “and in this study we found that Tb is influenced by Mb in both S. capensis and A. delalandii.” Ok, but what does this mean in practical terms? Conclude that. One way would be to highlight what kind of “specialisation” (more terrestrial?) and also to discuss why this was found in your study. Actually, many parts of the discussion sections are like this: “Tracy et al.. showed this… Navas showed that… and we found this.” For example, lines 348-367. The reader expects that the authors do the job of interpreting the study findings and conclude what is being discussed. Please review all the Discussion section.

Line 374: Where you showed that you expected this? Actually, expectations for Tb are lacking at the end of the Introduction section.

Lines 383-387: Prates and Navas 2009 data/paper weakly relate to what is being discussed. I suggest you delete it or even replace by another one.

Conclusion:

Lines 434-436: Check again if this sentence makes sense or should be rewritten. In addition, it is hard to understand from where this suggestion comes from, since you did not link with any of your findings in the sentences before. Again, what we learned from your findings that we did not know before? You should focus on that.


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: REDUCED P2X RECEPTOR LEVELS ARE ASSOCIATED WITH ANTIDEPRESSANT EFFECT IN THE LEARNED HELPLESSNESS MODEL
Review round: 1
Reviewer: 1

Output:
Basic reporting: -The article was written in clear and professional English. In addition, this study presents literature references consistent. However, there is an excessive number of references, which makes the article very speculative. Therefore references should be reviewed, prioritizing the most current references.
-Althougth the figures are relevant to the content of the article, of sufficient resolution, and appropriately described and labeled, the reorganization of the figures would contribute to a better understanding of the results. I suggest that the figures of the behavioral results and the western blot analyzes be separated and placed in the order in which the experiments were performed. For example, figure 1 behavioral results and figure 2 western blot analyzes.  In addition, representative western blot images should be added to the article, the lack of these data is an important limitation of this study.
Experimental design: -The experimental design of this study was well delineated to respond to the proposed objective.
-Given that the experimental groups present a different sample size, I suggest that the n of each group in each experiment should be clarified in the materials and methods or in the legends of the figures in order that this study has enough information to be reproducible by another researcher.
Validity of the findings: -The authors provided robust and statistically adequate data. A minor point is the lack of information about how the authors evaluated the normality of the data, since they used a non-parametric test.

-An important point is about the conclusion of study.
Although the results of this study can support the hypothesis created by the authors. The conclusion did not appropriately stated. Please provide a conclusion connected to objective of study and limited to those supported by the results.
Additional comments: -Although speculation is welcome in this jounal, the discussion of this study is too speculative. In particular, the fifth paragraph of the discussion (lines 233-250) that needs more connection to the data of the article

-The least important points:
 1-Some abbreviations appear without being spelled in the first use, for example: HRP and PPADS. Please ensure that uncommon abbreviations were spelled out at first use.
2- Do not acknowledge funders in the Acknowledgments session, there is a separate Funding Statement for that.
3-The sentence ..."and the values for the P2RX7 were normalized by the GAPDH value in each sample.." should be modified by ..."and the values for the P2RX7 and P2RX4  were normalized by the GAPDH value in each sample.."


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

Input:
Title: REDUCED P2X RECEPTOR LEVELS ARE ASSOCIATED WITH ANTIDEPRESSANT EFFECT IN THE LEARNED HELPLESSNESS MODEL
Review round: 1
Reviewer: 2

Output:
Basic reporting: In the manuscript “Reduced P2X receptor levels are associated with antidepressant effect in the learned helplessness model” the authors have reported that the genetic deletion or pharmacological blockade of purinergic receptors P2RX7, induces antidepressant-like effect in preclinical models. Moreover, they observed that the repeated administration of imipramine reduced the levels of both P2RX7 and P2RX4 in the hippocampus.
  The study is very interesting, well written and show good results. However, there are some points that could be improved to make it suitable for publication in PeerJ. Therefore, I have given some suggestions which could help the authors to do it.

1. The raw data of the manuscript were not presented, in accordance with the Data Sharing policy.
Experimental design: 2.	The results would be clearer if divided into two figures, one referring to the Western Blot (WB) analysis and the other to the behavioral analysis.

3.	In the results of WB, please add the images of the membranes to the figures 1a, 1b and 1c. Moreover, would be appropriate carried out a loading control of protein i.e.: β-actin, GAPDH…

4.	The authors cite that P2RX7 is activated by high concentrations of ATP, which would have been supposed to be high. It would be interesting to quantify ATP levels in the analyzed tissues.

5.	Still regarding the purinergic system, what happens to the levels of the enzymes that degrade nucleotides? Changes in ATP levels could be result or cause changes in activity or expression of these enzymes. Please add data about this.  Experimental analyzes of CD39 levels, for example, would improve the work.

6. 	What is the reason for the differences between the receptor levels in different places in the brain? Please add an explanation about this in the discussion.
Validity of the findings: no comment
Additional comments: 7.	In the text, the results could be presented in the order in which the figures appear, starting at "a" or the figures could be rearranged.

8. I think that creating a figure, illustrating the possible mechanism by which antidepressants effects occur would be a way to highlight the results.

 9. In the discussion, line 239-241, there is a repeated sentence.


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

Input:
Title: REDUCED P2X RECEPTOR LEVELS ARE ASSOCIATED WITH ANTIDEPRESSANT EFFECT IN THE LEARNED HELPLESSNESS MODEL
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: I consider that the quality of the study has been improved and my  questions were considered, then I recommend publishing this manuscript.


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

Input:
Title: REDUCED P2X RECEPTOR LEVELS ARE ASSOCIATED WITH ANTIDEPRESSANT EFFECT IN THE LEARNED HELPLESSNESS MODEL
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors properly answered the questions raised and added some important information, improving the quality of the manuscript.
Experimental design: The methodology is adequate and meets the objectives.
Validity of the findings: This work is of general interest and contributes to increase knowledge about the role of antidepressant on attenuation of the P2RX7/P2RX4 levels and the possible mechanisms that contribute to their behavioral/therapeutic effects.
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: MANX SHEARWATER (PUFFINUS PUFFINUS) RAFTING BEHAVIOUR REVEALED BY GPS TRACKING AND BEHAVIOURAL OBSERVATIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: I generally found the article very well written and easy to read. I have some thoughts about the structure which I describe in my general comments.
Experimental design: I feel the research questions about environmental effects could be better linked to some of the ideas the authors mention about the purposes of rafts to assist predator avoidance, socialising/preening or acting as compasses indicating the direction of foraging patches.
Validity of the findings: The methods and statistics seem to be extremely robust.

As mentioned above, In terms of interpreting the findings I would like to generally like to see a link between the environmental variables such as wind, and the other speculation about the purpose of rafts. Currently these ideas seem somewhat disconnected.
Additional comments: I was extremely interested to read this MS studying the factors influencing rafting behaviour of shearwaters. The authors have collected an impressive amount of GPS data and combined it with behavioural observations to answer questions about how environmental variables affect rafting behaviour. The manuscript is clearly written and easy to read. However, I feel the paper would benefit from some links to broader questions about group behaviour. This could be achieved in part by better integrating the ideas about the anti-predation benefits of rafting throughout the paper.

While I found it very well written, I am not entirely sure about the current structure of the introduction. I would prefer to see a more general introduction. I feel that a slightly bolstered second paragraph possibly with some basic discussion about group behaviour in other taxa, potential adaptive benefits, trade-offs would make for a stronger start. The first paragraph could be repurposed elsewhere to demonstrate why seabirds are good to examine these types of questions.

Similarly, I would then like to see the linking of the potential environmental variables back to larger questions about the adaptive significance of these rafts. For example, if rafting behaviour is indicative of the direction that individuals will depart to or have returned from foraging patches, how might this be affected by wind? Would individuals be departing or returning downwind, causing changes to the distribution? How might this interact with the authors ideas about predation risk? Etc.

I think the hypotheses about predators should also be introduced earlier and perhaps developed a bit more, it feels a little last-minute in the introduction at the moment.

The experimental and statistical methods are very clearly presented. However I feel a little more detail on the partitioning of GPS tracks between rafting and flight would be beneficial.

Details on the number of maximum number of GPS devices simultaneously deployed  would be useful to include in the results.

My comments on the discussion are similar to those about the introduction. While it is well written, I would like to see links to some broader speculation about the adaptive significance of rafting behaviour. This could be achieved by expanding on some of the ideas about the purpose of the raft being for socialising/preening or predator avoidance.


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: MANX SHEARWATER (PUFFINUS PUFFINUS) RAFTING BEHAVIOUR REVEALED BY GPS TRACKING AND BEHAVIOURAL OBSERVATIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript provides valuable new knowledge about rafting behaviour on seabirds by combining observational and GPS-tracking on Manx shearwaters, as well as wind conditions. Authors conclude size of the rafts and their location is influenced by wind speed and time of the day, although wind direction does not seem to play a role on rafting locations.
The manuscript is very well written, in an unambiguous and professional English. It is also well structured and main ideas are clear and well explained.
Introduction is clear and well structured, although there are some things I think that can be improved.
First paragraph is too general to me. Taking into account this manuscript has a clear focus regarding rafting behaviour, I would start talking about rafting in the first paragraph (now you have to wait until paragraph two to see the focus of the manuscript).
Second paragraph is very good and key to understand why seabirds perform rafts. However, perhaps you could also include a brief explanation on which groups or species are known to perform rafts and which ones not.
In lines 88-89 you say “… the importance of various environmental and biotic drivers on rafting behaviour…” Since this is the focus of the manuscript, I would better describe which are these environmental and biotic drivers that have been suggested to affect rafting behaviour. I would briefly explain them and then later focus on the wind, which is the one you actually look at. I would also better explain why you think wind might be key for your species, to justify why you did look at wind and not at other environmental or biotic drivers.
Both in the objectives and particularly in the discussion you say you used “environmental data” to understand rafting behaviour. However, to me, if you only look at wind, I am not sure you can claim to work with “environmental data” in a general sense, because it seems you worked with other environmental factors also affecting rafting behaviour. I would say “wind conditions” instead or something like this.
In the objectives, you talk about whether raft distance from shore changes through time. However, nowhere in the introduction you have explained this hypothesis. I would include this idea in the introduction so that it fits with the objectives. Indeed, it is a very interesting result!
Experimental design: The experimental design of the manuscript is well explained and with well defined and supported questions. Authors identify the gaps of knowledge they are going to fill in in a clear and ordered way. Ethics on the manuscript are correct.
Methods are detailed, clear and well explained, although I have a few comments. In the models with GPS data, did you take into account the individual? I do not know if it may play a role, but it can be included as a random factor in models. Also, did you think about including the moonlight intensity as a driver? I do not know if it can be important, but as it is important regarding the entrance of birds to the colony, it may also play a role with rafting behaviour.
L 130-132: I would simply say “Rafts observed at the 2000 m boundary and beyond were excluded in our analyses because…”
L178: So you only applied a “land-filter”? Didn't you do another filter (for instance velocity) to remove erroneous locations?
L184: “… Manx shearwaters flight speeds…” so you only used velocity to define rafting locations? Cannot birds also be intensively foraging with very low speed? What about the turning angle in these locations? I am thinking about EMBC approach (Garriga, J., Palmer, J. R., Oltra, A., & Bartumeus, F. (2016). Expectation-maximization binary clustering for behavioural annotation. PLoS One, 11(3), e0151984.).
Validity of the findings: Results and discussion are detailed, well explained and well supported by statistical tests and figures. I have a few comments.
L231: “around Skomer Island” you mean in the radius you looked at? Or in general?
L289-293: I am sorry, but I do not fully understand the relationship with the dual foraging strategy and the size and number of rafts. Could you explain it in another way?
Conclusions are very good, but they go beyond what it is explained in the introduction. To make the manuscript “circular” it would be a good idea to also talk about the importance of understanding rafting behaviour in conservation in the introduction. In this way conclusions will match with the introduction.
Fig. 1. I would mark in the island where exactly the observational points were. I would also include that this figure comes from observational data, and that observations were made in the 2 hours before sunset.
Fig. 2. I would include the number of birds in the figure caption. Also, I do not know if it is necessary to have colour in this figure (you can make the star black with white contour or something like this and you do not need to use the colour).
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: MANX SHEARWATER (PUFFINUS PUFFINUS) RAFTING BEHAVIOUR REVEALED BY GPS TRACKING AND BEHAVIOURAL OBSERVATIONS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Highly professional structure and clarity throughout.
Clear logical development of aims that are explored in the study.
Referencing is appropriate, but could be improved in places: sometimes a single, species-specific source is used to support the wider value/evidence base for an approach or concept: more general reference might be added (e.g. a review to complement a specific paper). Example is L68 Dean et al (2012), maybe complemented by broader scope or sources.
L249 Can the nature/directionality of the effect of wind be clarified here? If some benefit to land shelter or exposure is present, pls add specific detail.
Fig 2 demonstrates the 'halo' effect, but how the tracks provide evidence for this might be clarified in the body text or the figure legend... that the tracks are further from land by day and closer by night may not be so obvious to the reader
Experimental design: The study is well-designed, research questions clearly articulated and addressed.
High standard of technical expertise and ethics.
The knowledge gap is clearly identified.
Methods appropriately reported.
Is the inclinometer approach novel, or inspired by uses in other fields? Some clarification here would be valuable.
Validity of the findings: Clear rationale for the study and interpretation of results provided.
Elegant and concise.
Additional comments: I enjoyed reading this paper, thank you for the opportunity to read it. I liked the combination of approaches of tracking data and the more 'old school' inclinometer, the neat logical framework and the clear, concise writing.


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

Input:
Title: THRESHOLD-LIKE ASSOCIATIONS AS A FUNCTION OF DISTURBANCE
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: The research context is based in the educational and social sciences. It is unclear how it falls within the bounds of Biological Sciences, Environmental Sciences, Medical Sciences, and Health Sciences. However, the methodology is rigorous and described sufficiently, and the research question is relevant and meaningful. There is a potential for the methodology to be useful within the scope of the journal.
Validity of the findings: See general comments.
Additional comments: The current study presents simulations of possible relations between creativity and intelligence to test conditions under which the threshold theory would or would not be supported.  The argument is that something other than the assumed necessity of IQ for creativity could produce a threshold-like outcome, especially when sample sizes are large (e.g., N = 400+) and the true correlation is low (e.g., < .25). The other variable, called a disturbance, is due to individual differences other than in IQ.

I think this is a very interesting study that highlights that threshold hypotheses of any kind can be produced under circumstances other than the assumption that one variable is necessary but not sufficient for a second variable. In the case of creativity, IQ is considered as necessary for creativity but not sufficient: that is, as IQ increases, creativity also increases to a certain point (threshold) of IQ, after which creativity does not increase. However, I have some concerns that it is not tied enough to the research on creativity. My concerns revolve around the question, what might the disturbance be? I highlight these below.

1.  The authors mention a few possibilities in passing (e.g., low motivation, illness, linguistic confusion). But these mentions are too general for an enhanced understanding of creativity. How might these possibilities be related to creativity specifically? Or IQ? Some concrete examples of a variable that would affect both creativity and IQ would be helpful.

2.  Why choose skewed distributions for the disturbing factor? Again, some concrete examples of what these might represent would be helpful.

3.  Why would we find the threshold with some creativity tasks but not others, as some previous researchers have found? How does this relate to disturbances? More generally, the simulations, though impressive, do not explain why some studies find a threshold-like relation and some don’t. Is the claim that in some studies there are disturbances but not in others?

4.  One of the hallmarks of a threshold theory is that the relation between two variables in qualitatively different above the threshold than below it. If I’m understanding correctly, the authors claim that this is illusory – a fluke of the individual differences in disturbance. What does this mean for creativity theory more generally?

5.  It seems remarkably easy to find evidence for the threshold theory, especially when the correlation between IQ and creativity is low (i.e., < .25), no matter the sample size or skewness of the disturbance –the probability is above .05 in all these cases. Why don’t we find it more often in our creativity research?

6.  Other minor comments.
(a)  Something is missing from the first sentence of the introduction.
(b)  It would be easier to understand the extent to which the simulations present evidence of a threshold if the black dots in Figure 3 were in front of the gray dots.
(c)  When there is no correlation between IQ and creativity, as shown in the left side of Figure 4, there is essentially no threshold, even though there may be statistically. No sample was above IQ = 165.


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: THRESHOLD-LIKE ASSOCIATIONS AS A FUNCTION OF DISTURBANCE
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is well-written, concise, easy to comprehend, and makes appropriate reference to other works. The figures support the text in an excellent manner. The data are accessible.
Experimental design: The design of this simulation study is – as far as I can tell as a psychologist, not statistician – sound and appropriate to investigate the proposed hypothesis. The methods are described transparently and in sufficient detail to understand the simulation results. However, for a more comprehensive picture, I wonder whether a baseline simulation for true positives (sensitivity analysis) might be useful. The probabilities of detecting false and true positive could be compared, then.
Validity of the findings: Alternative models explaining the emergence of threshold-like distributions should be considered. The current model focuses on disturbance as a variable which attenuates the true scores. However, this is only one of many possible theoretical assumptions, and others would be plausible as well. Particularly, the threshold effect is consistent with the differentiation hypothesis (cognitive abilities are more differentiated, and thus less correlated, at higher levels), as proposed by Spearman (and confirmed in large samples by Deary).
I understand that the simulation is well suited to reproduce bivariate threshold-like distributions, and I very much appreciate the idea, but it should be acknowledged that there might be many other ways of reproducing such distributions. Whether attenuation of the true scores or different g-saturation of true scores, for example, are better suited to explain the observed associations would either require (a) different simulation models being compared against each other and/or (b) empirical research (varying disturbing or motivating factors, for instance). In my view, this study provides a great starting point for this.
Additional comments: First of all, I apologize for my delay in this revision process!
The manuscript under review reports on a simulation study of disturbing factors in threshold-like associations detected with segmented regression. The hypothesis that disturbing factors such as low motivation could generate such associations among two correlated variables is confirmed.
I am very happy to see that methodological research is devoted to the use of segmented regression in psychology. I find the proposed idea and simulation very interesting. My main point is that the interpretation should more explicitly acknowledge that different models could produce such distributions.


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: THRESHOLD-LIKE ASSOCIATIONS AS A FUNCTION OF DISTURBANCE
Review round: 2
Reviewer: 1

Output:
Basic reporting: Basic reporting is excellent.
Experimental design: The experimental design is sound.
Validity of the findings: The findings are valid.
Additional comments: The authors addressed all my concerns in their revision. I will say, in relation to a comment from the authors, that I have difficultly finding evidence for a threshold in my lab.


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: THRESHOLD-LIKE ASSOCIATIONS AS A FUNCTION OF DISTURBANCE
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors addressed my points from the previous revision - I am happy with the present form of the manuscript. Though the authors did not follow my suggestion to include simulations targeting the sensitivity of the method, I understand this goes beyond the scope of the present work.

As a very minor point, I would like to note that observing a few individuals with IQ ~ 60 in a sample of N ~ 300 is actually in line with the expected frequency based on a normal IQ distribution. The authors mention this as evidence for disturbance at different occasions, but it could also be seen as a sign of a valid sampling that includes individuals from all IQ levels (which is usually not the case in student samples). Maybe the authors could consider this perspective in their final manuscript.

I think this work provides important methodological considerations for creativity researchers, and more generally, for those investigating threshold-like associations, and will stimulate future research in a constructive way!


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: CLASSIFICATION OF RNA BACKBONE CONFORMATIONS INTO ROTAMERS USING 13C′ CHEMICAL SHIFTS: EXPLORING HOW FAR WE CAN GO
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1) No issues with writing.

2) In line 35-36, the authors should consider citing the work of Das and coworkers: ‘Structure determination of noncanonical RNA motifs guided by 1H NMR chemical shifts’, which describes the use of 1H chemical shifts to guide determine/prediction of RNA structure.

3) Figure 5 was reproduced from a dissertation, and though it was properly cited, the authors should consider producing their own version of this figure.

4) Raw data, QM input scripts, and machine learning scripts should be made available (currently missing).
Experimental design: 1) Original primary research within Aims and Scope of the journal?

Yes. RNAs play importance biologically roles and to understand how they function, we must understand their structure.

2) Research question well defined, relevant & meaningful. It is stated how research fills an identified knowledge gap?

Yes. The research aimed to assess the extent to which 13C chemical shifts can be used to classify RNA rotamer states. Answer to this question could help develop methods to maximize the amount of structural information about an RNA that can be extracted from NMR chemical shift data.

3) Rigorous investigation performed to a high technical & ethical standard?
Yes.

4) Methods described with sufficient detail & information to replicate?
Yes.
Validity of the findings: No comment
Additional comments: In line 35-36, the authors should consider citing the work of Das: ‘Structure determination of noncanonical RNA motifs guided by 1H NMR chemical shifts’, which the describes the use of 1H chemical to guide determine/prediction of RNA structure.

When describing the experimental datasets, the authors should include the number of conformations as well as the total number of set of {C1’,C2’,C3’,C4’C5’} CS data

For the DFT calculations, much of the justifications of the methods used are based on calculations carried out proteins. The authors should explore work by Case and coworkers to get some guidance on choosing of DFT functionals and basis set for computed NMR shielding in nucleic acids. For example, see AFNMR: automated fragmentation quantum mechanical calculation of NMR chemical shifts for biomolecules, and the references therein.

The impact of using the 3-21G basis set for some of the nuclei and extended 6-311+G(2d,p) basis set for the result is not fully discussed or should be justified by showing that the added computational expense of using the extended 6-311+G(2d,p) basis set for all nuclei does not yield significantly different results.

The lack of good performance for the experimental vs. theoretical, compared to theoretical vs. theoretical, was attributed to problems with atomistic models. Another reason for the poor relative performance is inherent errors in the computed chemical shifts. To test this, authors should consider adding noise to the theoretical database and then carrying out a theoretical vs theoretical+noise analysis. By adding noise on the order of the expected errors between actual and theoretical chemical shifts, they will be able to test how errors in the computed shifts affect the performance of the classifiers.

As noted by the authors, the experimental database used to train the classifiers was sparse, compared to the theoretical database. The author should consider using an imputation strategy to impute missing 13C ribose chemical shifts from existing 13C ribose chemical shifts, and thus, increase the number of RNA in their dataset. Currently, there are > 70 RNAs for which both 13C chemical shifts and NMR structures are available in the BMRB and PDB, respectively.

Raw data such as coordinate files, and QM calculation and machine learning scripts should be made available (currently missing).


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: CLASSIFICATION OF RNA BACKBONE CONFORMATIONS INTO ROTAMERS USING 13C′ CHEMICAL SHIFTS: EXPLORING HOW FAR WE CAN GO
Review round: 1
Reviewer: 2

Output:
Basic reporting: The text is well written in very good English; some minor issues, which are likely to be typos, pointed out in the comments to the authors. Literature references are adequate. Article structure and the form of tables and figures have no issues. The results presented support the conclusions; however, a table/figure with the geometric parameters/pictures of sample structures from the families dinucleotide conformations considered in this work would be desirable.
Experimental design: No issues in this part. The paper fits to the Aims and Scope of the journal, the research questions are well defined and adequately addressed. The methods used are relevant and explained sufficiently.
Validity of the findings: A table/figure with the geometric parameters/pictures of sample structures from the families dinucleotide conformations considered in this work would be desirable for readers' benefit.
Additional comments: This is a good paper which undertakes a difficult taks of the classification of RNA backbone conformations based on 13C NMR chemical shifts. The Authors divided the conformational space of dinucleotides into 46 possible conformations (based on the backbone angles) of which 38 were found in the experimental structures.. The conforomers were taken from the experimental structures and generated from the dihedral-angle distribution. Subsequently, the 13C chemical shifts were computed and correlated with conformer geometry to determine families using a menu of machine-learning methods. Careful statistical assessment of the validity of the resulting classification was done. The delta(i-1) and delta(i) angles related to the puckering of the flanking ribose rings were found to be the best descriptors that could be translated inito chemical shifts. Interestingly, the global-fold properties (A-non A helix, A-helix-everytning else) were found not to be related to the chemical shifts.

In my opinion this paper contains very useful results, because the classification enalbles a researcher to estimate the type of backbone conformation just by measuring 13C chemical shitt, which is relatively simple. I have a couple of minor suggestions:

1. A picture with the representatives of the 4 families of conformers classified based on delta(i-1) and delta(i) summarized in Table 1 or a table with the dihedral angles corresponding to those representative would be useful.

2.  Were the theoretically-generated conformers energy-minimized? If so, by what method? It not, was the set of generated conformers pruned to eliminate those with atom clases?

3. page 4, lines 93-95:
"Considering that the phosphate group in RNA is close to the nucleus of interest (as it happens with the
94 acidic groups) we can assume, without losing generality, that neutral rather than charged phosphate group
is a better approximation for the computation of the 13C′ 95 CS in the RNA suites."
The general conclusion is correct but the rationale of using protonated phosphate groups is rather that they are surrounded by counter ions (mainly the sodium ions). The phosphate group is rather unlikely to be protonated at physiological conditions.

4. I found a couple of minor grammar errors, e.g.:page 4, lines 89-91 (corrections in caps):

"Results based on the analysis of 139 conformations of ubiquitin
90 at pH 6.5 (Vila and Scheraga, 2008), INDICATE that use of neutral, rather than charged, amino acids is a
91 significantLY better approximation of the observed 13Ca CS in solution for the acidic groups"


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: CLASSIFICATION OF RNA BACKBONE CONFORMATIONS INTO ROTAMERS USING 13C′ CHEMICAL SHIFTS: EXPLORING HOW FAR WE CAN GO
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
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: A NEW SMALL DEVICE MADE OF GLASS FOR SEPARATING MICROPLASTICS FROM MARINE AND FRESHWATER SEDIMENTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors Nakajima et al. developed an extraction device to recover microplastic particles from sediment samples. Microplastics are a ubiquitously distributed particulate contaminant that by now has been detected in various environmental samples (incl. deep sea and the polar regions of the Earth), food and recently also humans. One of the major research activities in the topic of microplastics is towards and standardization and harmonization of existing analytical approaches. That being said, this work clearly falls within an up-to-date topic. However there are a few fundamental concerns that in my opinion prevent this manuscript to be published in its current state.
i) The overall manuscript reads more like a report rather than a high quality and impact research manuscript. It contains outdated literature, for example, the authors claim that only 1% of the plastics have been accounted for and the rest is missing. That information is clearly out dated or at least does not cover the whole story (for example, https://iopscience.iop.org/article/10.1088/1748-9326/aa9500).
ii) Another point this direction is the number of papers that are cited. Of course this should not be the sole criteria but point to the superficial character of the work. This is especially relevant since the analytics of microplastics is one of the few aspects with a considerable development over recent years.
Experimental design: iii) The authors use NaI for the density separation of the microplastics from the sediment and obtain nearly 100% recovery for almost all of their samples. It would be interesting to consider the densities of the polymer particles and take this into account for the discussion of the obtained results.
Validity of the findings: iv) I am missing more objective discussion of the limitations of the developed device. In comparison to glass funnel, where only very small amount of sediments can be used, the here developed device can handle a variety of different sample amounts (l. 180-185). While this may be true, looking at the device in its current form only comparably little sample amount will be handled by this device. Given the so-far expected small concentrations of microplastics in sediments, the sample volumes of orders of magnitudes higher will have to be extracted to reach robust results in the fields. This clearly limits the applicability of this technique.
Additional comments: Finally, although not being a native English speaker myself, I would highly recommend the authors to have their manuscript proof-red. Some of the sentences are somewhat difficult to understand or seem grammatically incorrect (examples are in lines 22, 26, 34 etc).
I hope the authors find my comments useful to further improve the quality of their manuscript.


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

Input:
Title: A NEW SMALL DEVICE MADE OF GLASS FOR SEPARATING MICROPLASTICS FROM MARINE AND FRESHWATER SEDIMENTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Minor comments:

1.	The authors should improve the English language throughout the manuscript to ensure that an international audience can clearly understand the text. Some suggestions can be found below:
L. 21: "on" the ocean's surface
L. 25: write "resuspension" instead of "re-suspension" throughout the manuscript
L. 34: "This new device is easy to clean and portable, yet enables rapid separation of microplastics from sediments, and these microplastics remain uncontaminated so can be used for further chemical analysis." (divide this sentence into two sentences for better understanding)
L. 38: well investigated
L. 42: , which are referred to as "the missing plastics".
L. 44: locations
L. 45: about how much plastic is accumulating in ocean sediments,
L. 58: onto
L. 65: "through" is not a suitable word in this context
L. 117: use "sand" instead of "soil"
L. 119: "during" instead of "in"
L. 120: that there were
L. 172: "low" instead of "lower"

2.	Regarding the title: why is the method only suitable for marine and not  for freshwater sediments?

Major comments:

3.	The introduction is definitely too short and lacking important information.
Please, define microplastics (MPs).
L. 43: Are there just fragments and fibers in the deep sea? What about films, spheres??
L. 47: How could your method help in investigating the impact of MPs on marine animals? Also, please provide more references from the literature here on MP effects on animals (e.g. the ones living directly in or on the sediment).
L. 50: When introducing current methods for MP separation from sediments, you are missing important references: e.g. Felsing et al. 2018. A new approach in separating microplastics from environmental samples based on their electrostatic behavior. Environmental Pollution.; Grbic et al. 2019. Magnetic Extraction of Microplastics from Environmental Samples. Environmental Science & Technology Letters. There are also some recent reviews dealing with this subject which you could cite.
Fig.1: legend: f: microplastics in the supernatant in the upper tube are poured by rinsing the internal walls of the tube.

Fig.1: I suggest to exchange the positions of pictures a and b so that the first picture shows the upper and lower plate separately.

Fig. 2: write "polyethylene" instead of "polyethene"; "µm" does not have to be written in italics.

Fig. 3: I do not understand what you mean by your y-axis labelling. Also, percentage is misspelled. I suggest labelling the y-axis using the term "recovery rate". Please, include the detailed grain sizes in the x-axis labelling.
Experimental design: LL. 54-57: Name the densities of the different salt solutions and why they are suitable for extracting MPs. Also, refer to the MP densities.
L. 64: " There is an upside-down funnel-shaped microplastic separator with a shut off valve (MPSS), but it is a large metal device and is thus heavy and expensive (Imhof et al., 2012)." Please, rephrase that sentence. Being a large metal device is not a justification for being expensive.
L. 67: It is SMI and not MSI.
L. 93: why did you choose a NaI solution? Again, refer to the MPs' densities here.
L. 96: You write that you used a magnetic stirring bar to mix the sediment for 20 min. I would like to know which type of magnetic stirring bar you used for that. From my experience, most magnetic stirrers possess a plastic (PTFE) coating which can easily be abraded by the sediment grains during mixing. Hence, you should find a lot of small plastic fragments from the magnetic stirrer contaminating your sample when mixing the sediment sample with the stirring bar.
L. 101: Did you also filter your distilled water before using it to remove any contaminants like you did it for the NaI solution?
L. 102: Did you cover the Petri dish with aluminum foil as well?
L. 114: why did you choose those MP sizes? Are they the most common MPs in deep sea sediments?
L. 117: did you really grind the sand?
L. 119: "These added microplastics were distinctive, both in color and shape, ensuring that only the added plastics were counted in the final enumeration." Which colors and shapes did they possess so that you could easily identify them as plastics by eye? You could provide photos. Also, MP verification by µFTIR analysis would have been useful.
L. 120: Which preliminary experiment are you referring to? Please, describe it in more detail.
L. 125: Again, which magnetic stirrer was used? Did mixing the sediment with the magnetic stirrer reduce the size of the added microplastics through microplastic abrasion?
L. 128: where did you collect the reef carbonate sediment and how did you grind it into fine, medium and coarse fractions?
L. 130: provide information on PVC density.
L. 132: are you again referring to wet weight when speaking about the sediment mass? If so, then please write that into brackets.
Validity of the findings: LL. 135-139: How did you test for normality of your data before performing the ANOVA?
L. 143-147: Move that section so that it appears after your results.
L. 151: You are referring to polymer density here but throughout your whole manuscript, you are not introducing the plastics' densities once. Please, provide information on the densities of the plastics that you used for your experiments.
LL. 151-152: Please, explain your statistics again in more detail (e.g. name the different levels analysed by the One-Way ANOVA)
LL. 151-152: write "particles" instead of "particle"
LL. 164-167: Please, discuss why there was no difference between the different sediment types (fine, medium, coarse).
L. 167: "." after "(Fig. 3)"
L. 216: Please, explain which contamination is avoided here.

Regarding the Supplementary data file:

1)	PE is polyethylene and not polyethyrene.
2)	You are providing MP collection rates in supplementary data 1 as ratios and in supplementary data 2 as percentages. I suggest using percentages throughout the whole data file as it should be consistent. When looking at the supplementary data for PE (B9) you write that you collected 38 MPs out of 40 MPs which would mean that you collected 95 % of the MPs with which you spiked your sample. However, when presenting the data you write that that equals 1.0 (100 %) which is quite misleading as, of course, only 40 collected MPs would mean that you recovered 100 %. Please, change that to avoid any misunderstandings. Also, you report the same collection rate of 0.9 for a recovery of 37, 36 and 34 MPs out of 40 MPs. Of course, the recovery rate is lower if you collect 34 or 36 MPs compared to your recovery rate if you collect 37 MPs. Please, adjust the precision of your calculations throughout the whole data file.
3)	Do you mean PVC instead of polystyrene for supplementary data 2??
4)	There are often graphic symbols together with Japanese symbols in the data file. Please, change that so that the readers can fully understand your data.
Additional comments: The authors present a small device which they developed to facilitate the separation of microplastics from marine sediments. Although I can see that the device they propose offers some advantages, such as easier cleaning and the prevention of clogging of the device by larger sediment grains, I do have some major concerns regarding the manuscript. Most importantly, the manuscript is very short and only insufficiently addresses the current developments in microplastic research. This is corroborated by the limited number (only 10 references!!!) of references from the literature. Furthermore, the authors should look at their data analysis again. In my opinion, the method they are presenting should rather be published as a Technical Report or Short Communication.


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

Input:
Title: A NEW SMALL DEVICE MADE OF GLASS FOR SEPARATING MICROPLASTICS FROM MARINE AND FRESHWATER SEDIMENTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The revised manuscript clearly shows that the authors have taken the reviewers' comments into account. They have improved their manuscript by adding recent literature and by proof-reading their manuscript.
Experimental design: The experimental design has been well described by the authors.
However, there are still three things (e.g. regarding the statistical analyses) that should be addressed by the authors before the manuscript can be published.

1) First of all, what do the authors mean when saying that the 30 g is the "wet" weight of sand in L. 151 in the revised manuscript? Was the sand soaked in NaI solution?

2) Do the authors really mean "mean extraction efficiencies" in L. 177? I assume that they used all the data on MP recovery rates (from replicate 1 to 5 per polymer type) for their analyses/ANOVAs.

3) Also, the authors should report the results of their ANOVA before presenting the results of their post-hoc tests in L. 181 and 182.
Validity of the findings: The authors have successfully clarified their data analysis in the supplementary material, but they still need to clarify their statistical analysis (see comments).
Additional comments: The revised manuscript clearly shows that the authors have taken the reviewers' comments into account. By adding recent literature and clarifying their data analysis the manuscript has been significantly improved. However, there are still three things that should be addressed by the authors before the manuscript can be published (see comments). Once these issues have been addressed, I am convinced that the manuscript can be published.


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: MERCURIC POLLUTION OF SURFACE WATER, SUPERFICIAL SEDIMENTS, NILE TILAPIA (OREOCHROMIS NILOTICA LINNAEUS 1758 [CICHLIDAE]) AND YAMS (DIOSCOREA ALATA) IN AURIFEROUS AREAS OF NAMUKOMBE STREAM, SYANYONJA, BUSIA, UGANDA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The context of fish and yam selection were not mentioned - was it because of locals diet ?
Experimental design: Author did not mentioned the following
1. What are the percentage of recovery for target metals in this experiment?
2. Any information on CRM?
3. How does the author perform the QA/QC for this experiment?
Validity of the findings: No comment.
Additional comments: Line 63 – What is ASGM and how dies it differs from ASM?

Line 51 – ‘Other safe gold recovery methods” – how do you sure the other method is safer?

Line 290 = How do you explain the high levels of THg recorded in water compared to sediments for the corresponding sluice in relation to environmental conditions such as salinity, resuspension, pH, redox potential etc?

The author mentioned that fish samples were not so aged – how does this relate to people consuming of fish?  Could be that their are not consuming this fish due to the small size?

Why the author study yam? What are the significant?

Line 147 – Were the fish samples are of edible size to be consumed by locals?

Why choose this species of fish? What is their relation to the human health?

Line 170 – Please put these info into table for easy reference

Line 268, 314 Mean ± standard deviations decimal points consistency

Line 274 – Please define 'slow flow' in your text – have you measured the flow rate?

Line 322 – Authors should put all these info into a table for an easy reference

Line 363 – Why the level differs since fish is a mobile organism and would not be staying in the same area. Are those study area separated and prevent migration of fish ?

Line 357 – Should put all these info into a table for easy reference

Line 374 - Fish samples were not so aged; - then does this reflect the fish been consumed by locals?

Line 375 – Why select this species of non-piscivorous fish ‘O. nilotica is non-piscivorous.’?

Line 383 = “95% of Hg in most fish exists as MeHg”
Then why are you not measuring MeHg?

Line 420 – the sentence containing ‘Thus, it can be deduced that O. 420 nilotica in Namukombe stream is endangered.’ is a hanging statement.
Author must explain endangered due to what, in a single sentence.

Line 481 – “water in Namukombe stream is  contaminated with up to 1.21±0.070mg/L of Hg which is above US EPA maximum permissible  limit for Hg in drinking water.”

Why are you comparing raw versus treated water?

Figure 1 – no demarcation on the location/position of the river and mining area found.
Explain your Recovery Percentage, What is your CRM  and how do you conduct the QA/QC for this research.


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: MERCURIC POLLUTION OF SURFACE WATER, SUPERFICIAL SEDIMENTS, NILE TILAPIA (OREOCHROMIS NILOTICA LINNAEUS 1758 [CICHLIDAE]) AND YAMS (DIOSCOREA ALATA) IN AURIFEROUS AREAS OF NAMUKOMBE STREAM, SYANYONJA, BUSIA, UGANDA
Review round: 1
Reviewer: 2

Output:
Basic reporting: This study reports an interesting study involving Hg pollution in an ecologically sensitive area of Uganda. Sufficient literature have been consulted and cited and also good number of environmental samples were collected and studied for Hg which is a toxic heavy metal. Introduction is well written and justifies the purpose of the study.

However, the manuscript is not written in good English. For example, L353- All the fishes from the stream did not exceed the maximum WHO….. Should be better to write as- Any fish sample from the stream did not ……

In some instances basic writing etiquette not followed Line 63: Expand ASGM at a first instance of mention. Is this synonymous with ASM mentioned in lines 57 and 58? If so, change ASM to ASGM such that abbreviations are used consistently to avoid any confusion. Authors seem to have used yam species D. alata for their study. However they have used the text ‘yams’ right from the title. It is not clear and mentioned what the other species of yam used was? If only one species used reword ‘yams’ throughout the manuscript.

There is only one figure (map) presented. This figure is not descriptive and labelled. For example, authors could have indicated sluice locations on the map.

Raw data of lab analysis is not shared.
Experimental design: Article fits into the aims and scope the journal. Research question is well defined and relevant but study design have flaws or not clearly explained. Methodology section is not explicitly and clearly described sampling site and sampling method and how sampling stations were determined.  Sufficient description is lacking to repeat or replicate the study elsewhere. Authors should indicate and label up sluices, mid sluices etc in Fig 1. Otherwise, readers cannot understand relative location of the sampling stations. Also the sampling stations of yam material should be clear and well described. For example, I cannot comprehend how did authors found a yam sample at exactly ‘0 m’ from a sluice? Does ASGM activities carried out in a food garden or crop field? If so, where did the water samples come from? Besides, how did you confirm that Hg contamination/ pollution are not from a natural source? Authors should have taken few samples before the ASGM area to confirm. Without these samples from uncontaminated areas, this study findings are just status reporting and cannot pinpoint THg found is due to the ASGM activities.
Validity of the findings: Regarding analysis of data-Have you checked for the normal distribution of the data? Generally, the data from such studies won’t show normal distribution. As a consequence, the parametric test that was used is not appropriate for such data.  As raw data is not provided could not be verified. What type of standards was used and what type of quality check measures were undertaken in lab analysis? No mention of quality checks in the manuscript. There should be a brief explanation. Conclusions are well stated and no issues.
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: MERCURIC POLLUTION OF SURFACE WATER, SUPERFICIAL SEDIMENTS, NILE TILAPIA (OREOCHROMIS NILOTICA LINNAEUS 1758 [CICHLIDAE]) AND YAMS (DIOSCOREA ALATA) IN AURIFEROUS AREAS OF NAMUKOMBE STREAM, SYANYONJA, BUSIA, UGANDA
Review round: 1
Reviewer: 3

Output:
Basic reporting: The paper deal with an interesting issue that is few investigated in the studied site. I think it is an important subject that will be of interest to the readers of the journal. However, I suggest improving the scientific writing to better understand the scientific problem (putting in a global perspective). Some grammatical errors need to be fixed.

Suggestion:
- Lines 57-86: The problem with the use of Hg in gold mining is worldwide. I suggest putting this problem in a worldwide perspective, indicating that this same problem occurs in many sites. After that, the text can conduct the reader into the study area. Currently, the paper is very regional.
Many papers can be used for this purpose. For example:
Chibunda, R.T., Pereka, A.E., Tungaraza, C., 2008. Effects of sediment contamination by artisanal gold mining on Chironomus riparius in Mabubi River, Tanzania. Phys. Chem. Earth 33, 738-743. https://doi.org/10.1016/j.pce.2008.06.051.
Donkor, A.K., Bonzongo, J.C., Nartey, V.K., Adotey, D.K., 2006. Mercury in different environmental compartments of the pra river basin, Ghana. Sci. Total Environ. 368, 164-176. https://doi.org/10.1016/j.scitotenv.2005.09.046.
Feng, X., Dai, Q., Qiu, G., Li, G., He, L., Wang, D., 2006. Gold mining related mercury contamination in Tongguan, Shaanxi Province, PR China. Appl. Geochem. 21, 1955-1968. https://doi.org/10.1016/j.apgeochem.2006.08.014.
Gerson, J.R., Driscoll, C.T., Hsu-Kim, H., Bernhardt, E.S., 2018. Senegalese artisanal gold mining leads to elevated total mercury and methylmercury concentrations in soils, sediments, and rivers. Elem Sci Anth 6 (11), 1-14. http://doi.org/10. 1525/elementa.274.
Goix, S., Maurice, L., Laffont, L., Rinaldo, R., Lagane, C., Chmeleff, J., Menges, J., Heimburger, L., Maury-Brachet, R., Sonke, J.E., 2019. Quantifying the impacts of artisanal gold mining on a tropical river system using mercury isotopes. Chemosphere 219, 684-694. https://doi.org/10.1016/j.chemosphere.2018.12.036.
Limbong, D., Kumampung, J., Ayhuan, D., Arai, T., Miyazaki, N., 2005. Mercury pollution related to artisanal gold mining in north Sulawesi Island, Indonesia. Bull. Environ. Contam. Toxicol. 75, 989-996. https://doi.org/10.1007/s00128- 005-0847-0.
Lino, A.S., Kasper, D., Guida, Y.S., Thomaz, J.R., Malm, O., 2019. Total and methyl mercury distribution in water, sediment, plankton and fish along the Tapajós River basin in the Brazilian Amazon. Chemosphere, 235, 690-700. https://doi.org/10.1016/j.chemosphere.2019.06.212
Marrugo-Negrete, J., Benitez, L.N., Olivero-Verbel, J., 2008. Distribution of mercury in several environmental compartments in an aquatic ecosystem impacted by gold mining in Northern Colombia. Arch. Environ. Contam. Toxicol. 55 (2), 305-316. https://doi.org/10.1007/s00244-007-9129-7.
Miserendino, R.A., Guimaraes, J.R.D., Schudel, G., Ghosh, S., Godoy, J.M., Silbergeld, E.K., Lees, P.S.J., Bergquist, B.A., 2018. Mercury pollution in Amapá, Brazil: mercury amalgamation in artisanal and small-scale gold mining or land- cover and land-use changes? ACS Earth Space Chem 2 (5), 441-450. https://doi. org/10.1021/acsearthspacechem.7b00089.
Experimental design: The sampling design need to be better described. It is impossible understand exactly what was done. What do upstream, middle stream and downstream mean? Distance (0, 10, 20 and 30) from what?

I pointed some suggestions:
- Line 133: What kind of plastic was used?
- Lines 133-138 and 153-159: The method blank and its THg results need to be detailed (perhaps in sup. material) – example: bottle blank, equipment blank, blank of the filtration and analytical blank.
- Line 136: What kind of filter was used?
- Line 136: How much acid was used to preserve samples?
- Line 138: Were water and wasterwater assessed? Only the procedure of water collection was described.
- Line 142: What kind of plastic was used?
- Line 148: This section can present a brief analytical method of fish samples.
- Line 150: Some Hg species can be lost after 5 h at 550 C. The discussion needs to consider that.
- Lines 153-155: Sediment, fish and yam: Were these samples oxidized with BrCl? How much BrCl was used? How much time? At room temperature? The description of analytical method needs to be improved.
- Line 152: How was accuracy assessed?
- Lines 216-218: How were EF, PLI, RI and HG calculated? Whats about their results?
- Lines 230-231: How was the background defined?
- Lines 242-249: They are not results. They are method.
- Lines 244-248: How were heterocedasticity and normality evaluated? Table 1 shows spatial comparison (between 0, 10, 20 and 30 sites) – this statiscal analysis is not described here.
- I suggest adding the detection limit of the methods.
Validity of the findings: - The discussion seems consistent with the results, but a deeper analysis could not be done. It is not possible to evaluate the general context of the discussion because it is not possible to understand the experimental design.

- The results need to be better described. Na forma atual, os resultados são somente a apresentação de tabelas.
Uma sugestão: Lines 268-271, 313-315, 317-320, 352 and 394-398: this is result (not discussion). Nao tem 0 (abaixo do limite de detecção). O que é variou de tanto a tanto com desvio? Não dá para entender os valores.

- The results need to be better described. In the current format, the results are just the presentation of tables.
Suggestions:
Lines 268-271, 313-315, 317-320, 352 and 394-398: this is result (not discussion).
There isn't 0 (results are below detection limit).
It is difficult to understand the values. Are they mean? Min-Max?

- Conclusions and recommendations: It can be summarized.
Suggestions:
You can remove results.
In further study – I suggest putting suggestions of studies that can fill gaps of the evaluated processes or about new important questions in the area of study. For example: "evaluate the mercuric content of the different organs": I understand the importance of this. However, why is it important for gaps raised in the study? "Lead and arsenic": why? Are they released in the mining activities studied here?

- ADD: There are many chemical species of mercury in sediment samples. The contact with polluted sediment will not necessarily transfer the mercury to person. This needs to be incorporated into the discussion. The present study assessed only THg. It is impossible to know if Hg is in a chemical species that will be absorbed by the dermal contact.
Additional comments: Title
- It can be shortened to be simple and straightforward.
Example:
The words “surface”, “superficial” and “(Dioscorea alata)” can be removed.
“Nile tilapia (Oreochromis nilotica Linnaeus 1758 [Cichlidae])” can be replaced by “fish”.

Abstract
- Line 29: "pollution and contamination". It was not clear what the difference between the two. I suggest putting only one.
- Lines 34-35: I suggest adding the sample size of each sample type (e.g., water, fish).
- There is no 0. I suggest presenting the results "<detection limit".
- It is difficult to understand the results:
THg in water from 0 to 1.21. Min-max? Mean? Is 0.04 the standard deviation?
The same occurs for fish and yams.
Sediments: is 0.14 the mean? The maximum value?
Please, clarify what the values of the results mean.
- Line 46: 91.7% is not "some".

Introduction
- Line 63: What does ASGM mean? The acronym has not been defined previously.
- Lines 71-72: This statement needs a reference.
- Line 96: The mercury released by goldminig supplies the ecosystem with anthropogenic mercury. The mining activity does not transmogrify mercury.
- Line 113: I suggest using "sediment" instead of "sediments".
- Lines 112-116: It could be divided into three sentences because the objective of the study has 3 different ideas. Each could be presented in one sentence.

Discussion
Lines 321-322: “other global studies”: Are they in mining sites? Are they in natural sites? What is the quality of environment assessed in those studies? It is important to understand if they are comparable with the studied site.
Lines 337-338: Sites worldwide vary naturally in THg concentrations in sediments. A value considered as normal in non-contaminated sediments can not be applied to anywhere in the world. It has to be used very cautiously and is not suitable for a wide range of naturally enriched environments.
Lines 351, 438, 440 and other: "Linnaeus 1758 (Cichlidae)" may be cited only once (when the species name is first cited in the text-line 210). Throughout the paper, "Linnaeus 1758 (Cichlidae)" need not be written. I suggest writing only the species name.
Lines 356-362 and Table 1: The mercury concentration in fish, sediment and yam are more or less low. The cited studies also have low concentrations. I suggest to report the results of the present study and those of literature in ug.kg-1 instead of ug.g-1 to facilitate the visualization of the values.
Lines 367-368: I suggest "epithelial absorption" instead of "epithelial ingestion".

Figure 1
The figure needs to be improved. Examples:
I suggest putting a letter in each square and saying in the legend what they mean.
The three maps need to contain geographic coordinates, scale and the compass rose.
What do the symbols on the map mean? (e.g., lines, purple x)
Do the names refer to the states? Limits of basin drainage?

Tables
The caption needs to be improved. A caption must be self-explanatory. All acronyms in the table need to be explained in the caption or in the footnote.

Table 1
e.g.
SE- standard error?
SD – standard deviation?

Table 2 and 3
e.g.
EDI?
THQ?
If there is no result of 30, do not put this line. Why is there no result of 30?

Table 4
e.g.
ADD?
THQ?

The same for other tables.

General:
- Always separate unit of measurement from the number: e.g.: 30 m, 0.04 μg. Only % needs to be without space (e.g., 90%).
- There are many acronyms in the paper and this confuses the reader. The authors could evaluate which acronyms are really needed in the paper (e.g., frequently cited expressions) and which ones could be removed.


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: MERCURIC POLLUTION OF SURFACE WATER, SUPERFICIAL SEDIMENTS, NILE TILAPIA (OREOCHROMIS NILOTICA LINNAEUS 1758 [CICHLIDAE]) AND YAMS (DIOSCOREA ALATA) IN AURIFEROUS AREAS OF NAMUKOMBE STREAM, SYANYONJA, BUSIA, UGANDA
Review round: 2
Reviewer: 1

Output:
Basic reporting: Authors have now extensively revised the manuscript and have addressed most of my concerns. I see a large improvement in the clarity of the manuscript. Authors have added some nice figures. One concern I have in this version is use of the term "sluicing". For instance, sluicing referred by authors in Fig 2a is actually 'panning' and 'not sluicing' in this part of the word. I suggest authors to revisit and recheck appropriateness of the word they  used and if possible to define and explain this a bit in introduction section.
Experimental design: Fine and understandable now. The concern is the one i raised on the earlier version. How did authors sampled yams at a distance of 0 m up sluice of the stream? This is not clarified and in the abstract it is written still as "Consumption of D. alata grown at 0 m up sluice of Namukombe stream may pose deleterious health risks as reflected by the health risk index of 0.985 being very close to one". In the revised version with a new figure (as Figure 4), and the ground situation as shown in the Fig 2a does not match the statement above.  I think authors have taken yam samples within 0-10 m from sluicing location. If this is the case, should be explained properly.
Validity of the findings: Statistical methods are now clearly explained.
Additional comments: Revised well.


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

Input:
Title: MERCURIC POLLUTION OF SURFACE WATER, SUPERFICIAL SEDIMENTS, NILE TILAPIA (OREOCHROMIS NILOTICA LINNAEUS 1758 [CICHLIDAE]) AND YAMS (DIOSCOREA ALATA) IN AURIFEROUS AREAS OF NAMUKOMBE STREAM, SYANYONJA, BUSIA, UGANDA
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: I think the authors did a good job. The new draft presented resolutions for the topics raised or they were properly answered. I recommend this new version 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: EFFECTS OF THE LOSS OF ESTROGEN ON THE HEART’S HYPERTROPHIC RESPONSE TO CHRONIC LEFT VENTRICLE VOLUME OVERLOAD IN RATS
Review round: 1
Reviewer: 1

Output:
Basic reporting: English definitely needs to be revised. Typos, spelling mistakes, missing spacing and non-meaningful sentences should be corrected

May abbreviations are using before being spelled out and explained
Experimental design: OVX and AR surgical procedures are not detailed in the methods sections. These details should be added.

Since multiple surgical procedures were performed in the current study, each time "sham operation " is mentioned, this needs to be clarifies: sham relative to which surgery

Were placebo pellets administered in control groups for E2-supplemented animals?

What is E2 levels in E2 supplemented animals and their control? These measurements should be included

Rationale for looking at genes expression of specific genes is not clear?

Explain why "Cyclophilin A" was used as a housekeeping gene? and not more commonly used ones?
Validity of the findings: In figure 1: It is not clear what stars are referring to in these 6 panels. The legend states that this is relative to sham groups, but sham group individual results are not displayed in the figure! It is fundamental to include this data set as a third group for each panel. Although the average would be one, still showing individual data will show variability.

It is not clear what is each p value included above panels refer to? and statistical test?

The type of statistical comparison tests used in figure 1 should be included in the legend

The authors relate their OVX effects as an effect ovarian hormonal depletion, which may not be true. OVX is associated with other hormonal differences so authors should be more accurate in describing their findings especially given that E2 supplementation did not restore all OVX-induced changes
Additional comments: Group codes are not explained in the abstract so it is hard to understand and follow the abstract