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: DIFFERENTIALLY EXPRESSED SERUM PROTEINS IN CHILDREN WITH OR WITHOUT ASTHMA AS DETERMINED USING ISOBARIC TAGS FOR RELATIVE AND ABSOLUTE QUANTITATION PROTEOMICS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Please have a look at Minor concerns: 1 to 3.
Experimental design: Please have a look at Major concerns: 1 to 5.
Validity of the findings: No comment.
Additional comments: Asthma is one of the most common chronic noncommunicable diseases, which is a chronic inflammatory disease of the lower airway that is commonly encountered by the otolaryngologist. Asthma is a serious global health issue and asthma guidelines recommend a stepwise approach to management with goals to achieve control and minimize future risk. It is a good idea to investigate the protein composition and expression levels in serum samples obtain from children with or without asthma. In this manuscript, Dr. Fang and colleagues looked at the role of the differentially expressed proteins (DEPs) identified by using isobaric tags for relative and absolute quantitation (iTRAQ) combined with liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses in the pathogenesis of childhood asthma. The study provides the conclusion, which showed that numerous DEPs that may play important roles in the pathogenesis of childhood asthma. Those proteins may be novel biomarkers of childhood asthma and may provide new clues for the early clinical diagnosis and treatment of childhood asthma. Although, the current study is interesting and well but there are some major concerns that need to be addressed.

Major concerns:
1. “Experimental design & Validity of the findings” In general, the research idea of this manuscript is novelty. However, the biggest defect is the lack of sample quantity, which makes it difficult to judge whether the conclusion is universal or not. If the authors increase each group of samples to 10, where possible, the results should be more applicable. However, considering the high cost of iTRAQ technology, I recommend that the author select 1 or 2 from the 37 DEPs for ELISA verification, and expand the samples to about 50+, which may significantly improve the impact of this study.
2. “Materials & Methods – Clinical information and serum sample collection” Line 103-112. “The included samples were collected from four children with asthma (experimental group) and four children without asthma (control group).” When grouping, did the authors take into account factors such as age, gender, and body mass index (BMI) of patients in the balance between the two groups? Because the physiological factors mentioned above also have a certain influence on serum protein, considering the small sample size of this study, these factors must be matched to exclude the relevant influence, please explain in the article. P.S. I saw that the author showed no difference in age and gender between the two groups in the results section. Please analyze BMI also.
3. “Materials & Methods – LC-MS/MS analysis” Line 147-150. “Protein fold changes (FC) of at least 1.5 were obtained, and P < 0.05 was considered a statistically significant difference. A” Generally, in high-throughput analysis, the threshold value of FC is defined at 2. Why the author chooses 1.5, please clarify the reasons and references.
4. “Table 1” Page 45. The data showed that G6PD was one of the most significantly down-regulated proteins. This is a very interesting finding. As an important gene related to G6PD deficiency disease, the detection of G6PD activity should be widely used in children's hospitals and general hospitals in mainland China. Can the author make a retrospective study, collect and analyze relevant data, and observe whether the activity of G6PD is down-regulated in the blood of children with asthma? The research value of this paper can be further enhanced by appropriately expanding the sample size.
5. Through PubMed searching, the keywords are "asthma" and "iTRAQ", and I find that there are very few relevant studies. There is a lack of research on the detection of serum in children with asthma, so I think the authors should try to add as many samples as possible.

Minor comments:
1. Figure 3B. The author should indicate which group “B1-4” and “A1-4” belong to?
2. Figure 4. The text in the picture is too small. Please increase the font size.
3. All Figure. The vector image exported by R language can be modified with drawing software (Canvas or AI) again. Please choose the appropriate font size for the convenience of readers. In addition, author should keep the horizontal and vertical ratio in the picture zooms in and out.


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: DIFFERENTIALLY EXPRESSED SERUM PROTEINS IN CHILDREN WITH OR WITHOUT ASTHMA AS DETERMINED USING ISOBARIC TAGS FOR RELATIVE AND ABSOLUTE QUANTITATION PROTEOMICS
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: This manuscript by Ming Li found 46 DEPs in serum samples of children with asthma vs. children without asthma. Verified some proteins that have been reported by others, and also provided several new potential proteins. These newly discovered proteins maybe some alternative targets in future studies of childhood asthma.
Here are a few questions and comments:
1. Line 84-86 “……selective chemokine ligand 5, hematopoietic prostaglandin D synthase, and neuropeptide S receptor 1 were involved……” line 88 “……fatty acid binding protein 5……” and line 272 “Complement component 1s……” Do all these descriptions refer to specific genes? Please mark their official symbol accurately.
2. Line 269 mentions “Inflammatory reactions can also promote the healing of injured tissues.” In the results of the manuscript, lines 184-185, the description of the control group is “children with trauma ……” Will the trauma here affect the experimental results? Maybe some discussion needs to be added.
3. Figure 3A, please accurately describe or set the color of the volcano plot. The description in figure legends “red dots” and “green dots” should be “yellow dots” and “blue-green dots” respectively.
4. Figure 8 need to adjust because some have exceeded the maximum value of the Y-axis.
5. Although “……Children with asthma typically present with eosinophilic asthma and allergy……” (line 77), the pathology type of children with asthma should be provided so that readers can clearly understand the possible impact on the experimental results.
6. line 149 “……>1.5 represented upregulated proteins and a FC <0.667 represented downregulated proteins……” however, the description in Figure 3 is “FC> 1.5” and “FC <1.5” as a significant change. And the volcano uses log10FC, and the heatmap uses log2FC, please explain clearly.


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: DIFFERENTIALLY EXPRESSED SERUM PROTEINS IN CHILDREN WITH OR WITHOUT ASTHMA AS DETERMINED USING ISOBARIC TAGS FOR RELATIVE AND ABSOLUTE QUANTITATION PROTEOMICS
Review round: 1
Reviewer: 3

Output:
Basic reporting: 1. Several studies that tried to identify differentially expressed proteins of asthma have been published (as listed below) and one also focused on childhood asthma. Could the authors explain the difference and significance regarding the design and results of their study compared to those studies?
1) Pilot-Scale Study Of Human Plasma Proteomics Identifies ApoE And IL33 As Markers In Atopic Asthma. J Asthma Allergy. doi: 10.2147/JAA.S211569.
2) Screening Serum Differential Proteins for Childhood Asthma at Different Control Levels by Isobaric Tags for Relative and Absolute Quantification-based Proteomic Technology. Zhongguo Yi Xue Ke Xue Yuan Xue Bao. DOI: 10.3881/j.issn.1000-503X.2017.06.014
3) Induced Sputum Proteome in Healthy Subjects and Asthmatic Patients. J Allergy Clin Immunol. doi: 10.1016/j.jaci.2011.07.053.

2. Some of the references are old, and the authors should also refer to studies of top journals in the field.
Experimental design: 1. The authors should describe the amount of protein they extracted from the blood sample as well as the amount they applied in each tests respectively.

2. The statistical analysis applied in this study should be mentioned in method.

3. In method, further explanations in terms of how the analyses of GO, KEGG, COG were carried out together with their meanings in the study should be described, instead of only focusing on their definitions.
Validity of the findings: No comments
Additional comments: In this study titled “Differentially expressed serum proteins in children with or without asthma as determined using isobaric tags for relative and absolute quantitation proteomics”, the author tried to differentially expressed proteins (DEPs) expressed between children with or without asthma. Among these DEPs in the result, 12 proteins were significantly upregulated and 34 proteins were downregulated. The author further analyzed these DEPs with GO, KEGG and COG to explain the meaning of identified DEPs in asthma.

However, several issues need to be addressed before further consideration for publication.

1. Serum samples derived from four children with asthma and four children without asthma were collected in this study, which is quite a small sample size compared with other similar studies. Could the author give their reason or evidence to support that this sample size is sufficient enough?

2. For those four children with asthma, were they hospitalized due to asthma exacerbation, or just with a history of chronic asthma? Furthermore, since their blood was collected the other day of their hospitalization, they were supposed to be treated with antiasthmatic drugs. Did the authors notice the impact of antiasthmatic drugs on DEPs, and the difference of DEPs during asthma exacerbation, which the authors should try to explain?

3. Should the authors explain the reasons and evidence why they chose to analyze the blood of the patients instead of sputum or samples derived from bronchoalveolar lavage?


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: DIFFERENTIALLY EXPRESSED SERUM PROTEINS IN CHILDREN WITH OR WITHOUT ASTHMA AS DETERMINED USING ISOBARIC TAGS FOR RELATIVE AND ABSOLUTE QUANTITATION PROTEOMICS
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: Asthma is one of the most common chronic noncommunicable diseases, which is a chronic inflammatory disease of the lower airway that is commonly encountered by the otolaryngologist. Asthma is a serious global health issue and asthma guidelines recommend a stepwise approach to management with goals to achieve control and minimize future risk. It is a good idea to investigate the protein composition and expression levels in serum samples obtain from children with or without asthma. In this manuscript, Dr. Fang and colleagues looked at the role of the differentially expressed proteins (DEPs) identified by using isobaric tags for relative and absolute quantitation (iTRAQ) combined with liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses in the pathogenesis of childhood asthma. The study provides the conclusion, which showed that numerous DEPs that may play important roles in the pathogenesis of childhood asthma. Those proteins may be novel biomarkers of childhood asthma and may provide new clues for the early clinical diagnosis and treatment of childhood asthma. After the modification, the data quality and language description are improved. Although there are still some deficiencies (sample size is small) in this study, there are few relevant explorations in this field. This study is a good case worthy of publication and guidance for further 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: DIFFERENTIALLY EXPRESSED SERUM PROTEINS IN CHILDREN WITH OR WITHOUT ASTHMA AS DETERMINED USING ISOBARIC TAGS FOR RELATIVE AND ABSOLUTE QUANTITATION PROTEOMICS
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Some minor issues need to be corrected by the author.
1. Please describe the clustering method used in the cluster analysis in Materials and Methods.
2. CCL5 should be chemokine (C-C motif) ligand 5 or C-C motif chemokine ligand 5. Please read the references carefully.
3. The Official Symbol of Complement component 1s should be C1S. Please pay attention to the wording of gene names to distinguish different species.


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

Input:
Title: CRYPTOSPORIDIUM SPP. AND GIARDIA DUODENALIS EMISSIONS FROM HUMANS AND ANIMALS IN THE THREE GORGES RESERVOIR IN CHONGQING, CHINA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Good use of scientific literature.
Paper structure and set up is okay.

The use of English could be improved.
I could not open the raw data, unclear which software is needed.
Experimental design: The research is interesting and relevant.

However, the paper is unclear in the description of exactly which data on what scale are used where and why. Insufficient detail to exactly replicate the research.

-	Are calculations really done on a 0.5 degree grid basis? Because the supporting information gives the animal and human population data per district. If the calculations are really done on a grid basis, it should be explained how the results were aggregated to districts (figure 5 and 6).
-	Furthermore, a 0.5 degree grid is very coarse for a model for an area of this size (the area would only be about 6 x 10 grid cells, according to figure 5 and 6).  The original Hofstra et al. model was a global scale model; then a 0.5 degree grid is justifiable, but if more detailed data for a smaller region is available then it is not justifiable in my view, a smaller grid should be chosen.
-	It is unclear from the paper and the supporting information where the data on urban / rural populations come from, and how these are spatially used in the model.
-	It is unclear from the paper what data were taken from the original Hofstra 2013 study, and what data are local data for China. For some this is in the supporting information, but not all.
-	Why was the year 2013 chosen for the study? Should be explained.
-	Line 199: first mention of calculations on a grid basis, this should be introduced earlier.
-	Line 203: What are ‘ breeding days’  and why is this variable included in the model? This variable was not in the original Hofstra 2013 model that this research is based on.
-	Line 208: Is all manure treated in China? Because this is not standard practice in other places around the world, some more information on this would be helpful.
-	Line 230-235: the authors have not thought of this approach themselves, but taken this approach from Hofstra et al. 2013, this should be credited.
-	Line 399: what does ‘disposed harmlessly’ mean? This assumption should be explained (in the methods section)
-	Line 399-400: What assumption did you make for your calculations about the percentage of manure applied to land? Should be explained in methods section
-	Line 408: did you use this number of 1.84 tons in your calculation? If yes, should be explained in your methods section. If not, why not, and what number did you use?
-	Line 429 – 431: But if these four studies provide data on Giardia in sheep and rabbits in China, and in fact you provide these data in Table S6, why were these data not incorporated in your model (0 in Table S4)??

-	Table S1: the livestock data should be given for the individual animal species instead of livestock total. Which are presumably the different animal species added up? Without the separated data, this research is not reproducible.
-	Table S1: Where do these data come from? Reference?
-	Table S1: The human population density and livestock density data in Table S1 are given per district. However, the original model by Hofstra et al. 2013 used gridded human and livestock population density data (on a 0.5 x 0.5 degree grid). It is unclear from the paper whether the calculations were done on a grid basis or district basis. The results are only shown in maps on a district basis, but the text does mention grids here and there (e.g. line 199 mentions explicitly that calculations are done per grid cell). As is, this research is not reproducible.
-	What are the units of Table S2 in the supporting information?
Validity of the findings: More discussion regarding impact and novelty would be welcome.
Results and discussion are unclear or erroneous in places.

-	The results are presented in the text with four significant digits. This suggests an unrealistic amount of certainty that is not justifiable with this modelling approach.
-	The population that is classified as non-source means a population that is connected to a sanitation type that produces no emissions to the environment, in the original Hofstra model. However, the text (lines 257 – 269) suggests that emissions are calculated for non-sources, this is very confusing.
-	Line 280: are these (oo)cysts per grid cell, per year, per district? No unit given.
-	Line 301-302: ‘the data were limited by subjective and objective factors’. Unclear what is meant here.
-	Interpretation of what the outcome of the sensitivity analysis means is lacking
-	Line 321-322: why is it that people in these regions obtain this treatment? Explanation and interpretation of the patterns in figure 8 is lacking.
-	Line 383: This statement that the model generally produces estimates around 1.5-2 log higher than observations is made about the surface water oocyst concentrations model by Vermeulen et al. 2019, NOT the oocyst emission model by Hofstra et al. 2013. It is therefore incorrect and very misleading to apply this statement to this research!
-	Line 388: give numbers on the concentrations of Crypto and Giardia that are found in the WWTPs in this area, that would be informative for the reader
-	Line 419-421: misleading, because you based your model on these models, they are not completely ‘different models’
-	Line 421-424: the work by Liu et al. 2019 sounds like a very relevant work. Can you discuss the assumptions that Liu et al. made regarding the oocyst production and emission in this watershed, and contrast these with your own assumptions?
-	Line 432-437: No, you cannot validate your model with surface water values because you did not calculate surface water values, only emissions. If you wanted to calculate surface water values, you should use your emissions as input for a hydrological model!
-	Line 453: you did not include actual runoff data in your model, only an assumption on a runoff fraction. This should be made clear to the reader and discussed in the paper!
-	Line 458: not just a routing model, but any hydrological information! A routing model is only a part of hydrological modelling.
-	The main conclusions from the scenario analysis should be incorporated in the conclusion section

-	Figure 1: reference for these data should be given!

-	Figure 2: this figure is adapted form Hofstra et al. 2013. Credit should be given!

-	Figure 2: Why is the arrow from Rural to Surface water grey? Because as I understand it, you are calculating this?

-	Figure 3: Strange y axis scale, bit difficult to interpret. (Especially the strange jumps from 5x10^13 to 6x10^14 to 1x10^15??)

-	Figure 4: Incorrect explanation of non-source, as non-source is not an emission category. This figure actually does not show emission categories, but the fraction of the population having a sanitation type that does or does not produce a certain type of emissions.
Additional comments: Examples where the use of English could be improved are listed here. This list is not exhaustive, it is recommended that the entire text be checked.
Plural vs singular / present – past tense:
-	Line 139 showed
-	Line 245 human  humans
-	Line 214: presented  present
Vague wording or too long sentences:
-	line 91-95,
-	line 95-99,
-	line 260-262
-	line 395-397
-	line 400-403
-	line 410-412
-	line 416: ‘it is deserved to’
Incorrect words:
-	line 51“ are the largest animal emissions”  “ account for the largest animal emissions”
-	line 111 whereas should be removed
-	line 115 ‘the’ before China should be removed
-	line 116 preponderance is strange
-	line 118 lead  leading
-	line 289: them  those
-	line 316: ‘the’ should be removed
-	line 328: ‘also’ should be removed
-	line 350: ‘from the increasing pollution’  ‘the’ should be removed
-	line 437: ‘in actual’ should be removed.
Other textual comments
-	Line 54: how can urbanization be ‘ improved’ ?
-	Line 65-66: Strange summary. The sentence suggests that children are examples of immunocompromised people, and that AIDS patients and neonatal animals are examples of malnourished individuals.
-	Line 69: Hofstra et al. 2013 is not a primary reference for this statement
-	Line 73: not all animals are a reservoir of Crypto and Giardia!
-	Line 89: does ‘ host’  here refer to animals, humans or both?
-	Line 134-136: Text seems to suggest that the figure shows emissions, but it does not
-	Line 140: ‘two pollution sources’  ‘ two types of pollution sources’
-	Line 212-213: ‘ range of the parameter that can take’  ‘ range that the parameter can take’
-	Line 230: insert ‘of’ after ‘understanding’
-	Line 321-323: this is an assumption in a scenario and not a fact, should be made clear
-	Line 361: commas around adequately should be removed
-	General: inconsistent spelling of feces / faeces


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: CRYPTOSPORIDIUM SPP. AND GIARDIA DUODENALIS EMISSIONS FROM HUMANS AND ANIMALS IN THE THREE GORGES RESERVOIR IN CHONGQING, CHINA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper entitled ‘Exploring Cryptosporidium spp. And Giardia duodenalis emissions from humans and animals to the Three Gorges Reservoir in Chongqing, China’ provides a useful approach to better understand the environmental flows of the protozoans in this specific area. The paper is comprehensive as it includes human and livestock loads estimations, a sensitivity and a scenario analysis for the year 2050.

This paper essentially is a local application of the Global Waterborne Pathogen (GloWPa) model. This model is cited throughout the paper (all Vermeulen and Hofstra references). However, to enable comparison with other GloWPa model results after publication, this paper should highlight better that it is mostly based on the GloWPa model. Right now it reads as if the authors have developed their own model.

Although being comprehensive and useful, I do also feel the paper in its current form has significant flaws. It will require quite some work to bring it to the level of a good publication. My main criticism is on the methodology and the discussion. I added the minor and textual points below and have added the methods and discussion comments in the other categories below.

Minor points
Several references are out of place. Make sure that you check them again and only really cite the correct literature. E.g. Hofstra et al 2013 is not the source to cite that diarrhea is the third leading cause of death. There are burden of disease studies or WHO documents reporting this. O’Neill et al 2015 is not the source that discusses the lack of available livestock SSP data (and are they indeed still missing or have they in the mean time become available?).

It is important to explain why the loads are relevant. What you say is that this model can contribute to risk assessment (line 460). However, risk assessment is based on the amount a person ingests. How do the loads that you calculated relate to this?

Line 250-251 For human … of total emission reach the TGR. How do  you know? How about decay along the way?

Line 279-280 Total annual animal source … emissions. Why are these animal sources spread over larger areas? I see for both human and animal emissions the full map coloured? I don’t understand.

You need to look at the units throughout the text. E.g. line 285 what are the units? (Oo)cysts per district or grid? Or? The units are in more locations unclear.

Line 403-408 What water pollutants did that Census of pollution sources look at? Would you expect the results be different for protozoans? Also, what does this text mean for your results. Should in your case livestock emissions have been larger than human emissions?

Line 444 you mention that Chongqing is an emission hotspot. How do you know? You only studied this area. How will it compare to other areas?

Figure 1 caption what is ‘sewage disposal’?

Figure 1 figure: now that you added rural and urban to the figure (compare to the figure in Hofstra et al 2013), the figure is inconsistent. Are animals, humans, rural, urban, land, WWTP and surface water really all model components as you mention in the caption? In case you do want to add in urban and rural, consider splitting the box Humans up in two, or add rural population and urban population and remove the box with humans.

Figure 3: why not use a log-unit scale. In the current way, the cattle Crypto emissions can not be quantified.

Figure 4: why did you scale this figure to 100%. Why not immediately show the importance of the individual categories? Also, is the y-axis title correct? Shouldn’t it be loads rather than population?

Figure 7: you show differences between the scenarios. In the text you will need to put these differences into perspective. What does it mean that the emissions are almost halved for Scenario 1? How does halving compare to increased log reductions during treatment or the logs difference in the sensitivity analysis?

Figure 8: The difference figures do not clearly show positive and negative. Make this more obvious.

Language

The paper uses emissions and loads interchangeably. However, in line 136 ‘load’ is defined. I would suggest the authors use load throughout the paper and remove the word emissions.

The English language used in the paper is mostly reasonable. However, still quite a number of mistakes are made and therefore I would recommend checking by a native English author or editor. I have picked up on a few mistakes, but this list certainly is not exhaustive:
Line 51 have the largest instead of are
Line 115 remove the before China
Line 122 estimateS
Line 135 humanS
Line 137 end up IN THE surface water instead of on
Line 141 systems THAT reach the TGR
Line 143 in rural areas that feces enter surface water… rephrase (not sure what you want to say)
Line 145 extensive lit review that only found 3 papers? Change wording of extensive.
Line 153 humanS
Line 155 and diffuse emissions. The emission categories…
Line 164 were also reference to … rephrase
Line 191 animalS
Table S4 should be clear on having infected livestock.
Table S5 and 6 can be merged. Also spell average correctly
Line 212 range of the parameter that can take… rephrase, not sure what you want to say
Line 221 2050 WERE based
Line 227-228 does SSP3 emphasize regional progress? Also in times of war? Not sure…
Line 228 strongly instead of highly
Line 230 understanding OF the
Line 251 Humans were responsible for ….% of the oocyst emissions. Line 253 same comment for the cyst emissions.
Line 256 add manure to subtitle
Line 324 urban residentS ARE responsible
Line 344 urban areas instead of residents
Line 351-352 China is one of the regions with Crypto…
Line 360 important instead of a significance
Line 361 remove commas twice
Line 363 aimed AT
Line 386 densely populated urban areas instead of high population density urban
Line 416 consequence, manure treatment, such as ….. on oocyst should be studied in more detail ….
Line 453 Therefore is out of place. The sentence does not follow logically from the previous sentence.
Experimental design: Methodology

The methodology of the paper should be further elaborated. There are many open ends and unclear interpretations. In addition, in its current form, I feel the paper is not reproducible. Examples:
-	Why is the year 2013 chosen as baseline?
-	The authors have chosen to use human and livestock loads and then split these up in connected, direct and diffuse loads. It is not in all cases clear what these categories involve. For example, in line 157 the authors mention that faecal waste has been collected and used for irrigation as fertilizer. It is unclear to me which faecal waste is meant here. Additionally, in the explanation of the livestock loads there is no mention of dealing with the different categories differently, but Figure 4 does split them up. It is unclear how this split up has been developed. Finally, the methodology does not discuss non-sources. The results and discussion section do. What is this non-source category?
-	Further on the faecal waste. Is the the faecal waste from pit latrines (only latrines, not from septic tanks, or the waste from sewer pipes?) directly used on the land? How about storage in the pits before emptying. Would this influence reduction of pathogens in the faecal waste? To solve this point and the previous one, the paper would really benefit from a TGR area-specific explanation of the sanitation situation and livestock manure management.
-	Why is the HDI relevant? You are working in one country with only one HDI?
-	From Table S2 it seems like the current sanitation fractions used are the same across the districts. Is that indeed the case? This could make quite the difference to the final results and maps.
-	The surface runoff fraction used (0.4) seems to be very high. Ferguson uses 2.5%, Vermeulen et al 2019 use even much lower values (2-8 log stay behind on the land). Motivate this fraction of 0.4.
-	Only from equation 4, I realised that some of the manure is treated before it is discharged into the surface water (is that my correct interpretation?). Is that my correct interpretation? What happens to the manure otherwise? Is there any manure storage? If so, would you need to consider losses during storage? Also, I happen to know that direct discharges from farms to the surface water regularly occur in China. Are these included? This is all not clear. Again, paint a picture of the situation in China and then explain which of these are included in in what way.
-	It is not clear to me why the breeding days of livestock species are relevant. Why is this included? Motivate.
-	The sensitivity analysis requires much more explanation. For example, the connected fraction is doubled. How can you get a connected fraction higher than 1? Additionally, the fractions together add up to one. When you change one, the others should also automatically change. How did you deal with this? Etcetera.
-	The scenario analysis requires a lot more motivation. The way it is currently discussed, shows a global scale interpretation of the SSPs. However, you are focusing on China alone. Did anyone already do a higher resolution interpretation of the SSPs for China? I would be surprised if nobody has done that yet. It is important to do a local interpretation of such large-scale scenarios, as they may mean something very different in different parts of the world. So how can you interpret the SSP1 and 3 scenarios for China and the TGR region specifically? Also, motivate better why it is OK to assume that there are no changes in excretion rates and prevalence (wouldn’t a better developed country have lower prevalence?). Finally, you currently assume that the livestock density increases at the same rate as the human population. Is that realistic? Motivate. In this way, all choices will have to be more carefully motivated.
-	In line 272, at once it is mentioned that spatially distributed maps are produced. How did that spatial distribution happen? And does a 0.5 x 0.5 lat lon degree resolution make sense in the case of this study, or should the resolution be higher, or can you also plot districts (maybe you have done that already, but that is not clear).
-	Are there animals in urban areas? Line 265…?
Validity of the findings: Discussion

The discussion of the paper is weak. Many holes are picked in the own work, but the results are not put in perspective of earlier results or the own sensitivity analysis. The GloWPa model is seen as a different model (line 419), but at the same time, it is seen as the same model (at least, that is what I think?) in line 382-384 (‘the model’ on line 383). Nevertheless, results of both models for the same areas are not compared. Why not? This is an easy first step. You have more spatial detail in your model inputs, so your model is potentially the better model. Right?
There are opportunities for more comparison. E.g. the Xiao et al 2013a paper is mentioned on line 369. Measured concentrations are mentioned. However, how can they be compared to your results? You try to make a calculation and arrive at a load, but it is unclear to me 1. how you get to this load and 2. how I can compare this load to the concentrations of Xiao. That is a missed opportunity.
The discussion is full of texts like ‘it is reasonable that our estimated emissions [] exceeded actual loads of waters in the TGR within an order of magnitude’ (line 382) how do you know it is within the order of magnitude?!), the TGR-WP ‘comprehensively’ (line 425) simulated emissions (how do you know? What tells you this?), ‘Although there are many shortcomings, our TGR-WP model can contribute to understanding’ (line 438-440, what makes you conclude this? You just mentioned quite a number of things that were not included in this model. You need more ammunition to make it convincing). The discussion really requires better comparison with available data and much better argumentation. You did a sensitivity analysis. Use this analysis to put your results into perspective!
Additional comments: See text above.


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: CRYPTOSPORIDIUM SPP. AND GIARDIA DUODENALIS EMISSIONS FROM HUMANS AND ANIMALS IN THE THREE GORGES RESERVOIR IN CHONGQING, CHINA
Review round: 2
Reviewer: 1

Output:
Basic reporting: I think this is okay now, except for one comment:

I think you should not just refer to your model as ‘the GloWPa-Crypto model’ (as is done in the abstract), as this is the name of the original global scale Cryptosporidium model. You should make clear your model is an application/adaptation of the GloWPa-Crypto model, not suggest it is the same, as you apply it locally and not only for Crypto. Perhaps a derived name, such as ‘GloWPa-TGR-Crypto’. Or your own name with a clear reference, like ‘our TGR-Crypto-Giardia model is based on the GloWPa-Crypto model [ref]’.
Experimental design: -	You now mention in your discussion that you do a calculation using a hydrological model (lines 416-420). All calculations should be detailed in the methods section and reported in the results section, not in the discussion!! It is unclear now how you did this calculation, and what hydrological information you used. Applying a hydrological model is not something you can mention in one line in the discussion, this is a whole section of your paper at the very least if you actually do these calculations.
-	Vermeulen et al. is not the original reference for a hydrological model.
-	Furthermore, this hydrological model is on a 0.5 degree grid scale, while you calculate on a district level. Unclear how you would combine this with grid-based hydrological modelling.
-	Furthermore, table S11 suggests you also model decay in the surface water according to Vermeulen et al., if this is a calculation you do, it should also be detailed in the methods and results section.
-	I still do not understand what the addition of breeding days in equation 3 means. If you already have the number of animals, their manure production and the amount of (oo)cysts in the manure, then what does it add? What exactly does the number ‘breeding days’ represent? Is that the number of days between litters of offspring? The gestation time? The number of days young live before slaughter? Should be explained.
Validity of the findings: -	I do not have the Matlab sofware, so I have not checked the supplementary files.
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: EXOSOMAL CARGOES IN OSCC: CURRENT FINDINGS AND POTENTIAL FUNCTIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is a nice review from Dr. Zhao et al. that summarizes the current findings and potential functions of exosomal cargoes in OSCC and their potential clinical use.

The topic is interesting and well descripted.
The manuscript is well-written and timely. Moreover, the figures are clear.
Experimental design: The manuscript flows logically.

It is very well referenced, however I would suggest the authors to add Thery 2018 (PMID 28598415) particularly relevant in the context of extracellular vesicle characterization.

I also suggest the Authors to better explain the link between CAV1, exosomes, EMT and fibroblasts (lines 188-189)
Validity of the findings: Arguments are well developed
Additional comments: A great contribute that represents a good source for extracellular vesicles community.


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: EXOSOMAL CARGOES IN OSCC: CURRENT FINDINGS AND POTENTIAL FUNCTIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1)Exosomes in diseases especially in cancer attract great interests in recent years and numerous high-quality reviews have been published. However, the filed focused on oral squamous cell carcinoma (OSCC) has not been reviewed and the present article provided supplemental knowledge in this specific cortex.
2)The manuscript contains numerous counts of unclear, ambiguous, and unscientific English writing, as well as grammatic errors and misspellings.
Experimental design: Article content is within the Aims and Scope of the journal. Sources were adequately cited.
Validity of the findings: Conclusion identify unresolved questions and future directions.
Additional comments: Introduction
The first two paragraphs briefly introduced the classification, biosynthesis, sorting, etc. of exosomes, whereas which were repeatedly reviewed by previous manuscripts. Can be shortened.


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: EXOSOMAL CARGOES IN OSCC: CURRENT FINDINGS AND POTENTIAL FUNCTIONS
Review round: 1
Reviewer: 3

Output:
Basic reporting: - The manuscript required an in-depth English editing, as it is often hard to under the meaning of sentences
- References are fine
- The structure of the manuscript needs comprehensive revisions. Each paragraph has similar structure, which consists in a list of examples from literature for the majority of the text and there is not logic transition between different topics (they seems disconnected, stand-alone sections).
For example, in the paragraph 2: lines 122-145, list of proteins in exosomes, lines 145-167 list of effects of exosome proteins on immune cell functions, lines 168-184 list of effects of exosome proteins on metastasis, lines 185-189 effect of exosome proteins on EMT.
As review manuscript on OSCC, a more in-depth critical review and discussion on the role of exosomes in OSCC should be provided. Critical discussion and in depth description of the biological and cellular mechanisms regulated by exosome cargo are required. A particular focus should be directed to the clinical relevance of exosome cargo in OSCC (specific paragraph).
Experimental design: 'no comment'
Validity of the findings: 'no comment'
Additional comments: The manuscript requires a thorough revision.
The paragraphs need to be reorganized. Their structure requires an elaborated description of the topics with critical comments that are relevant to OSCC. The review manuscript has to deliver important and relevant information on the topic to the reader.


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: SERUM URIC ACID LEVEL IS CORRELATED WITH THE CLINICAL, PATHOLOGICAL PROGRESSION AND PROGNOSIS OF IGA NEPHROPATHY: AN OBSERVATIONAL RETROSPECTIVE PILOT-STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: Dr. Lu et al. studied the relationship between SUA and the clinical and pathological phenotype of IgA nephropathy. They evaluated 208 patients with IgAN  divided in 2 groups, with normo-uricemia and hyper-uricemia  and followed  up them for an average of 25 months. Patients with hyperuricemia had higher mean arterial pressure, increased levels of total cholesterol and triglycerides and lower eGFR. When IgAN biopsies were evaluated using MEST-C score,  the authors found out that patients with hyperuricemia had a higher scores of tubulointerstitial changes, arteriosclerosis, glomerular sclerosis and a significantly lower percentage of fibrocellular crescents. They concluded that the levels of SUA were associated with renal function level and pathological severity of IgAN and could have a prognostic relevance.
The language is  clear and the literature well referenced. .
Experimental design: The figures and the tables are well labeled and described and the results are clearly explained. Methods are sufficiently  described.
Validity of the findings: Several investigations have demonstrated the  detrimental impact of  SUA levels on the progression of IgAN,  could the authors  make clear the novelty of their findings?
Additional comments: I have some questions:
High blood pressure, increased levels of lipids and uric acid are some factors associated with metabolic syndrome.  What about hyperglycemia and insulin resistance?

Have  the patients received UA lowering agents, during the follow-up?

T score of patients with hyperuricemia was higher. This finding could be much more discussed.


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: SERUM URIC ACID LEVEL IS CORRELATED WITH THE CLINICAL, PATHOLOGICAL PROGRESSION AND PROGNOSIS OF IGA NEPHROPATHY: AN OBSERVATIONAL RETROSPECTIVE PILOT-STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The employment of the English language is good in general. although it could be improved.
References are adequately addressed, although the format is uneven.
The structure of the paper is adequate.
The results are tuned to the hypothesis.
Experimental design: The design is appropriate.
However, the power of the sampe has not been calculated for this sort of hypothesis to be proven. A 200-patient cohort is low.
Validity of the findings: The impact and novelty are low actually. I explain this topic to the authors.
Conclusions are well stated, but as the impact is low and lacks of novelty, in my opinion the paper offers nothing interesting.
Additional comments: This paper lacks of novelty. Thence, no high impact is expected.
As the authors hace demonstrated in a small cohort of 200 patients with biopsy-proven IgAN, high uric acid blood levels correate well with the chronic variables assessed. Hyperuricemia is a well-known cardiovascular risk factors for chronic situations are hypertension, left ventricular hypertrophy, hypercholesterolemia, CKD. We already know these findings.
With respect to IgAN, it is not unexpected to find that hyperuricemia is, again, correlated with chronic variables, as tubular atrophy and interstitial fibrosis, vascular damage, ang glomerulosclerosis.
Hyperuricemia is associated with CKD, and is a well-known predictor of renal outcome in CKD, glomerulopathies and IgAN.


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: SERUM URIC ACID LEVEL IS CORRELATED WITH THE CLINICAL, PATHOLOGICAL PROGRESSION AND PROGNOSIS OF IGA NEPHROPATHY: AN OBSERVATIONAL RETROSPECTIVE PILOT-STUDY
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is well written. Please check for mistyping errors (seed e.g. table 1 legend "cells count in per")
References are in part outdated. Morphological measures in IgA nephropathy should be analyzed in terms of modern neprhon number techniques (see e.g. PMID: 31534861 ). The tubular changes also could be interpreted in view of techniques based on fractal analysis (PMID: 29540159). The role of urates could also be discussed taking care of more recent reviews (see e.g. PMID: 29689561).
Experimental design: aims are clear.
Statistics are not adequate: please correct for multiple testing.
Validity of the findings: Findings are interesting
Additional comments: Please clarify in Table 1 notes, what is a "mesangial area"
Table 2: uric acid - please check in parenthesis the units (umol/l or g/l?)
	eGFR: please report in the table the equation used (CKD-Epi or MDRD etc)

Table 3: please verify the number of decimals reported in the table. For example, it is possibly not useful to report two decimals after the age (years) or to describe the weight (kg) or the MAP (mmHg) when the measure itself has a much lower precision

Figure 1 and 3: please use the same layout of Figure 2, reporting the values of each subject rather only the mean


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: SERUM URIC ACID LEVEL IS CORRELATED WITH THE CLINICAL, PATHOLOGICAL PROGRESSION AND PROGNOSIS OF IGA NEPHROPATHY: AN OBSERVATIONAL RETROSPECTIVE PILOT-STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: The language is clear and the literature well referenced.  The figures and the tables are well designed and described.
Experimental design: the results are clearly explained. Methods are sufficiently described.
Validity of the findings: the revised version  strenghtened the findings
Additional comments: The authors mostly addressed my concerns.


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: SERUM URIC ACID LEVEL IS CORRELATED WITH THE CLINICAL, PATHOLOGICAL PROGRESSION AND PROGNOSIS OF IGA NEPHROPATHY: AN OBSERVATIONAL RETROSPECTIVE PILOT-STUDY
Review round: 2
Reviewer: 2

Output:
Basic reporting: The items to be addressed in this box are adequate.
Experimental design: Adequate
Validity of the findings: This topics hace been improved in the new version.
Additional comments: In general, the authors have considerabily improved the quality of the paper after the reviewers inputs.
In their letter to the editor and reviewers, after my comment that the number of patents in low, they comment that this study is "a pliot study", and that they plan to do a prospective trial.Thus, this statement must be addressed in the manuscript. Moreover, the title should be changed to: "Serum uric acid.....: an observational retrospective pilot-study".


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

Input:
Title: SERUM URIC ACID LEVEL IS CORRELATED WITH THE CLINICAL, PATHOLOGICAL PROGRESSION AND PROGNOSIS OF IGA NEPHROPATHY: AN OBSERVATIONAL RETROSPECTIVE PILOT-STUDY
Review round: 2
Reviewer: 3

Output:
Basic reporting: In the revised manuscript the authors have responded to all my previous concerns
Experimental design: The experimental design is appropriate
Validity of the findings: The findings are interesting and will prompt further research about the subject
Additional comments: The revised manuscript presents novel and interesting material that is worth sharing with the scientific community


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 22-AMINO-ACID PEPTIDE REGULATES TIGHT JUNCTIONS THROUGH OCCLUDIN AND CELL APOPTOSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Some more background introduction of occludin will be nice, especially known mechanisms of occludin in cell apoptosis and human fertility.
Experimental design: In statistical analysis of methods, the significant difference level was set as P<0.05. However, in every figure legend, the significant difference level was set as P<0.01. Please correct it.
Validity of the findings: In discussion part, authors explained “The western blot images in this study showed only one band for occludin, and thus, no degradation was found”. This is a completely wrong conclusion. Protein degradation could be affected by many ways, e.g., ubiquitination, SUMOylating, autophagy-related protein degradation, etc. Authors should re-write this sentence or do protein degradation assay to exclude this possibility.
Similarly, in discussion part, author stated “Therefore, SC and spermatid apoptosis might lead to a decrease in occludin expression.” However, at 47D, spermatogenesis had returned to normal, while expression level of occludin was approximately one-third the normal expression level. Why did the expression of occludin not return to normal? Authors should explain this.
Additional comments: In the paper, authors investigated the mechanism of 22-amino-acid peptide in regulating tight junctions through occludin and cell apoptosis. The study gives an interesting new prospective of a mechanism not deeply studied for male fertility. The topic presented is of interest and the data are quite important; however, the manuscript in this form presents several problems.
1. In figure 2A, why did authors use one β-actin for two different groups?
2. From figure 2 to figure3, it is too weird. Authors analyzed the expression of several microRNAs (miRNAs) that can target occluding, but did not show the results. I think that reporting this data is fundamental.
3.  Could miR-122-5p target other structural proteins of TJ?


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 22-AMINO-ACID PEPTIDE REGULATES TIGHT JUNCTIONS THROUGH OCCLUDIN AND CELL APOPTOSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors present the results that provide experimental support for the role of 22AA peptide in SC and spermatid apoptosis and in sperm density regulation. The figures are relevant but need some modifications that is mentioned in later section. I commend the authors for doing a good literature review with a well written introduction section. The experiments are mostly well executed with control; the exceptions are mentioned in general comments. Some minor additional experiments can be done to improve the overall quality of the paper.
Experimental design: The study is of importance and within the scope of Peer J. The research plan is well defined and relevant experiments are performed. Some modifications and addition of better picture can improve the clarity of the paper. Additionally, some minor experiments can be done to improve the overall quality of the paper to meet the standard of Peer J.
Validity of the findings: The study is fairly robust and looks statistically sound. Conclusions are well supported by results except the ones that I have mentioned in the general comments.
Additional comments: This is an interesting study and the strength of the study is the use of in-vivo model system. The manuscript is concise and clear. Although, there are certain topics that need attention as followed.

1. The abstract claims (line 7-8) that distribution of occluding was analyzed by immunofluorescence electron microscopy but I do not see any such data in the manuscript.

2. Fig 1 is a very important piece of evidence but there is some ambiguity in the data. The authors state that they counted sertoli cells and spermatids from this imaging data but the picture shows no specific staining that can be used to differentiate sertoli cells from spermatids. I only see blue staining (nucleus) and red (Bax). I see in the method section, the authors mentioned WT1 and Prm2 immunostaining but no such staining is clearly visible in these pictures (Fig 1). They should use images with better quality and contrast. I would also suggest to add different panels of single stain and then last one with merged. Additionally, The picture shows a single seminiferous tubule, I would recommend to add pictures with multiple seminiferous tubules in one field for each condition.
I would also suggest adding biochemical data to consolidate this imaging result. One experiment that can be performed to measure functional outcome of loss of sertoli cells is to measure Anti-müllerian hormone (AMH) level. AMH is produced by sertoli cells in early developmental stage. If there is loss of sertoli cells, a drop in AMH level would be expected. Considering the importance of this figure, the above mentioned points need to be addressed.

3. For Fig 2 –Fig 4, the figure sub labeling (A/B) can be placed at the left top of each figs.

4. Fig 2 shows quantification of WB result; the details of quantification methods including the software used need to be given in the method section.

5. Fig 3: This result is not really convincing. I see an increase in expression of miR-122-5p from 0 to 0.1! Is this minor increase physiologically relevant? This result can be excluded. The increase shown here is very marginal and the result is not adding any substance to the overall conclusion of the paper.

6. The results show that the effect of 22AA disappeared post 27D but the authors have not given much details of this incongruity in the discussion section. One line is written saying that the degradation of 22AA happens post 27D but I don’t see any data in the manuscript supporting this hypothesis. If any data exists, it should be included or else if the comment is based on previous observation that should be mentioned.

7. In table 1: The table is labeled as ‘Antibody types and sources’ but this table also enlists other reagents like PBS, so I would suggest labeling it as- Table of resources/Source of reagents.

8. Fig 1 legend: Consider changing the title of the legend to make it more objective.

9. There are typos that need to be corrected, some examples are as followed:
Line 42: remove ‘.’ before TJ
Line 60: at the end of the line ‘??’ is there. One’?’ to be removed.
Line 179: add a ‘-’ in t test.


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 22-AMINO-ACID PEPTIDE REGULATES TIGHT JUNCTIONS THROUGH OCCLUDIN AND CELL APOPTOSIS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The study investigates a relevant research question to understand the mechanisms underlying the regulation of tight junctions by 22AA. How 22AA affects the expression and localization of occludin? The manuscript covers appropriate content (literature, description of methodology and results) in specific sections.
Experimental design: Experimental design is well described now.
Validity of the findings: Findings appear to be valid.
Additional comments: Language errors need correction:
In vivo and in vitro should be mentioned in italic font throughout the text.
L33: adluminal
L34: lumen
L42: punctuation error
L314-319, L353-364, L369-373: These lines describe and mention the results which in my view is Repetition of results, it would be more useful to precisely discuss the finding here and further reduce the extensive text in discussion.


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

Input:
Title: A 22-AMINO-ACID PEPTIDE REGULATES TIGHT JUNCTIONS THROUGH OCCLUDIN AND CELL APOPTOSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Thank you very much for your response to my concerns. Well done!


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 22-AMINO-ACID PEPTIDE REGULATES TIGHT JUNCTIONS THROUGH OCCLUDIN AND CELL APOPTOSIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: It looks much better than the previous version. Few minor changes can improve the manuscript further.
Experimental design: Well designed
Validity of the findings: No comments
Additional comments: 1. I would suggest adding some of the short and long term medical consequences of the 22AA mediated effects.
2. Fig 1: Images need to be properly labeled. I would specifically suggest labeling control and 22AA peptide treatment panels.
3. The author should discuss in detail the reason of disappearance of the 22AA mediated phenotypes post 27 days.


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: HABITAT PREFERENCE, MOVEMENTS AND GROWTH OF GIANT MOTTLED EELS, ANGUILLA MARMORATA, IN A SMALL SUBTROPICAL AMAMI-OSHIMA ISLAND RIVER
Review round: 1
Reviewer: 1

Output:
Basic reporting: This paper describes a 2-year mark-recapture study of juvenile and subadult Anguilla marmorata in a relatively small stream in southern Japan. The focus of the work is on distribution, preferred habitat, home range, and age and growth. The findings add to a growing body of ecological knowledge for this and other tropical species of Anguilla, which up to now have been little studied.

The manuscript is relatively well researched and draws on an extensive list of references. Most references are more recent (post-2000); some older but relevant references on other species of Anguilla might be included, e.g. Gunning and Shoop 1962, Haro & Krueger 1991 (see citations in General Comments). The text contains few technical, grammatical, and stylistic errors, and I have relatively few comments/edits. The authors sufficiently review recent research with this and other subtropical Anguilla species.  To my knowledge this research has not been published elsewhere.
Experimental design: The experimental design follows that for a standard mark-recapture study. Tag retention and fish catchability are not quantified, but the tagging methods have been used with juvenile Anguilla before and are probably appropriate. Some of the statistics employed are new to me (e.g. random forest models) but seem appropriate given limitations on sample size, non-normality of data, etc. The authors do a good job of justifying the specific analyses used.

Catchability of eels using backpack electrofishing may be influenced by habitat (i.e., it is generally harder to detect and catch small eels in deep runs or areas with cover with electrofishing), which may have affected results somewhat (i.e., fewer small eels collected in deeper areas). However, there are no data to evaluate this potential effect. The recapture rate of eels in this small system, which seems to be easily comprehensively sampled, is quite small (48 of 339 fish = 14% recap rate; line 322). This might imply somewhat limited catchability using the electrofishing technique. Can the authors speak to this?
Validity of the findings: The study was performed in a lower portion of a relatively small river system only 500 m long; how representative as a general habitat is this for this species? I would imagine larger/longer river systems might have different size/age distribution characteristics. Can the authors make any comparisons between this and other riverine habitats for this species?

Eel density ranged from 0 to almost 0.7 eels/m2. How does this compare to other studies of density of tropical and temperate eels?

My understanding is that adults of this species can grow to a very large size (2+ m total length), but few eels over 600 mm TL were collected. Why were larger/older eels absent from the system? Could the stream size have been too small to support them, or do larger eels prefer other habitat types (e.g., larger rivers)?

The authors mention the potential for competition and habitat segregation of sizes in this river system (lines 436, 528). Is there any additional evidence for this? Are mottled eels the dominant fish species (in terms of number or biomass) in this system? If so, one might also look for evidence of cannibalism (which is common in Anguilla); the authors note that no glass eels and very few very small eels were collected throughout the 2 years of study.

Along these same lines, in temperate eels large runs of glass eels usually invade streams in the spring months and attempt to migrate as a far upstream as possible in their first year. If this is the case for mottled eels, one might expect large concentrations of glass eels accumulating below the waterfall 520 m from the river mouth (albeit perhaps during a very limited period of the year). Can small eels ascend this waterfall? If so, is there suitable habitat above the waterfall? If they cannot ascend, could they be consumed by larger eels or other predators? The authors make the claim that juvenile eels enter the river and establish themselves primarily in the riffle region 100-300 m above the river mouth but have no direct evidence of this. Because they were rarely observed, alternative routes and/or fates of glass eels entering from the ocean into this small stream should also be acknowledged.
Additional comments: Title: capitalize “island” if Amami-Oshima is indeed a specific named island

Line 38: change “main reaches” to “mainstem reach”

Line 57: change “is” to “was”

Line 86: change “most species have not been studied for their freshwater ecology” to "their freshwater ecology has not been studied"

Line 131: “…A. marmorata is highly dominant…”  - does this mean dominant among other Anguilla species or among other fishes in general?

Line 160: provide latitude and longitude of study site

Line 169: sentence not clear; reword

Line 171: state (if known) whether the waterfall is passable by eels

Line 173: omit “to”

Line 173: how long is the total length of the mainstem, including the portion above the waterfall?

Line 179: change “was” to “were”

Line 183: “entire river” – there was no sampling above the waterfall; reword

Line 190: “uppermost reaches - there was no sampling above the waterfall; reword

Line 192: omit "of every sampling time"

Line 201: Few A. japonica were collected; does this imply habitat segregation of the two species? A. japonica is infrequently mentioned in the Discussion; habitat segregation (or not) of the two species could be discussed more there.

Line 233: change “detected” to “quantified”

Line 367: change “was” to “were”

Line 396: I assume the remaining sentences in this paragraph refer to the three eels that made the longest movements.

Line 401: omit comma

Discussion: The Discussion section is rather long, but still interesting. The authors speculate a bit, but perhaps are warranted in this given how little is known for this species. Editor’s call.

Line 467: change “range” to “range of”

Line 551: A home range of 15.1 +/- 26.5 m is pretty small. The collections were made during daytime only, perhaps collecting eels only while in they were in their daytime refuges, which they may have had some fidelity to. Movements a night (e.g. ranging much farther from a daytime refuge) may be much more extensive; the authors should acknowledge this.

Fig. 5; why are no large eel results shown in Figs. 6C and 6D?

Figures: In the interest of visualizing the data, it would be nice to include in the main manuscript a figure for density vs. distance, for both small and large eels, perhaps also showing sediment and bank type (i.e., Fig. S2). I would suggest breaking up Fig 3 and retain Fig.3A as a stand-alone figure, then add a new figure that emulates Fig. S2, adding the predictive value and 95% CI to the rightmost panel of S2. The open circles for A. japonica can be omitted.

Suggested references:

Gunning, G. E., and C. R. Shoop. 1962. Restricted movements of the American Eel, Anguilla rostrata (LeSueur) in freshwater streams, with comments on growth rate. Tulane Studies in Zoology 9:265-272
.
Haro, A. J., and W. H. Krueger. 1991. Pigmentation, otolith rings, and upstream migration of juvenile American Eels (Anguilla rostrata) in a coastal Rhode Island stream. Canadian Journal of Zoology 69:812-814.


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: HABITAT PREFERENCE, MOVEMENTS AND GROWTH OF GIANT MOTTLED EELS, ANGUILLA MARMORATA, IN A SMALL SUBTROPICAL AMAMI-OSHIMA ISLAND RIVER
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is readable, and the study appears to be conducted by experts on the subject matter.
Experimental design: Methods are well described and statistical analyses appropriate. I have several major issues with the Methods section.

(1) Growth: Given the irregular sampling intervals that range 3-14 months, it is challenging to interpret growth rate data. Authors wisely acknowledge the issue on line 582-591, and relegate data to Appendix Figure S5. On Figure S5, it seems growth rates appear to be higher when intervals contain spring, which I suspect may be the season with the highest growth rate. This is a major, if not fatal, shortcoming, which at this point is hard to offer a constructive suggestion.

(2) Home range: I don't think that this phrase is used appropriately in this manuscript, as estimating home range requires location data at finer temporal scales (i.e., kernel density estimates). The phrase in fact is not defined clearly in the manuscript, but it is referred to as "long-term linear home rage", which I take to mean cumulative distances for individuals recaptured more than once. Four recapture events over > 2 years are not simply sufficient for home range estimation, and I advise that authors refrain from using this phrase throughout.

(3) Movement: Authors conclude that movement is limited because 47.9% were recaptured in the same 10m section, but I wonder if the short study area (450m) provides a classic example of missing emigration from the area. The finding that smaller individuals traveled longer distances than larger individuals (Line 384-385) could just a symptom of the issue, where larger, mature individuals moved to the ocean. This would result in detecting only those that stayed in the study area (underestimating long-range movement). This potential criticism is further substantiated by the fact only 48 out of 396 individuals were recaptured, although it is possible that low recapture rates are due to other factors such as morality and detection rates by electrofishing. I suggest that authors discuss the potential impacts of the study design on underestimating movement, and even better, demonstrating why this would not be an issue in this study.

(4)  Electrofishing: I wonder whether electrofishing was effective at capturing animals that burrow into substrates in brackish waters. I have a mixed experience with electrofishing when water conductivity is high, and species like juvenile eels and lamprey ammocetes appear quite elusive. It is particularly problematic when electrofishing capture probability varies by habitat. For example, on Figure 6(A), eel density decreases with depth. Is this because eels are harder to catch at deeper habitats? I suggest authors demonstrate that electrofishing capture probability is not habitat dependent (ideally) or at least report the range of conductivity along the study river.
Validity of the findings: The key take-home message of this manuscript is: "Given the long life, slow growth, strong site fidelity, and size-dependent habitat preference of A. mamorate, the long-term maintenance of diverse natural riverine habitats are important to conserve this eel species (Line 64-66)." This blanket statement (sounds like a cliche) is likely right, but when examining data presented in the paper carefully, I wonder if the statement is sufficiently back up by data.
- long life: true, but not a focus in this paper (no data presented).
- slow growth: yes, there were a couple of individuals that did not grow at all for months, but there were others that grew much faster. What is striking seems growth variation among individuals, not necessarily slow growth.
- strong site fidelity: this is questionable (see Comment 3 above)
- size-dependent habitat preference: Given that large eels were habitat generalists, this applies more to small eels that occupied riffles/runs.

I wonder whether an alternative take-home message, supported more strongly by data in this paper, would be appropriate. For example, authors emphasize many similarities of the giant mottled eel to better-studied temperate eels. Would you be able to say that many conservation strategies that apply to temperate eels apply similarly to tropical eels, etc?
Additional comments: A few other, more minor comments:

- line 211: The total sample reported in Abstract (line 41) and Results (line 312) is 396 individuals. But here, 228 PIT tagged individuals + 105 VIE tagged individuals = 333 individuals?

- line 219: Was there only a single measurement of depth and velocity per 10m section? This should have been taken at 3 or so points across multiple transects per section. At the least, you are measuring meso-habitat characteristics, instead of micro-habitat characteristics, because you cannot exactly know where eels were within a 10m section.

- line 408-412: It is challenging to discern in this study (and many others) whether individuals that moved ended up growing faster, or faster growing individuals decided to move to different locations.


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 MODEL OF POPULATION DYNAMICS WITH COMPLEX HOUSEHOLD STRUCTURE AND MOBILITY: IMPLICATIONS FOR TRANSMISSION AND CONTROL OF COMMUNICABLE DISEASES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is well organized and the methods and results are presented in sufficient detail. However, the presentation of some Figures could be improved (see minor comments below)
Experimental design: The study aims to account for fluid household structure, which is an important topic and of epidemiological relevance. The methods are sufficiently described with the replication code reposited online. Overall, the model structure looks plausible and well executed. I only have a few suggestions that the authors might wish to explore as additional/sensitivity analyses.
-	Dwelling occupancy was randomly chosen according to the set of probability p_i’s, which means some dwellings may have an extraordinary number of occupants due to randomness. Hoewver, this might not happen in reality because people may avoid already occupied dwellings or make arrangements when to use them. In that case, the number of occupants in each dwelling would be more evened out than under the random-allocation assumption.
-	As the authors adopted LHS, it may be informative to show correlations between the parameter values and the outcome (final size/effectiveness) to assess the sensivity of the results to the parameter assumptions.
Validity of the findings: Conclusions seem fair and valid. Analysis was thorough and looks robust enough, although I suggest some additional sensitivity analysis as above.
Additional comments: Minor comments:
-	 L79: please add context of citation. Does Morphy 2007 suggest there is overcrowding (what is definition?) in Aboriginal population?
-	L172-173: I think the representation using a set notation {} here is unclear (a set is usually not ordered). How about explicitly presenting scenarios as a combination of N and H, for example, (N, H) = (2500, 358), (500, 80),… ?
-	L182: ditto; () instead of {}.
-	Fig2: The readers cannot assess if this is consistent with the observed data in Musharbash 2008. Could there be a comparison between simulated and observed distributions (e.g., bars vs red lines)?
-	Fig2c: The x-scales seem random: I would suggest to use the consistent scale as much as possible for comparability.
-	Fig5: The 4 curves and shades overlapping each other makes the figure very difficult to read. Could there be any alternative way, e.g., separating fluid and stable occupancy curves in different panels?
-	L269-271, 274-276: I would say there was a slight difference with 100% effectiveness and not difference with 50% for Baseline and Lower q scenarios according to Table 2. Also, 100% effectiveness may be rather unrealistic given that timely prophylaxis at time of infection of an idex case would be rarely possible, especially when the model considers a remote population where medical resource might not be fully available.


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

Input:
Title: A MODEL OF POPULATION DYNAMICS WITH COMPLEX HOUSEHOLD STRUCTURE AND MOBILITY: IMPLICATIONS FOR TRANSMISSION AND CONTROL OF COMMUNICABLE DISEASES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article passes on these counts, although I have concerns about the accuracy of figures 2 and figure S1, outlined below. Some minor typographical errors have also been identified in the general comments.

An improvement is recommended for figures 2c, S1c . The plot titles indicate the final numbers of core, regular, and on/off residents, but the distribution of cumulative nights stayed for each class would be more interesting. Since each bar represents a unique individual, I suggest colour-coding and grouping according to residential class within each plot.
Experimental design: The paper passes these aspects.
Validity of the findings: Findings appear sound except for figure 2 and figure S1.
1. Figures 2a and 2b seem identical to figures S1a and S1b, respectively. This is strange since figures 2c and S1c are mutually distinct although still very similar. If this is accurate then it requires comment or explanation, and if it is to be expected from the model then displaying figures S1a, S1b is redundant and they should be removed.
2. Similarly, the plot titles in figures 2c and S1c are surprisingly matched even though the plots are not quite. This requires explanation or comment.

In all other regards the paper meets these criteria.
Additional comments: I also make these suggestions/corrections:
1. The tick-labels on the y-axes vary inexplicably between plots in figure S1c. Every multiple of 50 should be indicated on every y-axis, as they are in figure 2c.
2. Page 8/11, line 275, “protected” should be “protecting”, “the core household of a case” would read better as “a case’s core household”.
3. Page 10/11, line 13, “finding” should be “findings”.


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 MODEL OF POPULATION DYNAMICS WITH COMPLEX HOUSEHOLD STRUCTURE AND MOBILITY: IMPLICATIONS FOR TRANSMISSION AND CONTROL OF COMMUNICABLE DISEASES
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: My comments have been fully addressed and I believe the manuscript is ready for publication.


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

Input:
Title: A MODEL OF POPULATION DYNAMICS WITH COMPLEX HOUSEHOLD STRUCTURE AND MOBILITY: IMPLICATIONS FOR TRANSMISSION AND CONTROL OF COMMUNICABLE DISEASES
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The paper is in a much better state and is much more readable.
It is ready for publication after a missing space is inserted before CI in "95%CI" on line 9 of the caption for figure 2.


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: ANTIMICROBIAL RESISTANT GENE PREVALENCE IN SOILS DUE TO ANIMAL MANURE DEPOSITION AND LONG-TERM PASTURE MANAGEMENT
Review round: 1
Reviewer: 1

Output:
Basic reporting: The aim of this study was to determine how the combination manure and litter application along with 3 different grazing procedures affects AMR gene levels in soil. The study is well justified in that there are few studies highlighting the effects of pasture management on AMR gene abundance. The figures are mostly effective, but a few modifications are necessary. The sequences are available on NCBI based on the links given on the website, but the links and accession numbers are not in the text.

Fig 1 explains the experimental design clearly and was helpful to reference while reading through results.
Fig 2 is also well labeled, except number of samples for each mean abundance (n) should be specified. It would be preferable to achieve a similar resolution as Fig 1 and 3, where there’s no obvious pixelation.
Fig 3 is clear, but it could be a helpful reminder in the legend to note how many of each of the soil samples (n) were used for shotgun sequencing.
Experimental design: The use of 3 different pasture management practices allows for a comprehensive study and were described well, especially in fig 1. Sampling the soils and the manure and litter was informative, but would have been stronger if performed in the same year. A few more details in the stats and shotgun metagenomics are needed for clarification.

Lines 183-185: Was Tukey’s HSD also applied for multiple comparisons?
Lines 188-191: For clarification, which time point was chosen for CG and H, and was a pre- and post- application soil sample chosen for RBR and RBS each? It could be clearer to just list explicitly what the 6 samples are (for example, post-application zone 1 CG, post-application zone 1 H, post-application zone 1 RBR, etc.)
Lines 200-202: Is a positive hit 85% of the query read aligns with a subject AMR gene, or is a positive hit 85% of the subject AMR gene aligns with the query reads? Does the 85% refer to coverage or identity? What is the minimum length of a read?
Validity of the findings: There is an interesting story in the results, especially with the interaction effects, but more statistical methods need to be described to verify the analyses for the QPCR data. For example, how were the multiple comparisons made, and what were the detection limits for each of the genes? For values not detected for certain genes in the samples, were those values excluded, or were they substituted with a number at or below the detection limit?

Lines 215-216: It would be clearer to note the units after the numbers. Rather than listing the mean log gene copy numbers measured from all the treatments, it could be more informative in the text to state by how many logs the grazing treatments increase the ermB compared to RBS. Same comment with lines 217-218.
Line 219-220: If there are no significant differences between treatments, it is necessary to state the means or log difference of those treatments.
Lines 233-235: These statements as they are structured appear like CG has significantly the greatest abundance of the 3 genes compared to the other treatments, but then the figure has only the star to indicate that RBS is significantly the least abundance of the genes. If CG is also significantly greater in gene abundances than the other grazing methods as well, it would be clearer to change the stars in the figure to connecting letters. Or if it’s a more interesting story, the text may also be reworded to note that the grazing methods that were coupled with manure and litter application were all significantly greater in gene abundances than RBS by over x log. This comment applies to all 3 genes and the other 2 factors.
Lines 242-244: This paragraph describes detection and non-detection more than the abundance of each gene. This paragraph would be more useful in the beginning of the results to help give context of how many samples were used for comparisons by ANOVA.
Lines 245-247: Remove this sentence fragment and just skip to “Among these 93 samples…” and then use the absolute number of samples instead of the percentages in the list.
Line 250: change “ collected following” to “, one following”.
Line 251: insert “one” between “and” and “in”.
Lines 253-265: This paragraph is very interesting in that it highlights the main story told in the Q-PCR data, but it also seems redundant. If the first 3 paragraphs could be reframed as suggested in an earlier comment, this paragraph could be excluded.
Lines 280-281: How were the abundances of sul1 and intI1 not significantly different between years when they were detected in one year but not the other? The statistics section of the methods also needs to have a statement of how non-detectable values were incorporated into the analysis if this is the case.
Lines 285-289: These numbers are already listed in the table, so these sentences can be omitted.
Lines 323-324: In general, specific CTX-M genes can be difficult to detect using PCR methods since many similar genes are hybrids of 2 or more other CTX-M genes. In the future, Birkett et al. 2007 could be a good resource for detecting a range of CTX-M genes.
Lines 342-345: This citation is a useful reference when discussing the importance of many classes of AMR genes in soil and the connection to human pathogens, but it does not connect well to the point that blaCTX-M-32 was not detected in soils in this study. Are there other studies that point out blaCTX-M-32 in soils or manure?
Line 392: Bacteria from soil samples?
Line 423: While interesting to point out, this is not pointed out in fig 2, but using letters instead of the stars would help depict this. Did the RBR practice also result in greater gene abundance than H as well since cattle manure was used here too?
Table S1: The legend appears missing.
Additional comments: The use of “AMR genes” and “ARGs” seems to be used interchangeably, but it looks like “AMR genes” is used more often, so for consistency, use only one term.


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: ANTIMICROBIAL RESISTANT GENE PREVALENCE IN SOILS DUE TO ANIMAL MANURE DEPOSITION AND LONG-TERM PASTURE MANAGEMENT
Review round: 1
Reviewer: 2

Output:
Basic reporting: - Line 30: typo on Antibiotic resistance- please fix.
- Line 40 (Abstract): What type of residues? If not focusing on this in the study, it would probably be better to remove, or if not, to clarify what type of residues the authors are referring to.
- Line 58-59: Saying as a conclusion in the abstract that poultry litter may minimize AMR genes might be a stretch given what this study showed. I would suggest removing that idea.
- Line 64: AMR is defined as antibiotic resistance, but usually AMR is referred as antimicrobial resistance. I would suggest defining AMR as antimicrobial resistance.
- Line 71: on what type of soil was that application? I would suggest adding that information.
- Line 72: Please add a citation for 'ARGs can be found naturally'.
- Line 79: un-degraded is one word: undegraded.
- Line 83: Here, the authors talk about poultry litter for the 1st time but there were no previous studies in the background/Intro section that referred to poultry litter and ARGs. It would be nice to have some context about poultry litter the same way the other manures were mentioned.
- Line 97: any specific manure? If so, I would suggest being specific in the aims about the type of manure the authors evaluated.
- Line 643 (References): typo on the reference re: the word 'antibiotics'.
Experimental design: - Lines 105 and then line 109: It is a bit confusing to first read the experiment had 9 watersheds and then refer to only 3 watersheds in line 109. I would suggest clarifying this.
- Line 115: Please provide a reference to 'the best management strategy'.
- Lines 119-123: This portion is a bit confusing. I would suggest either removing some of it if it is not relevant, or adding a bit more detail to the figure to show what it means by shoulder, upper, etc.
- Line 127: Did this commercial farm give antibiotics to the broilers? That would be good to mention, and if known, it would be ideal to mention which specific antibiotics.
- Line 131: I would suggest removing the plural in 'collections'. The sample collection section could be reworked a little bit to be clearer about the type of samples, timing, and number of samples of each type.
- Lines 135-136: Please clarify if the soil samples were taken manually or with a robot of some sort.
- Line 140: Please clarify what the authors mean by in-house piles.
- I am a bit confused about the timing of sampling of poultry and cattle manure. Why was the cattle manure collected in 2018-2019 and poultry in 2016-2017?. Also, it is unclear how many times cattle manure samples were collected.
- Line 150: when the authors say "cattle manure, or poultry litter..."- so not all samples were analyzed the same way? Or should that 'or' be 'and'?
- Why were these four genes in particular chosen? Some explanation related to the decision behind choosing these genes would be good.
- Line 165 is redundant. I would suggest removing it.
- I appreciated the authors giving lots of details on the steps from the qpcr and the manipulation of the data. It is not as common to find all these details. This makes it more transparent and reproducible.
- Line 184: is the log transformed data referring to each of the genes? I would suggest explaining this/expanding on it. Was each one of the 4 genes analyzed used as a dependent variable in the analyses, or were the 4 genes analyzed altogether?
- Line 189: Why were those 6 samples chosen for the metagenomics analyses? Some explanation about this would be good.
Validity of the findings: - The results section is a bit long. I would suggest the authors trying to summarize the results further. Some suggestions below.
- Figure 2 has low resolution- the text is a bit blurry. Also, I could not see in the figure the A, B, C for each panel of figure 2. Also, I am a bit confused about the quantities mentioned for each gene and each part of the figure. In the tex, the quantities are referred to as micro log gene copy numbers per dry weight while in the figure the y axis is log only and it is hard to know the quantities exactly only inferred by the figure. Please clarify and be consistent with the text and figure.
- Lines 257-258: "The gene...": Please reword/fix this sentence.
- The paragraph that goes from line 253-265 can probably be integrated in the previous results. Some of the information is redundant and it makes the results too long.
- Supplementary Table 1 would need a repeated heading as it extends beyond one page.
- Line 271: I would suggest removing the word 'these'.
- Line 277: Here please clarify the units again as mentioned before for the micro gene- is this log or gene copies and in micro?
- Line 296: How was multi-drug resistance defined? How many resistance genes?
- Figure 3. Please capitalize betalactams to be consistent with the other headings.
Discussion:
- Line 305: Please rephrase "AMR genes is an evolutionary phenomenon" to something like " AMR, including AMR genes is a naturally occurring phenomenon", something along those lines.
- Line 325: add ' of' before the word ' AMR genes.
- Line 326: add either 'to' or 'in' before 'fertilized soils'.
- Line 343-344: I do not understand this sentence. There seem to be two ideas- finding the ctx-m and beta-lactamase genes and that they might be the same as the ones found in human pathogens. But finding these in soil does not necessarily imply they are in human pathogens. Please clarify the ideas.
- Somewhere in the intro and/or discussion it would be good to mention if these experimental watersheds receive the input potentially from other sources- human or other animals, etc.
- Line 354: Please also make a point to comment about the use of antimicrobials in your experimental design under your comparison sites and when talking about the animals.
- Line 361: remove the word 'that'.
- Line 361- 362: Given only 2 years of data, I would suggest not making strong conclusions about the differences related to the temporal component. I think more years would be needed to compare that factors.
- Lines 376-380: Please rephrase this section.
- Lines 390-397: This paragraph should be better integrated into the results of the experiment. It seems like floating there with two different ideas but are not well connected to the previous paragraph.
- Lines 400-403. Please connect the idea about the use of beta-lactam antibiotics and the finding of beta-lactamase gene in your experiment., As it is, it reads as two different ideas not well connected.
- Line 416: change 'characterize' for 'characterized'.
- Line 418: remove the word 'these'.
- Line 421: This idea of live and dead bacteria is the 1st time it appears. I would suggest talking about this during the discussion, or removing it if not relevant.
- Supplementary Table 2: Please try to make the table more consistent. For example, Reason for the drug used includes deworm, parasites- what does it mean parasites vs deworm?; for Amount, also include route of administration and duration if that information is available.
Additional comments: This study presents an interesting contribution related to the potential dissemination of antimicrobial resistance (AMR) genes in the environment, specifically in soil. There is still a need for more studies evaluating AMR in soil. I have made specific suggestions and comments for this section. Overall I feel the paper can be more concise. I think once the concerns have been addressed, this paper will be worthy of publication and will add an important contribution to the field.


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: ANTIMICROBIAL RESISTANT GENE PREVALENCE IN SOILS DUE TO ANIMAL MANURE DEPOSITION AND LONG-TERM PASTURE MANAGEMENT
Review round: 2
Reviewer: 1

Output:
Basic reporting: The grammar improvements have made this manuscript a smoother experience to read, and their conclusions indeed make sense given their data and past research. Most of the suggestions here are minor, but figure 2 still needs improvement.
Line 100: insert “to” before “balance”.
Line 104: insert “to” before “evaluate”.
Figure 2: The boxplots are much more informative than the bars from before. However, differentiating between the stars indicating significance and the dot plots that indicate the outliers is difficult visually. It would still be more informative to include the exact results of the Tukey’s differences in the figure. JMP is usually pretty clear with providing connecting letters to indicate exactly which pairwise comparisons are significant, and the letters would also be easier to see than the stars.
Table 3: For the 3rd column, is this the mean quantity ± standard deviation?
Experimental design: Lines 149-150: When was poultry litter applied? Just wondering how much time passed between application of litter and sampling.
Lines 183-184: Thank you for including how you worked with values below detection. What were the detection limits per gram of soil?
Lines 208-210: Thank you for indicating that the 85% referred to coverage. For the queries that aligned with the subject AMR genes, did they have a minimum %identity?
Validity of the findings: Line 211: “pre-“ and “post-“ should replace “pre” and “post”
Line 212: I would change “detection” to “abundance” since ANOVA is really comparing the means between the pre- and post-litter application, so all the values that the test is comparing are detected
Line 248: I would change “abundance” here to “detection” in this case, or “proportions of detected to non-detected samples”
Line 302: insert “,” after phenomenon
Line 336: Change “found” to “detected”
Line 343-344: I would just say ctx-m-32 wasn’t detected rather than there was no difference between the manure and litter, or even omit that fragment since it was already stated in the paragraph above
Lines 361-362: It would be preferable to reword “relies” here since it’s not the only way to characterize AMR genes. Culture-independent methods are vital for many AMR genes from organisms that are hard to culture, but culture-dependent methods also have importance in AMR research (McLain et al. 2016, JEQ)
Lines 377, 379-380: Jacobs et al (2019, JEQ) could be a valuable reference here regarding AMR gene movement via storm water runoff
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: ANTIMICROBIAL RESISTANT GENE PREVALENCE IN SOILS DUE TO ANIMAL MANURE DEPOSITION AND LONG-TERM PASTURE MANAGEMENT
Review round: 2
Reviewer: 2

Output:
Basic reporting: I feel the authors improved their background and justification of the paper. It now reads more clearly.

Minor comments:
- Line 64: typo before the word 'fundamental'- a instead of an.
- Line 101: I think there is a missing word between effort and balance. Please check.
- Line 115: Materials and Methods
- Line 204: it would be"using Tukey’s honestly significant difference test"
- Line 220: Correct Bos taurus
- Line 271: Please correct/modify the x before zone. Same for lines 283-284. Please express this using words instead of the x.
- Line 314: typo in applications
- Line 318: add 'the' before lowest

Figure 2. I would suggest saving this figure in higher resolution to see better (unless this was something from the pdf review and it has better quality than what it looks like). Also, please include either a legend about the colors used that differentiate each group in each panel (A, B, C) or else add what the colors mean to the figure legend text.

Figure 3. Please define acronyms like MLS in the legend.

- Line 438-439: Please try rephrasing this sentence to remove "Take the.."
- Line 453: Please check the 'from' and 'in' together.

If possible, it would be nice to see in the suppl. materials the specific AMR genes that were found through sequencing for each antibiotic class, or a link to the data.
Experimental design: The research question and goals are more clearly defined. The methods read much better now, and I have no further concerns for this section.
Validity of the findings: No further comments.
Additional comments: The authors improved the manuscript based on the reviewer's comment, and I understand how much work this is, so I have only minor comments for the authors at this point.