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: SPECIMEN ALIGNMENT WITH LIMITED POINT-BASED HOMOLOGY: 3D MORPHOMETRICS OF DISPARATE BIVALVE SHELLS (MOLLUSCA: BIVALVIA)
Review round: 2
Reviewer: 2

Output:
Basic reporting: As I mentioned in my previous review, this work is clear an unambiguous, well written and with a correct language. This reviewed version answered my questions and comments and expand some previously confussed points.

Separation between Results and Discussion, and expansion of the discussion about the extension of the methodologies are satisfying developed. Added sentences and references contribute to explain the methodologies and usages, and help to understand the topic for a wider audience (and add relevance to this research).

The new version is more concise and more complete and the figures now acompannied better to the main text.
Experimental design: I have not new comments about experimental design.

Sentences and explanation given to this topic are clearer than the previous version. I am happy with this new version.
Validity of the findings: As I mentioned in my previous review, this research shows novel results, referred to a known problem for whose use morphometrical tools with frequency (a broad distributed field). So, the conclusions are very well established.
Additional comments: Modifications suggested were well answered and new addition contributes to a more clear manuscript. I agree with the responses to all my comments or suggestions.

Because the subpanel labels on Fig. 1 I was able to more easily follow the flow of the manuscript. Thanks for this addition.

Expantion on abbreviatures of museum collection are correct, but the Table S1 have more abbreviatures than the mentioned in the Acknowledgement section. My previous missing comment (sorry for this error) in Table S1 indicates this point. May be the Table S1 could provide these data. Collection material is the primary basis of this study in my opinion, and the proposed and discussed methodologies are very useful for future collection-based research.

New section "Applications to other model systems with accretionary growth" adressed with my previous comment about the extension of the problem and dicussion. I was happy to read it.

Newly detailed rephrased references in the text to Comparative Morphometrics instead Geometric Morphometrics approach seems to me a very good assess to this work, expanded and illustraiting in a better view your work.


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

Input:
Title: IDENTIFICATION OF ALUMINUM-ACTIVATED MALATE TRANSPORTERS (ALMT) FAMILY GENES IN HYDRANGEA AND FUNCTIONAL CHARACTERIZATION OF HMALMT5/9/11 UNDER ALUMINUM STRESS
Review round: 1
Reviewer: 1

Output:
Basic reporting: I think the Figure legend is too simple, some key points should be given, e.g. in Figure5, the Al concentration and how the treatment was conducted.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The manuscript by Qin and colleagues describes 11 ALMTs from Hydrangea, their physicochemical properties, phylogeny, conserved motifs and protein structure were analyzed. Moreover, three members were selected to study their Al tolerance in yeast. To some extent, these results help us to improve recognition of ALMTs from Hydrangea.
The manuscript could be improved by addressing the following points:
Major Concerns:
In M&M, lines 106-109,
1) Why 15 mM Al2(SO4)3 was used? Generally, the plant growth will be inhibited at micromolar level of Al3+. So, the author should cite related literature or give the reasons.
2) Al3+ will complex with some anions (e.g. PO4, OH-, SO4, EDTA…) in Hoagland’s nutrient solution at pH 4.5. What is the Al ion activity in Hoagland solution? The author should calculate it by GEOCHEM-EZ.
3) Line 229-230: HmALMT7 was up-regulated in flowers? The error bar is too large for HmALMT7 expression in flowers (also HmALMT9 in leaf, HmALMT8 in CK root). The qRT-PCR experiments (HmALMT7, HmALMT8, and HmALMT8 expressions in Figure5) should be redone. I also suggest the author makes an analysis of variance between CK and aluminum stress (Figure 5).

Minor Concerns:
1) Line31, ‘tissue specific’ should be ‘tissue-specific’.
2) About the writing format of ‘ALMT gene family’, sometimes ‘ALMT gene family’ (e.g. line 35, line 67, line 72, line92, line213…), sometimes ‘ALMT gene family’ (e.g. line90), please check the whole MS.
3) Lines 66: the sentence should be ‘the secretion of malic acid and citric acid was controlled by aluminum-activated malate transporter (ALMT) and multidrug and toxic compound extrusion transporter (MATE), respectively.’
4) Line73: ‘scALMT1’ should be ‘ScALMT1’.
5) Line 188: only one gene (HmALMT11) in Cluster III…, the word ‘gene’ should be omitted.
6) Line212: …under Al Stress, ‘Stress’ should be ‘stress’.
7) Line214-215: ‘…leaves, stems and flowers harvested from the control and treated plants’ (refer to Figure 4). What treatment has been done?
8) In Figure 5, in order to be consistent with the whole manuscript, I suggest the author replaces aluminium with aluminum.
9) Line281: The right parenthesis symbol is missing.
10) Lines 287-288: HmALMT2 was significantly up-regulated in the roots than other tissues? According to Figure4, HmALMT2 was significantly up-regulated in the stem rather than roots, please check.
11) Lines 290-291: only one gene (HmALMT2) was uniquely up-regulated in roots and down-regulated in other tissues after Al treatment (see Figure 4). Figure4 or Figure5?
12) Line 303: ‘In a conclusion’ should be ‘In conclusion’.
13) The file named ‘Supplemental_Table_S3’, however, the title in this table is ‘Table S2 The information of 3D structure prediction’. Please check.


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: IDENTIFICATION OF ALUMINUM-ACTIVATED MALATE TRANSPORTERS (ALMT) FAMILY GENES IN HYDRANGEA AND FUNCTIONAL CHARACTERIZATION OF HMALMT5/9/11 UNDER ALUMINUM STRESS
Review round: 1
Reviewer: 2

Output:
Basic reporting: In the manuscript entitled “Identification of Aluminum-Activated Malate Transporters (ALMT) family genes in Hydrangea and functional characterization of HmALMT5/9/11 under Al stress” (#71046), the authors identified 11 candidate ALMT family members from the transcriptome data in hydrangea. The amount of data analyzed is large and the results obtained are abundant.

I believe that this article can meet the requirements published in PeerJ after major reversion.
Experimental design: well
Validity of the findings: no comment
Additional comments: 1.	The organization of paper writing needs to be further improved.
2.	Some normative writing needs attention, such as Table 1: PI should be pI, and italic.
3.	Line 56, Reference formats need to be proofread, e.g. Bernhard Wehr et al. 2003; Line 70, 70 Ligaba et al. 2006; Ligaba et al. 2012 should be Ligaba et al. 2006; 2012.
4.	The conditions for plant growth are not well defined, and need to be further defined.
5.	Figure 6 lacks statistical analysis.
6.	Overall, major text in the discussion looks fine. However, a better discussion will help improve the manuscript.
7.	Reference needs further examination. Retain the relevant and recent literature/report.
8.	Before being considered for publication, the manuscript will be corrected by a fluent English speaker, alternatively please use one of the commercial English language editing services available.


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

Input:
Title: IDENTIFICATION OF ALUMINUM-ACTIVATED MALATE TRANSPORTERS (ALMT) FAMILY GENES IN HYDRANGEA AND FUNCTIONAL CHARACTERIZATION OF HMALMT5/9/11 UNDER ALUMINUM STRESS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In this Ms, the authors have answers all questions carefully. However, some questions should be addressed:
Minor Concerns:
1) In M&M, why the plant Al treatment was conducted in soil with 15 mM Al2(SO4)3  irrigation rather than hydroponic culture? Generally, the hydroponic conditions are stable and easy to control.

2) In your response, the final pH of Hoagland solution after adding 15mM Al2(SO4)3 is between 2.5-3. The solution is strong acid. In the MS, Line 106-107, Hoagland solution (500 ml per pot) with or without 15 mM Al2(SO4)3 was used to irrigate…, Line 214-215, the control [Hoagland solution without Al2(SO4)3]…. My question is the pH of the control treatment?  2.5-3? 4.0-4.5 (usually used for Al treatment)? or normal pH (~5.8)? The author should indicate this in M&M part. Similar question is presented in Line 234, …grew better than control (pYES2.0) on SG-U medium. The possible effect of pH on the phenotype/growth should be excluded.

3) I note that the expression of HmALMT8 in Al-treated root is nearly 8-fold compared with CK in previous version, it markedly declines (less than 2.5-fold) in the revised version (Figure 5). It varies greatly between the two versions. What are the possible reasons?

4) Line 226, Line 289 OsALMT7 and -9, Line 297, the ‘and’ should not be italic.

5) In reference lists, line 327, the ‘Vitis vinifera’ should be italic.

6) Table 1 (including the Note below the table): PI should be pI, and italic.


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: IDENTIFICATION OF ALUMINUM-ACTIVATED MALATE TRANSPORTERS (ALMT) FAMILY GENES IN HYDRANGEA AND FUNCTIONAL CHARACTERIZATION OF HMALMT5/9/11 UNDER ALUMINUM STRESS
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: no comment


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

Input:
Title: IDENTIFICATION OF ALUMINUM-ACTIVATED MALATE TRANSPORTERS (ALMT) FAMILY GENES IN HYDRANGEA AND FUNCTIONAL CHARACTERIZATION OF HMALMT5/9/11 UNDER ALUMINUM STRESS
Review round: 3
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors answer all questions I concerned very well. I think the manuscript should be accepted for publication in PeerJ.


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

Input:
Title: DELETION OF TWO-COMPONENT SYSTEM QSEBC WEAKENED VIRULENCE OF GLAESSERELLA PARASUIS IN A MURINE ACUTE INFECTION MODEL AND ADHESION TO HOST CELLS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article is wordy and contains many grammar mistakes. The abstract has many spelling and grammar mistakes and should be reviewed. In the introduction, there is a whole section that is repeated (line 69 to 81 is identical to lines 59 to 69). Introduction should be reorganized and focused on the subject of the study.
Review M&M section and turn it more readable. Some section of M&M seems to be copied from laboratory protocols (mainly section 2.4, 2.6, 2.8 and 2.9) and are hard to follow. Therefore, the reader might get confused about what exactly has been done. These sections should be carefully rewritten.
Excess of information of equipment could be removed. The “natural transformation technique” indicated on line 119 and 124 should be described.
Results section should be shortened to indicate the results. Keep discussion for the discussion section.
Discussion.
Please start with your main finding and discuss the meaning of your data considering how they altered G. parasuis pathogenicity. Start at line 442. Everything prior to that (e.g lines 401 to 441) might be incorporated on your discussion as you go through your finding. Otherwise, most of it could be removed.
Experimental design: Satisfactory. M & M section could be improved.
This reviewer could not find the tables with the description of the primers used in the experiment.
Validity of the findings: Result are novel and add valuable information to current knowledge.
Additional comments: none


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: DELETION OF TWO-COMPONENT SYSTEM QSEBC WEAKENED VIRULENCE OF GLAESSERELLA PARASUIS IN A MURINE ACUTE INFECTION MODEL AND ADHESION TO HOST CELLS
Review round: 1
Reviewer: 2

Output:
Basic reporting: English needs to be improved. Many grammatical and spelling mistakes. Many sentences need proper reframing. Some sentences in the text are repetitive
The quality of the figures is bad. No resolution. Legend key needs to a full explanation of figure. Some figures does not reflect in terms of what authors report (microscopic images).
Experimental design: Methods need proper explanation
Some images quality is really bad
Some of them I don't see any difference what authors are saying
Validity of the findings: N/A
Additional comments: Authors need to analyze data properly. The quality of the figures is bad. Microscopic experiments need to be redone. I don't see any difference in the Wt vs mutant strains. Need to cite proper references. English needs to be improved, it does not read like publication quality.


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: DELETION OF TWO-COMPONENT SYSTEM QSEBC WEAKENED VIRULENCE OF GLAESSERELLA PARASUIS IN A MURINE ACUTE INFECTION MODEL AND ADHESION TO HOST CELLS
Review round: 1
Reviewer: 3

Output:
Basic reporting: This is a very well designed project and have reported some very interesting findings on the role of QseBC in bacterial virulence. I like how the authors presented their findings in a step-wise manner and ensure the logics is correct.

However, the authors can use a lot of help on their language editing and proof reading. The manuscript's current writing has many grammatical flaws and the flow of some sections are not correct. I highly recommend the authors to invest in a good editor to re-write the manuscript so that it's in professional English.

One example:
the 3rd paragraph of Introduction is not well written: there is no intrinsic flow within the paragraph and content is repetitive (e.g., row 59-61 and 69-72, there are many more repetitive sentences in this paragraph)
Experimental design: Overall good and logical design.

Normally method section should be written in passive voice for reporting purposes. The manuscript currently has the method and approach written as instructions in many places. Please have a scientific editor to rewrite this section to ensure the readers are not confused with the methods
Validity of the findings: The findings are interesting and the authors provided both in vitro and in vivo data to support that QseBC is crucial for bacterial virulence.

However, as with many other sections, the result section needs to be re-written and properly proofread.

Two minor comments on the figure:
- Figure 4A: the lines are hard to read with the same color and similar pattern. Suggest using different color or add different shapes (solid circle, solid triangle. Empty triangle) to data points so that the distinction is clear
- For immunofluorescence images, I would recommend the author to increase the contrast so that the DAPI  and CY3 staining comes out more clearly. Currently it is a bit challenging to see.
Additional comments: I want to emphasize that this manuscript has very high potential. Understanding the English is not the native language of the authors, I want to make sure that great academic work is not punished by linguistic limitations. Please have a professional editor rewrite the manuscript and ask a few international colleagues to quickly proofread before submitting again.


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: CHARACTERIZATION OF LC-MS BASED URINE METABOLOMICS IN HEALTHY CHILDREN AND ADULTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Raw data not shared, nor a link provided in the manuscript

General:
•	It would be more "correct" to use the term "sex" instead of "gender" since the term "gender" (esp. in today's culture) is used more broadly to denote a range of identities that do not necessarily correspond to the established ideas of male and female.
•	The authors consider consistency in terms of word-use throughout the manuscript, since they use the word “sex” in line 51+103, but “gender” elsewhere. This should be reviewed for other wording choices as well: “primary school” (lines 81, 210, 235, etc.) vs “second school” (line 449)
•	“geriatric” is a better word choice than “old”.
•	Define at first mention (blue highlight): BMI (line 51), TCA (line 54), TOF (line 57), HRLC (line 69), FDR (line 130), MSI (line 142); 15d-PGJ2 (line 408)
•	Avoid personal pronouns/terms like “we” (line 58).
•	Please see the PDF edits for some minor grammar/linguistic corrections: lines 23–25, 29, 47, 81, 162, 189, 237, 251, 278, 279, 283–284, 329, 340, 346, 359, 363, 368, 403, 406, 421, 425, 435, 451.

Abstract:
•	The authors state “…gender-dependent urine metabolites are much greater in adults than in children” (lines 25-26), but this is in contrast with their summary which states “…urine metabolites showed larger gender differences in children than in adults” (no line numbers available, page 4)
•	Please confirm the age of the adult cohort. In the summary the authors state “…aged 20-78”, but in the abstract (line 24, and also line 67 in the introduction) it is “…70”.

Introduction:
•	The authors indicate stated what knowledge gap in the field they aim to fill, but should elaborate HOW this would be useful for, for example, future disease biomarker discovery? (line 74)

Methods:
•	S2.4: The need for missing value replacement, transformation, scaling and a 50% filter needs to be referenced (lines 125-126). The authors might consider the work of Luies and Loots (DOI:10.1007/s11306-016-0969-x) who describes all of these approaches.
Experimental design: Methods:
•	S2.1: How were the children informed on the study, in which case parents/guardians need to approve participation?
•	S2.1: Were there any incentives to participate, financial perhaps?

•	S2.2: How were the 50 samples used to compile the QC selected? The authors state that these were representative across the groups (line 98), does this mean that 8-9 samples were RANDOMLY selected across the six groups (mentioned in lines 81-82)?
•	S2.2: Analysis was done over 12 days, how many batches were involved? How were these batches comprised, e.g. did they represent the entire cohort of various ages?
•	S2.2: Did they analysis include ‘leading QCs’ (e.g. 3-5 injection prior to the first samples daily to ‘warm up’ the LC and thereby avoiding potential batch effects?

•	S2.3: Why was positive ESI mode selected? Did the authors also consider negative ESI mode?

•	S2.4: Please state the software version used (line 121).
•	S2.4: The need for missing value replacement, transformation, scaling and a 50% filter needs to be referenced (lines 125-126). The authors might consider the work of Luies and Loots (DOI:10.1007/s11306-016-0969-x) who describes all of these approaches.
•	S2.4: Referencing is needed for lines 127-133 that actually describes these statistical approaches, should the reader be interested.
•	S2.4: The sentence starting in Line 133 (“100 permutation tests were used to…”) needs to be rephrased as sentences should not start with a number. Consider “Permutation tests (n=100) were used to…”
•	S2.4: Please confirm that the symbol in line 136 should not be greater than OR EQUAL TO 2, instead (≥)
•	S2.4: The description of “VIP” (line 136) needs to be corrected to “variable importance in the projection” (this does not stand for variable importance plot).

•	S2.5: “…and imported to MS method for targeted data dependent analysis” (line 142) is confusing and needs to be rephrased. Is there perhaps a missing word(s) here?
•	S5.2: What percentage isotope similarity (line 146/147) was considered sufficient?

•	General: The conclusion makes reference to batch effects being an issue (lines 446–447), how was this corrected in the current dataset? There is no mention of this in the methods section.
Validity of the findings: Results:
•	S3.1: How dit 663 samples result in 730 injections; were 67 QCs injected?
•	S3.1: Principle component analysis was abbreviated as PCA in line 131. This abbreviation should be used in line 162.

•	S3.2: The first 2 sentences of this section should be switched (lines 167-169).
•	S3.2: Was the PLSDA not used as well?
•	S3.2: The terminology (males vs boys; females vs girls) can cause some confusion. For example, do the authors refer to females as an entirety in line 179? In such case, they should clarify and denote this as "female/girls". If not, the terminology is correct as is. Also consider the same question for “males” in line 181.
•	S3.2: Can the authors elaborate on what other type of environmental factors they refer to in line 196?

Major comments:
The data interpretation is scattered between Sections 3 and 4, making this somewhat repetitive, with a lot of ‘jumping between sex vs gender trends’. The Results section should simply list the results only (what the data showed based on what stats they did), without ANY explanations provided for any of the metabolic changes. This would be Sections 3.1–3.3 only, which still need to be re-evaluated/adapted to remove any explanations (e.g. lines 194–197; 226–227). In Sections 3.4–3.5 the authors started to explain some of their findings (e.g. lines 238–255). This should all form part of the Discussion section; hence these type of discussions need to be moved to/combined with the Discussion section. Consider the following feedback for all of these sections in light of such a restructure:

•	S3.4: The first 2 sentences (lines 231–234) are redundant and repetitive. It should be removed and/or only mentioned in the Results section.
•	S3.4: (line 242) The authors can exclude either Fig 4 or Table 2 from the manuscript, since these both give the exact same information — the reader would not miss out on any NEW information in the absence of one of these. Since Table 2 is easier to interpret for a reader who doesn’t understand heat maps, I would recommend removing Fig 4. (This Pathway Analysis part can remain part of the Results section, leaving the rest of this interpretation/discussion for Section 4) — Combine S3.4 interpretations with the discussion; combine the results parts with Section 3.

•	Section 3.5 is somewhat repetitive of Sections 3.3 (last paragraph) and 3.4; this section should rather be combined with the rest of the Discussion, to expand on what is stated there. This way, the authors will not need to repeat the mentions of previous sections before providing and/or discussing the key variation further.
•	S3.5: Why would this trend in lines 268-272 occur?
•	S3.5: Did pantothenate/VitB5 (lines 273-276) decrease with age? Do we then need less B5 as we age?
•	S3.5: Why would this trend in lines 283-287 occur?

•	General: The authors should consider each of their differentiating metabolites/pathways (for sex and age) separately and interpret these on an individual level. For example, “…steroid hormone biosynthesis was found to be age dependent, although this difference was found in girls rather than boys, partly resulting from later sexual development in boys” (lines 225-227). Here, the authors give an (although very brief/superficial) EXPLANATION of the increased steroid hormone biosynthesis observed. There is no in-depth or even a generalised explanation, such as above, for their statement in lines 219-222: “In the children population, the metabolites involved in (1) histidine metabolism, (2) riboflavin metabolism, (3) pantothenate and CoA biosynthesis and (4) fatty acid biosynthesis were found to change with age in both boys and girls”; why would these different pathways (labelled 1–4 only for the purpose of my feedback) be affected? The “changes” referred to, were these up- or down-regulated? Why? The explanation provided in lines 277-282 is also very nice and it makes sense for the reader. Such explanations for all of their observed trends would enhance the article’s usefulness and impact significantly.

Discussion:
•	S4.1: Why do females have higher Trp levels (line 304)?
•	S4.1: The authors mention that uric acid is higher in boys than girls (line 315), and this contributes to a higher prevalence of hyperuricemia. Can they, however, speculate on WHY this would be the case (physiology/biochemistry)?

•	In S4.1 the authors state that Trp is likely higher in girls (line 303/304, therefore contributing to higher levels of its catabolism products (5-OH-indoleacetic acid). Yet, in S4.3 (lines 368–369), they state that Trp-metabolites (5-methoxytryptophan) were higher in boys. Similarly, line 321 higher acylcarnitine levels in males, but in line 402, these are reported as higher in girls… These statements seem in contrast with each other…

•	S4.3: Why would arachidonic acid metabolites need to be higher in females (lines 409–412), is there a physiological need perhaps?
•	S4.3: With increased 8-OH-7-methylguanine detected in men (line 414–420), does this mean men is more prone to develop tumors/cancer?

•	General: The authors need to include more reasoning (why?) in their discussion(s). Simply stating that a compound is increased in males vs females and that this corresponds with previous results isn’t sufficient. [Please also see my ‘general comment’ for Sections 3 — these explanations can be included/moved to the discussion instead.] Such explanations for all of their observed trends would enhance the article’s usefulness and impact significantly.
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: CHARACTERIZATION OF LC-MS BASED URINE METABOLOMICS IN HEALTHY CHILDREN AND ADULTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is overall well written, despite some typographical and grammatical errors. The cited litterature is appropriate and the article is well structured.
Raw data are not shared. The peaktables used for statistical analyses should at least be accessible to readers.
Experimental design: - Regarding metabolite identification, is there MS/MS information for all metabolites displayed in the table 1? How are the MS/MS scores calculated? Are these the "scores" from the table S1? It is important to know which reference MS² spectra from the public database have been used (there are often several spectra per compound), and the instrument with which they were acquired should be specified. Figure S3 gives information on only a few compounds and it is difficult to compare spectra based on images alone. For important compounds, it is necessary to acquire reference MS² spectra in the laboratory with authentic standards.
In Table S1, some metabolites are detected in other forms than the protonated one. Some like Tyrosyl-valine or glutamine should be detected in the protonated form. Is there an explanation for this? On the other hand, glutamine appears twice in the table (in the L and D forms) and at two different masses and two very different retention times. This is very unlikely given the chromatographic column used.
To summarize, please carefully check the data in table S1 and validate the identification of metabolites of biological relevance using authentic standards.
- Regarding urine sample preparation, I understand that urine samples were not normalized to correct from diuresis variations. This should be discussed in the manuscript.
- Why was the MS detection only achieved using the positive ionization mode?
- Were the samples from each cohort analyzed all at once or in multiple batches?
Validity of the findings: This study could be of interest for those who are involved in research activities dealing with metabolism, physiology and biomarker discovery using metabolomics. However, the validity of the findings is highly dependent on the strength of the metabolite identification data.
Additional comments: - Table 2 and Figure 2: which metabolic pathway ontology was used by the authors? This information should be provided as footnote.
- Figure 2: please indicate the software used for designing this figure in the legend or in the methods section.
- Figure 3c and 5b: what is “num”? This should be clarified.
- Figure 4: the color code is missing.
- Figure 5a: the color code is missing. Are there any statistical differences? These two figures include data that were acquired in two separate experiments. Did the authors normalize the values before? How did they check that the signal intensities are comparable? This should be clarified at least in the figure legend.
- Table S4: what are the “factors”?
- The peak tables used for statistical analyses should be shared.


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: CHARACTERIZATION OF LC-MS BASED URINE METABOLOMICS IN HEALTHY CHILDREN AND ADULTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: No particular comments
Experimental design: - Concerning section 2.2: urine sample preparation: is the QC sample common to both cohorts or on the contrary, is there a QC sample for the pediatric cohort and another for the adult cohort? This should be clarified in the manuscript. The clustering observed in Figure S1a suggests that there is only one QC, common to both cohorts.
- Concerning section 2.5: Feature annotation and metabolite identification: the score value is calculated from mass error, isotope similarity, and fragmentation similarity. However, it is not indicated with which formula this score is obtained. Why is the maximum value of the score set to "60"?
Validity of the findings: No particular comments
Additional comments: In the excel file of tables S1-S8, there is a tab "sheet 1" which is not described, and which contains values which are not the same as those of table S1a. What does this tab correspond to?


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: TARGETED METABOLOMICS SUGGESTS A PROBABLE ROLE OF THE FTO GENE IN THE KYNURENINE PATHWAY IN PREDIABETES
Review round: 1
Reviewer: 1

Output:
Basic reporting: - I think this manuscript is easy to follow. However, some clauses in the Introduction should cite references. For example, in lines 39-40, you wrote "Genome-wide association studies have identified the fat mass and obesity-associated gene (FTO) as the first susceptibility gene of obesity. ". Then, lines 52-53 "However, 53 few studies have investigated the profile of metabolites based on the genetic variation of FTO. " also deserve references. Please add them.

- From the Introduction, I understood the rationale for analyzing the FTO gene. However, how did you select this FTO rs9939609 SNP?
Experimental design: - In the Subjects section, you described only a serum collection.  However, DNA extraction for genotyping purposes is usually performed with buffy coat samples. Did you collect buffy coats from these patients? It should be added to the Subjects section.
Validity of the findings: - In lines 124-125, you stated "BMI tended to progressively increase with the number of the FTO
rs9939609 A allele, but this did not reach statistical significance, as shown in Figure 1". Which was the p-value? Have you tested other genotypic models (allelic, dominant, and/or recessive)? By the way, I looked at your raw data. I did not find BMI values. Could you add them, please?
Additional comments: - I think you have enough results for further analysis. I would suggest testing regression models involving all significative variants aiming to build a fast tool (such as a nomogram). In my view, it would take advantage of your results.


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

Input:
Title: TARGETED METABOLOMICS SUGGESTS A PROBABLE ROLE OF THE FTO GENE IN THE KYNURENINE PATHWAY IN PREDIABETES
Review round: 1
Reviewer: 2

Output:
Basic reporting: Line 39. Needs a reference. Databases like the GWAS catalog website could also be used.
Line 20: The most updated name for Fat mass and obesity-associated gene FTO is alpha-ketoglutarate dependent dioxygenase gene.
Line 50-52, 53, 55, 155, also need references to according to studies.

No information or details regarding the specific genetic variation in the FTO gene is provided in introduction. For example, why among all the SNPs in the FTO gene this one in particular is chosen.
Moreover, no details about systemic inflammation which is in the next sections are one of the focuses of this study is provided in the introduction.

Some sentences as noted below have complicated phrasing.
I suggest better English proofreading for these lines 43, 44, 45, 48,49, 131, 154-7, 162-4, 167-70, 180-3.
Line 44. the word "role" instead of "action" would better describe the meaning. Also "and or comma" instead of "or" in lines 45 and 47.

Line 52. To reduce the ambiguity of sentences, it should be stated the phrase "physiological pathways" underscores which physiologic eras (Obesity?).

Line 65. Any details for the study cohort are directed to a previously described article in reference No.8. It is most acceptable to describe the significant aspects of the study cohort through the method section.

Also, the referenced paper for the population cohort, (Ref No. 8) presents the "EGAT 1" population, which is irrelevant to this study and is published in 2007.
The population studied here is "EGAT 3" which started in 2009.

There is a lack of description of variables in methods section. "Progression to diabetes 3 years later" first is seen on line 67 which its contribution in the study has not been articulated before.

There is no straightforward list of metabolites that are analyzed. They could only be found as column headers in supplementary materials. This list is best to be more accessible for one who is reading the article.

The diabetic definition is not consistent, progression to diabetes is first described as progression in 3 years, but in the statistical section 5 years is mentioned.

In the description of each table, it is better to include the name of the analysis of the statistical test in which it is done for those data and in Table 3 there is a typing error before 0.13.

Line 147. FTO G is misspelled and is not clear which of A or T alleles are meant to be here.

Line 173. The word "Phospholipid" is not clear to which of phospholipids is pointing to.

It is unclear why "Prediabetes" and "Progression to diabetes" were defined in the method section since these entities were not included in the downstream analysis in the results and discussion sections.  Providing further information about inclusion/exclusion criteria would address this issue.
Experimental design: Line 56. The description of the study's hypothesis should be more clarified. Also, "Functions" could be changed to "function"/"role".
The grouping of participants is not clear and should be indeed described in the method section.

It is not clear why the study focus is redirected to tryptophan after OPLS-DA analysis. Although the FTO gene pathway, the Kynurenine pathway, is associated with tryptophan and serotonin metabolism, the reasoning behind this approach of focus on Tryptophan is not well described through the study background.
Validity of the findings: Line 122. As in table 1. Most subjects in the study are female, not male. Although this info is inconsistent through the submission. In Table.1 males are 68, in Line 62 males are 9, and in abstract males are 70.

No information regarding the VIP scores for other metabolites is provided.
It is not clear if these three top metabolites are the top three sorted for highest in positives or highest in absolute scoring.

Furthermore, score plots of variable selection analyses are so informative which are missed here.

Table 3. The highest association coefficient from logistic regression is for Gender (0.99), which is 100 times bigger than the highest among metabolites. Considering the imbalance of the female/male ratio (1:8) of study participants it is best to subdivide the population among males and repeat the logistic regression only in the male group. This will increase the power for detecting the lowest association coefficients such as the observed numbers for metabolites here.

Line 141-142, For comparing the Pearson's correlation values, we need to transform the values in Fisher’s r to z transformation. Nor in the method statistical section nor in the results section it is mentioned if this transformation is applied here or not. Although even if this method had been applied, while one of the correlation p-values is not significant, any conclusion from this transformation is still limited.

Line 147. Although the P-value of the correlation is below 0.05. But r is 0.03 which is inconsistent with the presented significant association between CRP and metabolites ratio.

Line 177,178. Among the presented data and tests in this manuscript, no findings suggest the conclusion in lines 178, 186.
Additional comments: The limitations of the study are not implied and no recommendations for further investigations are provided. The potential confounder effect of fasting before blood sampling is not articulated.
Furthermore, Tryptophan itself, and the Kyrunilic pathway are associated with serotonin production. Also, Quinolinic acid, one end production of the Kyrunilic pathway could be tightly associated with psychiatric, mood, and eating disorders. These associations could cause bias if not considered in the study design of research on obesity.


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: TARGETED METABOLOMICS SUGGESTS A PROBABLE ROLE OF THE FTO GENE IN THE KYNURENINE PATHWAY IN PREDIABETES
Review round: 2
Reviewer: 1

Output:
Basic reporting: Dear authors,

Thank you for addressing my first concerns. However, I would like to comment on the following points:

- In the "Subjects" section, methods for obtaining buffy coats from patients should be added.
Experimental design: - Despite having few samples to produce a nomogram, I think it is necessary to do a multivariate analysis on Tables 3/4. Please consider applying this test to avoid overinterpretation.
Validity of the findings: - Thank you for clarifying interpretations of p-values and sending raw data. In addition, I think that mean+- the standard error could hide some data. For figure 1, I suggest changing this plot for a violin plot. It would improve the communication of your findings in a more transparent form.
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: TARGETED METABOLOMICS SUGGESTS A PROBABLE ROLE OF THE FTO GENE IN THE KYNURENINE PATHWAY IN PREDIABETES
Review round: 2
Reviewer: 2

Output:
Basic reporting: Authors did an excellent job improving the professional article structure and in their response to my previous comments, they have clarified the manuscript ambiguities and the issues in the context of clarity, data reproducibility are now addressed.
Experimental design: No Comment
Validity of the findings: No comments
Additional comments: No additional comments


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

Input:
Title: TARGETED METABOLOMICS SUGGESTS A PROBABLE ROLE OF THE FTO GENE IN THE KYNURENINE PATHWAY IN PREDIABETES
Review round: 3
Reviewer: 1

Output:
Basic reporting: Dear authors,

Thank you for having addressed my last comments. In their current version, I endorse this manuscript for its publication.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: No additional comments


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

Input:
Title: THE INFLUENCE OF A MANIPULATION OF THREAT ON EXPERIMENTALLY-INDUCED SECONDARY HYPERALGESIA
Review round: 1
Reviewer: 1

Output:
Basic reporting: In general, I consider your reporting style to be of a very high quality. I am in strong support of the open science approaches throughout the manuscript, in particular, the pre-registration of your analytical pipeline. I found the detail of your method to be excellent, and this has made the manuscript a much easier paper to review.

I have a few details that I would like you to review below:

Major points: 1) Within your abstract and introduction, you state that your approach will target "physiological changes within the dorsal horn", yet all of your outcome measures are focusing on subjective ratings of pain, anxiety or detection. I suggest that you are not, in fact, targeting the scope of your study towards spinal physiology, but are instead using SH as an experimental metric for examining the subjective impact of sensitisation of neurons within the dorsal horn. While perhaps a subtle distinction, I think it's important to separate your approach from spinal physiology.
2) Despite your transparency, I am a little unclear about your SH protocol. What were the instructions given to your participants to calibrate the points? Were they providing a point-rating based on detection, sensation change or pain? This has implications for the interpretation of the variable itself. Additionally, how did you calculate the area of an 8-sided polygon?

Minor points: 1) Odd formatting at line 117- use of italics is unusual, and I suspect a typo of actives<->activates
2) While you cite seminal work, I found that there were basic points (such as pathology not equalling symptomology, or clinical instances of pain-free behaviour) that were using very out-dated references. If possible, update your references to include more recent research, which is highly relevant to the arguments you are making
Experimental design: As stated, your discussion and methodological critiques were very well written and contained a number of well thought through ideas.

The only edit I'd recommend (or clarification I'd request) is that you proposed a theory of a failed manipulation due to "competition between threats", and that essentially, the threat was equally raised across both arms for the participants, rather than unilaterally. Have you tested the correlation between the ratings across arms for your sample? This seems like a good way to test whether it was the case, and based your scatterplots, appears likely.

Can you also clarify whether SH was collected by the same experimenter for every participant? High variability in SH measurements has been previously reported, with low inter-rater reliability. It appears that it was the case as your "assessor" completed all sensory tests. But I want to confirm this

Out of interest, I’d perhaps suggest that one concept that is missing which may tackle the issues you’ve encountered is to attribute the risk (within a between groups design) to the equipment, rather than the design itself. i.e. If the participants were to think that a piece of equipment was faulty, and had been performing irregularly, you may be able to impose risk which is outside of the umbrella of protection of the ethics and removes the trust element that exists between participant and experimenter. I don't require this to be added to manuscript, unless you decide so, but it could represent a solution to your issue.
Validity of the findings: I have no concerns about the validity of your findings, mainly due to the excellent description of methodologies and transparency of your design and approaches. In particular, I am pleased to see null findings being appropriately published.
Additional comments: I believe there are some naming issues concerning the supplimentaries. Line 550 refers to supplementary 5 for the results of your semi-structured interview, but this appears to be contained within supplementary 4. Likewise, on line 557 there is reference to supplementary 6, which was not included within my submission files. I suspect this will be a matter of formality, but I'd appreciate the opportunity to review these on re-submission

The reference section was irregularly formatted, different font type and size, and a few issues of punctuation and spacing

Again, out of curiosity, what was the rationale behind using an otoscope for a forearm inspection for threat? I did find this to be unusual from the off, and would have thought that most participants would be familiar with ear exams enough to suspect this was a sham inspection? You have addressed this limitation comprehensively, and I do not think this requires any more alteration to the manuscript.


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

Input:
Title: THE INFLUENCE OF A MANIPULATION OF THREAT ON EXPERIMENTALLY-INDUCED SECONDARY HYPERALGESIA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Typos etc.
•	There are several double spaces that have crept in throughout the manuscript. I suggest using the replace function in Word.
•	Line 112. The citation is in italics.
•	Line 226. Should be “using a two millisecond pulse”.
•	Line 743. Your references are font size 18. PeerJ’s guidelines say that text should be 12 points Times.
•	Line 443. “larger surface area but magnitude” – the “but”.
Experimental design: Rigorous investigation performed to a high technical & ethical standard.
Some more detail is needed when describing the method (see 4. Additional comments).
Validity of the findings: All underlying data have been provided.
Conclusions are well stated, linked to original research question & limited to supporting results.
Additional comments: Bedwell and her colleagues examined the influence of a threat manipulation on induced secondary hyperalgesia from high-frequency electrical stimulation (HFS). Their experiment was preregistered and well designed. They aimed to induce threat by telling participants that one arm was particularly sensitive to HFS, and that the other one was not, and measure the effect on secondary hyperalgesia from HFS. However, it appears that the manipulation failed and/or the unpleasantness of HFS washed out any effect of the threat manipulation. It is a shame that the manipulation did not work, as the research question the authors set out to address is interesting and important. They had also gone to great efforts to ensure the that the study was rigorously designed (e.g. the blinding was done to a high standard).

In general, I think this is a well conducted study. The main issue is the threat manipulation, as the authors already point out. I believe there are a few points that need addressing/clarifying prior publication.

Major:
1.	The discussion is lengthy (almost 3000 words). The authors raised many valid points and considered many different reasons why their manipulation may not have worked. I would suggest shortening the discussion and making their points more succinctly. Doing so will increase the readability of the manuscript, so that the interesting points that you raise are more likely to come across and not get missed out by your audience.
2.	It would be helpful to include more details about the HFS stimulation. For instance, how does your procedure compare to previous studies? Did you measure the electrical detection threshold on one arm only? I am familiar with some of Emanuel van den Broeke’s previous work with HFS where they measure electrical detection thresholds for each arm, and show that there is some intra individual variability (e.g. Filbrich et al., 2020, Cortex). From reading your manuscript I am under the impression that thresholds were only measured for one arm. Is this correct? Either way, more detail is needed.
3.	Related to my previous point, I think it would be helpful to include some of the information from Figure 4 in plain text in addition to its graphical presentation. It was not immediately clear to me how long after the HFS stimulation that the sensory testing etc. was conducted.
4.	Is it necessary to abbreviate secondary hyperalgesia? The text would be easier to follow if you spell it out.
5.	The effect size derived from your pilot data should be stated so that it is clear what magnitude of effect your study is powered to detect. Is it derived from the third stage of piloting (n = 6)?

Minor:
•	It was not clear to me if there why the sensory testing was repeated so many times (e.g. 3 times at baseline, 6 times during follow-up). Please could you clarify the rational for doing so.
•	Line 89. It would be useful to cite the three studies that you make reference to. Later you only go on to describing two of the three studies.
•	Line 129. It would be helpful to outline how threat will be manipulated and what will be compared. It is difficult for the reader to contextualize the outcomes and hypothesis without this information. It should be made clear exactly what conditions etc. that will be compared.
•	Line 244. How many pain ratings did participants give? Perhaps this information could also be depicted in Figure 4.
•	Line 249. There is no clear rationale for why you are using pain ratings as your primary manipulation check. Please clarify. I suspect this is because you did not include a direct (quantitative) measure of perceived threat from the HEFs, and so had to rely on proxy measures.
•	Line 258. How was anxiety measured?
•	Line 727. More detail is needed about the procedure used to determine the primary and secondary outcomes (i.e. mechanical punctuate stimulation and surface area of secondary hyperalgesia).
•	Line 325. Which arm was the individual threshold calibrated for?
•	Line 343. Did any participants withdraw? It would be useful to report the number that withdrew, even if this is zero.
•	Line 354. What was the motivation for measuring the surface area of secondary hyperalgesia three times?
•	Line 379. What is the advantage of using a mean pain rating for the two pinpricks (i.e. 128 and 256 mN) rather than having the raw ratings for each and adding Force (128 mN, 256 mN) as a fixed factor? You approach might be equivalent, or even superior. Either way it would be useful to add some detail so that the reader understands the reasoning behind this decision.
•	Line 392. PeerJ’s guidelines for citing unpublished work states: “Cite work unpublished, in preparation or under review as 'unpublished data'. Supply the author's first initial and surname, and the year of the data collection, in the text citation and do not include the citation in the reference section. Example: (A Castillo, 2000, unpublished data).” Please correct your citation.
•	Line 395. Was there a correlation between individually determined thresholds and magnitude of secondary hyperalgesia in your unpublished data?
•	Line 435. How were p-values calculated from your linear mixed model? E.g. did you use the Satterthwaite degrees of freedom (see Luke, 2017, Behavior Research Methods)?
•	Line 456. The information about what the box plots show etc. should be included in the legend of each figure containing a boxplot so that the figures can be interpreted on their own.
•	Line 480/Figure 5. Is it important to show the difference between HFS trains? This figure might be easier to read if you simply have condition on the x-axis (e.g. with a box plot). This point also applies to Figure 8.
•	Line 496. More detail in the figure caption would be helpful. For instance, what does a positive value show on the y-axis show?
•	Line 625. A citation might be needed here. Has it been demonstrated that knowledge of a monetary reward activates descending modulatory mechanisms?


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

Input:
Title: THE INFLUENCE OF A MANIPULATION OF THREAT ON EXPERIMENTALLY-INDUCED SECONDARY HYPERALGESIA
Review round: 2
Reviewer: 1

Output:
Basic reporting: Thank you for addressing my previous comments. I only have very minor comments that address some of the revisions made to the manuscript.

Figures 6 & 7 have a legend for the Alpha. I imagine this in unintentional, or possibly to distinguish the outliers from the box plots. If it is the latter, you can simply suppress the outliers of the box plot in ggplot with this line of code: "geom_boxplot(outlier.shape = NA)+". If, however, the different alpha levels are intentional, then their significance should be described in the caption.

Typos:
Line 253. You should define SPARS the first time the acronym is used (i.e. line 253 rather than 273).
Line 264. It is not entirely clear what "the procedure" refers to. Presumably this refers to your experimental protocol, as outlined in figure 2.
Line 482. There is a space missing: “the‘lmer’”.

*line numbers refer to the revised manuscript with tracked changes.
Experimental design: The authors provided an in depth response to my question about how the sample size was calculated (i.e. R2.13). I would like to see more of this justification in the manuscript. For instance, there is no mention of scale factor in the manuscript. As a consequence, it is not clear from the manuscript how the pilot data (n = 6/third phase) was used to inform the sample size estimate. This issue could be addressed by adding more detail to the main manuscript, or to include a full breakdown of your GLIMMSE calculation as a supplementary file.

I appreciate that this is somewhat pedantic, but I consider it important detail to ensure that your study can be replicated.
Validity of the findings: The preregistration is still under embargo. Otherwise I have no comments to add.
Additional comments: I congratulate the authors on their effort to revise the manuscript. I believe it has improved substantially and is appropriate for publication. I have raised a few minor issues that the authors might want to address to further improve the manuscript and its reproducibility.


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: REMOTE SENSING TECHNIQUES FOR AUTOMATED MARINE MAMMALS DETECTION: A REVIEW OF METHODS AND CURRENT CHALLENGES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The analysis provides an updated review of the efficacy of different remote sensing methods for detecting marine mammals.
The review and its introduction are deep and limited to the objectives, and they are sufficiently innovative.
The review's focus is of broad and cross-disciplinary interest, so is within the journal's scope.
Experimental design: • The review has been performed through automated bibliographic searches and can be replicated. Sources are appropriately cited.
Validity of the findings: Sources are appropriately cited.
The description of satellite data sources is not sufficiently developed. Because this is a review, the reader must access more information to compare and decide on the available methods and techniques for a more helpful review.
Satellite and UAS information could be improved, i.e., by adding a second table with each image's spatial and spectral resolution characteristics or identifying which satellite platform or UAS the data is being taken from (e.g., Worldview 2 is named in one case).
If the platforms are not identified in the papers analyzed, they should be made explicit in the review.
On the other hand, since Marine mammals species are very different in size, it would be necessary to identify the spatial resolution of each study.
Additional comments: About the title:
Replacing the title with one similar to:
"Remote sensing techniques for marine mammals detection: a review of methods and current challenges of automated approaches" could better reflect the content of the work.
 The topics analyzed are correctly exposed, but this review would be desirable to contain more information from each reviewed paper.
Particular comments:
In lines 442-449, the authors discuss the importance of analyzing the spatial resolution and the spectral bands of sensed imagery for better detection, but the manuscript does not provide data on this. This aspect could be improved i.e., by adding a second table.

Lines 472-473: Please provide references on studies of passive acoustic monitoring for submerged animals and satellite tracking for detailed movement patterns.

Although the original works do not explicit this, Google Earth comes from "processed" satellite images classified as such. Furthermore, there is a reference to which image is used in each Google earth view.


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: REMOTE SENSING TECHNIQUES FOR AUTOMATED MARINE MAMMALS DETECTION: A REVIEW OF METHODS AND CURRENT CHALLENGES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The introduction and background supporting the rationale for the review is weak. While it is stated who this review is targeted towards, there is little evidence provided as to why the paper is needed and how it differs from similar previous reviews in the field, i.e.;
•	Hollings et al., 2018
•	Wang et al., 2019 – Surveying Wild Animals from Satellites, Manned Aircraft and Unmanned Aerial Systems (UASs): A Review
•	Corcoran et al., 2021 – Automated detection of wildlife using drones: Synthesis, opportunities and constraints
The background needs more detail on the use of automated methods for detecting animals in satellite and UAS imagery (including terrestrial animals), the types of methods, and their increasing use along with their potential advantages. This would provide the rationale for the review (for example, relatively little use of automated methods in marine mammals) and the rest of the background can then be more briefly summarised. The readability and flow of the paper would be greatly improved if the background context emphasized that manual count of animals in satellite and UAS imagery is time and labor-intensive, but that automated methods of image analysis offer promising solutions.
Experimental design: The literature search does not appear to be rigorous. There was an important seminal paper missing:
•	Maire et al. (2015) – Automating Marine Mammal Detection in Aerial Images Captured During Wildlife Surveys: A Deep Learning Approach
As well as two other more recent papers:
•	Gray et al. (2018) - A convolutional neural network for detecting sea turtles in drone imagery
•	Woolcock et al. (2021) - Effectiveness of using drones and convolutional neural networks to monitor aquatic megafauna
Ideally, the literature search should be conducted with a database such as Web of Science or Scopus, in addition to Google Scholar. Inclusion of other keywords such as “drone”, “automat*”, and “detect*” etc may increase the number of related papers.
Line 156 – “…works that compared detections with imagery of different resolutions.” – it is not clear what this means and what studies this criterion excludes.
The reasons for the inclusion of studies of marine mammal detection using satellite or UAS imagery that did not include automated methods for detection is not stated and is unclear. Since the paper is primarily focused on the use of automated methods, it seems appropriate that the reviewed papers and Table 1 only include those that apply automated methods.
Validity of the findings: The broad recommendation of the potential use of thermal imagery for marine mammal detection should be cautioned. The thermal imaging study referenced (Seymour et al., 2017), although detects pinnipeds, is conducted primarily on a terrestrial background. The use of thermal imagery in in-water marine environments is not discussed in the paper.
Gonçalves, Spitzbart & Lynch (2020) incorrectly define the F1-score, I would not use this paper to support the argument that metrics are lacking.
In spite of my agreement that there is a lack of consensus regarding reporting metrics, I remain skeptical about the novelty and utility of the proposed evaluation metrics and what they will add to the field of automated detection. For example, Equation 1 is commonly known as miss rate or false negative rate (FNR), and it is no more informative than recall, since it can be calculated directly from it. In the same way, Equation 2 doesn't seem any more informative than precision since it can indirectly tell us the number of false positives or "overcounts" per correct detection (true positive). I am open to the idea, but there needs to be greater justification for why practitioners should use the proposed evaluation metrics over more commonly used ones such as recall, precision, and average precision - as evidenced across computer vision and remote sensing object detection studies.
Line 291 - “Table 1 suggests that this may not be true in the field of marine mammal detection.” – what exactly in Table 1 suggests?
Re: Table 1 - Dujon et al. (2021) reports confusion matrix in their supplementary material. Equations 1, 2 and 3 can be calculated.
Line 378 – “Overall, we concur with Hollings et al. (2018) argument that automation is a viable alternative to reduce the time and labor necessary to analyze remote sensing imagery,…”. The paper does not provide hard evidence of a reduction in labor or time in the analysed studies. Although, line 391 does reference Thums et al. (2018), which suggests time savings with automated methods - this could be moved further up and expanded on. A further comparison of manual analysis vs automated analysis in the context of time savings across the reviewed studies would be needed to support the findings of Hollings et al. (2018).
Additional comments: The paper seeks to review the literature for automated methods applied to detect marine mammals using satellite and UAS imagery. To my knowledge, the topic of this paper is novel, and it would be the first paper to synthesize and compare automated methods applied to marine mammals.
When I first read the abstract, I thought the description and summary of the paper sounded intriguing and well-organized, but as I read the full report, this feeling faded somewhat. I would suggest the paper has potential, however, revision through addressing at least the above comments should be considered before acceptance.
It is important, I think, to emphasize that manual counting of animals in satellite and UAS imagery is time-intensive and labor-intensive and that automated methods offer promising solutions. It is one thing to have these automated methods, but it is another to convince researchers and practitioners of their utility.
It is apparent there is limited research on automated methods for satellite and UAS imagery for marine mammal detection, and some further comments on possible reasons for this would be helpful 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: REMOTE SENSING TECHNIQUES FOR AUTOMATED MARINE MAMMALS DETECTION: A REVIEW OF METHODS AND CURRENT CHALLENGES
Review round: 1
Reviewer: 3

Output:
Basic reporting: This is an interesting and well approached review of the use of UAS and satellite imagery to detect and count marine mammals. It adds results of novel tools to previous reviews on these subjects.
I have only some minor comments:
Lines 66-67 should also mention the technique of counting animals while surrounding rookeries on boats, which is also very common.
Maybe the first study that counted pinnipeds from UAS imagery and compared them to those made by eye from boats is Adame et al. (2017). I think it deserves a mention in the Introduction.
Line 162. I think the authors should use an alternative strategy to communicate the patterns they claim from Table 1. I encourage them to run a simple metanalysis of the bibliographical results so they can draw such conclusions.
Lines 212-212: I think the imperfect detection of cetaceans deserves more discussion. This is important, because no matter how good a technique is detecting cetaceans at surface from remotely sensed imagery, some probability of detection should be possible to estimate to truly replace ship- and airplane-based surveys for cetacean density and abundance estimations. Is there any idea in the literature to deal with this? Is there any potential development in that field?
Line 225: I think the indices you mention need to be as main equations within the text instead of just footnotes.
Line 302: Please add a number next to the equations within the text.
Lines 302-306: I do not see the contribution of the authors’ proposed index. It is just the complementary proportion of the index explained before. Is it just semantics o is there a real reason for that?
Since the authors are proposing a new index of the proportion of animals missed by the counting technique and mention others previously proposed, I think they should present the application of those indices to the results of the studies they compiled in plot, so the reader can compare their performance.

Reference:
Adame K, Pardo MA, Salvadeo C, Beier E, Elorriaga-Verplancken FR. 2017. Detectability and categorization of California sea lions using an unmanned aerial vehicle. Marine Mammal Science 33:913–925. DOI: 10.1111/mms.12403.
Experimental design: 'no comment'
Validity of the findings: 'no comment'
Additional comments: 'no comment'


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

Input:
Title: MICROPLASTICS DO NOT AFFECT BLEACHING OF ACROPORA CERVICORNIS AT AMBIENT OR ELEVATED TEMPERATURES
Review round: 1
Reviewer: 1

Output:
Basic reporting: It is an interesting manuscript, a current thematic that considers the main disturbances on coral species: heating (bleaching) and marine debris (microplastics).
I suggest to review the discussion because the abstract, introduction and almost the entire discussion are all very similar. Reading one of these sections it is pratically the same. You need to bring new informations, raise new ideas, hypothesis on the discussion to improve it.
It will be very important to discuss about the potential of different types of microplastic, which probably should influence your results.
Experimental design: The experimental design did not consider the long-term effect of microplastic disturbance. This reflected in your results. Increasing temperature values (about 4 degrees) will have an immediate (few days) damage on corals, but increasing miscroplastic density will need longer period (probably months) to harm or impair the corals.
Validity of the findings: You need to show the artefact effect since your study was in a confined system and the ocean/sea is not. How could this interfere in your results.
Additional comments: What is the global oceanic average of microplastic concentrations? Please include these values and the references that mention it.


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

Input:
Title: MICROPLASTICS DO NOT AFFECT BLEACHING OF ACROPORA CERVICORNIS AT AMBIENT OR ELEVATED TEMPERATURES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper is written in very clear, unambiguous English throughout the text.  I feel the literature is appropriately referenced, especially into the second paragraph of the introduction.  Perhaps in paragraph #1, lines 49-50, when the authors talk about early studies, more than one reference (i.e., in addition to the Reichert et al. 2018) could be listed?  Additionally, might some of the equivocal studies (line 51) be listed?

The overall structure is solid.

Regarding figures:  On Figure 1 – does one want to mention the three panels that are significantly different (Nitrate, Nitrite & Ammonia?). Standing alone, a reader might recognize the potential, but I think having reference to that in the legend would be good.

Also, figure 2 – in color, it is appropriate.  In black and white print, it is very difficult to see both the actual 2 microplastic beads and also the red arrow.  Changing the arrow to black would solve the latter, perhaps adding small circles around the 2 beads on panel A would help with the second.  There might be instances when the paper is desired to be printed, and B/W is more common, and cheaper.

Figure 3 = again, does one add statistics to the panel?

Table 1 – Why include the zero-inflation model? Especially if not referenced to in the text anywhere?

While the raw data is indeed, shared, I have some concerns with it, in diving deeper.  First, it is somewhat difficult to follow: For example, it is never listed anywhere which tank is in which treatment. Digging deeper one can glean this information by sorting and organizing the data further, but it would seem helpful to perhaps maybe have 2 columns – one with TREAT TEMP (1 = 28; 2 = 32) and one with TREAT PLASTIC (1 = no plastic; 2 = plastic) or a column with four variables representing the 4 treatments:  1 = 28/no plastic; 2 = 28/plastic; 3 = 32/no plastic; 4 = 32/plastic.  Additionally, (and this is a bit picky, I’ll admit), on the TEMP data page, the label TANK is not consistent with the TANKS on the other pages (i.e., there are 32 tanks), rather, on the TEMP page, Temp should likely be WATERBATH or some such.
Experimental design: Methods:  It was not clear in the methods that the experiment was set up at two different times, one (11/2020) using the moderately heat tolerant corals and the other (6/2021) using the heat tolerant corals.  This was quite confusing at first when looking at the raw data.  Including the 2 time frames when the two different genotypes were done in the methods would be helpful.

Regarding bleaching data (paragraph starting line 165).  It says bleaching and percent surface area affected by tissue loss were estimated DAILY.  First, I take it “the area affected by tissue loss” in the text means “NECROSIS” on the data sheet?  How exactly was necrosis calculated?  Next: in looking at the raw data on bleaching tab in EXCEL, I don’t see daily estimates were made for all tanks, for example, from Tank 1’s in either the 2020 moderately heat or the 2021 data.  Indeed, I don’t see 17 measurements (the length of the study from Fig. 1) for ANY tank in either coral genetic study.  This implies the bleaching and necrosis data are NOT daily.

Finally, what is the exact difference in bleaching and necrosis?  You don’t really define in the methods what they mean/relate to each other.

I would next start a new paragraph describing the Zoox Density data (i.e., after “IMMEDIATELY FOLLOWING on line 168).  It might be helpful to provide a bit more background/ detail on some of the calculations here.  For example, it would SEEM to me that the total volume of Zoo (total homogenate volume) would appear to be affected by the amount of seawater spray and the amount of formalin?  Are either of those standard volumes?  I’d like a bit more info.  Also, as it appears that “foil weight” is important in the calculation of the coral surface area, more detail beyond referencing Marsh (1970) would be helpful.  Additionally, in the surface area column in the raw data (Z00x density page), it appears that the first experimental run (moderate tolerance), there is a formula used to calculate the surface area that depends on the foil weight.  However, for those data from the heat tolerant run, there is a number in the surface area column – no formula calculation.  IF one copies the formula used to calculate surface area for the moderate tolerant:  =(R2*100)/0.424. to the heat tolerant data, the calculated number is different.  This is problematic.

I am interested in the fact that actual microplastic ingestion was not truly documented.  I understand the photo the authors provided (Fig. 2) with the “before” (00:03) and “after” (00:13), with the one polyp associated with the pellet definitely changing shape from A to B implies ingestion.  But, for example, the other pellet that disappeared from A to B, we just assume was NOT ingested because no polyp change was observed?   I guess there is no normal way of quantifying actual ingestion by counting particles when water changes, or looking for particles when the polyps are processed?  Indeed, the sentence (line 156) “…ingestion was an assumed response due to evidence…” is interesting.  Might the lack of differences in microplastic effect here maybe be due to the fact that these corals are not really ingesting these microplastic particles much, and the assumption might be problematic?
Validity of the findings: I think, given some of the issues in the raw data, that perhaps a going over of the data analysis might be warranted.  I would also perhaps mention somewhere in the discussion the above mentioned “assumption” of ingestion of plastics.

After addressing calculation concerns in the database, and perhaps the assumption of actual consumption of microplastics by these corals, that microplastics are not affecting this species is an interesting result.  One always assumes that microplastics are bad.  As the authors state in the introduction, this isn’t a foregone fact – there are equivocal results in the literature.  The study does show experimental support for now just a 4 degree increase in water temperature negatively affects coral.  Thais is a legitimate experimental result.

Reasons for their somewhat surprising findings are supported well.  One might be interested in ideas about how to go about a deeper quantification of what is really happening here.
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: MICROPLASTICS DO NOT AFFECT BLEACHING OF ACROPORA CERVICORNIS AT AMBIENT OR ELEVATED TEMPERATURES
Review round: 2
Reviewer: 1

Output:
Basic reporting: the improvement on the revised manuscript is quite evident. I have just attached some minor corrections at the revised file attached.
Experimental design: already corrected
Validity of the findings: already corrected
Additional comments: the improvement on the revised manuscript is quite evident. I have just attached some minor corrections at the revised file.


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: MICROPLASTICS DO NOT AFFECT BLEACHING OF ACROPORA CERVICORNIS AT AMBIENT OR ELEVATED TEMPERATURES
Review round: 2
Reviewer: 2

Output:
Basic reporting: Concerns have been addressed.
Experimental design: Concerns have been addressed
Validity of the findings: Concerns have been addressed
Additional comments: I am pleased with the thoughtful comments/responses to the issues that were raised previously on the manuscript.  I feel they have adequately 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: COMPOSITION AND ENVIRONMENTAL INTERPRETATION OF THE WEED COMMUNITIES IN THE MAIN PLANTING BASE OF JUJUBE (ZIZIPHUS JUJUBA MILL. CV. ‘LINGWUCHANGZAO’), NINGXIA PROVINCE OF CHINA
Review round: 1
Reviewer: 1

Output:
Basic reporting: In general, the research content of this paper is novel. In this paper, the environmental factors and weed community diversity of the main planting bases of 'Lingwu Changzao' were analyzed by TWINSPAN and CCA. It is helpful to understand the relationship between weed distribution composition, distribution and environmental factors in jujube producing areas. The current version still needs some changes before Acceptance.
In the introduction, I noticed that the author mentioned the 'biodiversity protection' (Line 84-85), and I can't understand how it relates to this study. Is it to protect weeds?
Line 42, 'strategies. (He et al., 2020)'  the period needs to be removed;
Line 55 and 374, 'Pan et al., (2015)' 'Zhang et al., (2012)' the comma needs to be removed;
Line 62, please quote the correct article, this article is not suitable for quoting here. I recommend you read this “Guo, M., Zhang, Z., Li, S., Lian, Q., Fu, P., He, Y., Qiao, J., Xu, K., Liu, L., Wu, M., Du, Z., Li, S., Wang, J., Shao, P., Yu, Q., Xu, G., Li, D., Wang, Y., Tian, S., Zhao, J., Feng, X., Li, R., Jiang, W. and Zhao, X. (2021) Genomic analyses of diverse wild and cultivated accessions provide insights into the evolutionary history of jujube. Plant Biotechnol J, https://doi.org/10.1111/pbi.13480”;
Line 65 to 108, Ma et al. 2021 has three articles in the bibliography, please mark them with a/b/c to distinguish them.
Line 71 72, lack of references after 'transcriptome differences analysis of fruit of grafting and root tiller propagation';
Line 96 and 103, missing space before parentheses;
Line 394 to 504, journal names in references should have a uniform format, some abbreviations and some full names.
Fig.3 is missing a note, 'H, D, E, S' should be noted;
Fig.7 to Fig.16 were not appear in the body of the article. Are they useful? Please mark it in the appropriate place!
Experimental design: no comment
Validity of the findings: no comment
Additional comments: no comment


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

Input:
Title: COMPOSITION AND ENVIRONMENTAL INTERPRETATION OF THE WEED COMMUNITIES IN THE MAIN PLANTING BASE OF JUJUBE (ZIZIPHUS JUJUBA MILL. CV. ‘LINGWUCHANGZAO’), NINGXIA PROVINCE OF CHINA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The literature review and discussion should be improved.
Experimental design: Good
Validity of the findings: The current one is more on descriptive analysis. More in-depth reasoning should be applied to enhance the quality
Additional comments: Review for Peerj-71010-v0

The work contains some useful information, but the discussion was superficial which needs further in-depth elaboration and reasoning.

The analysis of the soil etc should finally be reflected in the fruit quality, which should be discussed further. For instance, whether the metal ions of the soil be finally linked to the contents in jujube fruit? For instance, Food Chemistry, 289, 40-48.

Many figures can be combined to make the data presentation concise and informative. The current ones are like presenting raw data. A key step is to extract the science behind rather than just simply presenting the data collected.

The composition and environmental interpretation should consider the approach of these influences which might be related to the metabolites, whether primary or secondary, but in total should be critical. Therefore, more metabolite investigations should be discussed further. For instance, Critical Reviews in Food Science and Nutrition, 61(9), 1448-1469.

More ecology reasons should be discussed on the effects of weed communities. For instance, Weed Science, 46, 467-474.


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: COMPOSITION AND ENVIRONMENTAL INTERPRETATION OF THE WEED COMMUNITIES IN THE MAIN PLANTING BASE OF JUJUBE (ZIZIPHUS JUJUBA MILL. CV. ‘LINGWUCHANGZAO’), NINGXIA PROVINCE OF CHINA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors addressed the questions quite well.
Experimental design: Good
Validity of the findings: Good.
Additional comments: The authors have improved the manuscript. The current version is acceptable 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: RABBIT HINDLIMB KINEMATICS AND GROUND CONTACT KINETICS DURING THE STANCE PHASE OF GAIT
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors use clear, professional language, and have written an appropriate article in style and format, that is self-contained with relevant results and hypotheses.
Experimental design: This experiment is appropriate for PeerJ in scope and aims and fills a gap in the literature of normal single limb lab rabbit kinematics. However, I do believe the authors could provide some further elaboration without collecting additional data.
Validity of the findings: The findings are valid, but require some additional elaboration and could benefit from additional analyses. Data were provided, but were not easy to decipher. An additional key or additional elaboration on column titles would be helpful.

The discussion requires more work, particularly in contextualizing the results with human biomechanics (or eliminating this comparison altogether).

Please also address issues with Figure 5. I might suggest that this figure be removed altogether (see my comments about using relative joint position without scaling and context).

The conclusions are appropriate given the results. However, the results and interpretation need some work before this manuscript is suitable for publication.
Additional comments: General comments:

The authors attempt to make links between human and rabbit mechanics, presumably for future work using rabbit models to clinical studies. However, these comparisons are very cursory and require a lot more discussion if this is going to be included. I also suggest the authors include a more significant summary of human biomechanics in the introduction to contextualize their discussion section. My suggestion is to consider dropping the human locomotor comparisons and only discuss their results in the context of other rabbit or half-bounding locomotor studies.

I also think that the authors need to either eliminate on human comparisons or remove them altogether. There are some factual errors and some glossing over comparisons that need to be addressed (e.g., rabbits don’t heel strike).

Finally, I would suggest the authors consider additional analyses that don’t require additional data collection. Why not look at stride length? Time of contact? Vertical force impulse? Time of contact and force impulse in particular would be useful for characterizing differences in limb loading when testing clinical interventions in future work. The authors also don’t quantify velocity, acceleration, etc.? Was this collected with steady state locomotion? Was there variation in joint angle that correlated with velocity? Were there differences between the animals? Some basic statistics may be warranted here. These analyses would make this a stronger manuscript with broader applicability.

Besancon et al. 2003 (Vet Comp Orthop Tramatol) and Lascelles et al. 2007 (Vet Record) provide examples of how others have used a pressure mat to characterize normal forces in an animal model. Additionally, previous work using other animal models such as rats have used swing and stance phase duration to characterize gait kinematics pre- and post-injury/intervention (e.g., Iwata et al. 2010 [Muscle Nerve].

Notes for future work:

I also recommend a different camera for future work comparing surgical interventions given that frame rates above 200 fps are required to detect compensatory gait patterns in rodents (Lakes and Allen 2016 [Osteoarthritis and Cartilage]). This number may be lower in rabbits, but my guess is that 60 fps may not be sufficient. Additionally, I would recommend including hip joint markers for a more complete picture of total limb kinematics.

Specific comments:

 [Lines 93-95] Is it standard to only compute appropriate sample sizes in clinical trials? IACUC regulations mandate reductions in animal numbers through a variety of means, including power analyses. Is there a separate justification for why you chose n=6?
[Line 131] Most studies seem to calculate the MTP angle using the opposite direction. It’s not a big deal since that’s the complimentary angle, but just to note for the future.
[Lines 132-134] The ground is defined as the horizontal plane that runs through the MTP joint, but is this just at mid-stance? Please provide some more detail.
[Lines 138] I’m unclear what you mean by “knee joint center position”. Please clarify.

[Line 158-159] While I appreciate that the authors are making the case for using rabbit models to orthopedics, I’m not sure that a similar foot strike pattern is necessary. Many other animals exhibit similar ankle angles at heels strike, with similar loading patterns. However, I would argue that half bounding kinematics may be more relevant to human running than in other typical animal models like rodents.
[170-171] Characterizing the change in distance of the knee joint in the vertical and anterior-posterior dimensions strike me as an unusual kinematic analysis. In particular, it’s not size-scaled and not standard so it’s difficult to compare it to other work in the literature. I’m also not sure what you mean by “knee joint center position”. Why not just use joint angle over stance phase? I suggest removing the segment position results.
[175-176] It’s not particularly surprising that vGRF is correlated with contact area. The more body weight that is supported, the higher the vGRF. In fact, it should. Please add some context for this result. Is it a check on data quality?
[189-190] What do you mean by rabbits “load their heel first”? While plantigrade, rabbits don’t heel strike. Contact is on their metatarsals and toes. The heel is at the ankle joint.
[191-192] What do you mean by “foot angle during stance is more negative during human walking”? Negative compared to what? Please use refer to angles being smaller or larger. There are also other significant differences in locomotor mechanics than just joint angle (e.g., striding bipedal gait, limb-mass mechanics, etc.). Please elaborate if you are going to try to relate your results with human mechanics.
[202-207] Please elaborate on what you mean by “constant ground-paw pressure may be a locomotor strategy employed by the rabbit’s sensorimotor system”. Additionally, what do you mean by constant paw pressure? Pressure wasn’t constant as exhibited by the %BW force trace (e.g., changes in vGRF).

[208-215] I appreciate that injured animals may have biomechanical compensation, but how can you have different limb segment positions if joint angles are the same? Chang et al. found that limb length recovery tracked kinematic recovery. If animals are loading one limb less than the other, this probably means increased joint flexion, which means decreased joint center height (i.e., compensations should be correlated with joint angle). Additionally, as commented above, reporting 2D distances of joint center is difficult to interpret, not scaled, and not standard.
 [217] I don’t agree that your system is ‘marker-less’. You used markers, just not reflective ones. Both reflective and ‘sharpie’ marks are still subject to skin movement over joint centers. There are marker-less systems for humans (e.g., Theia3D) which use multiple landmarks to locate joint centers.
[236] Though there are advantages to force plates, even with these trade-offs. One in particular for this study would be more precise center of pressure and impulse data.
[255] Again, I suggest removing comparison to humans unless a more thorough comparison is made. I don’t think it detracts from your study to keep contextualize this within rabbits or other animal models.

Figures:
[Fig 5] I find this figure really difficult to interpret. There is a grey shaded region showing the SD around the mean, but it’s really difficult to follow and interpret. Also, why are there no confidence bands around the ankle and MTP joints?


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: RABBIT HINDLIMB KINEMATICS AND GROUND CONTACT KINETICS DURING THE STANCE PHASE OF GAIT
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript clearly describes a well-designed study to characterize certain aspects of rabbit gait.  It is well organized, includes appropriate references to the literature and provides generally clear figures to document methods and findings.  The study is well motivated and discussion includes consideration of limitations.

The inclusion of raw data is helpful for further investigation of the study's results, however it would be helpful if the data workbook included more detailed headings, with explanation of some of the extraneous data provides at the bottom of columns.

For example, in the data set, two sets of "pressure" data are provided.  Is one the raw data from the sensor and the other the calculated data?

It would be helpful if units were provided for each type of data.
Experimental design: The study's experimental design is well-defined and within the scope of the journal.  The results are primarily of use as a "baseline" dataset for comparison for future studies, but that is quite valuable to the research community.  It also describes methods for others to consider, with sufficient detail (in general) for replication.

One area that warrants further description is the selection of the five trials used for analysis.  It seems clear from the dataset that multiple trials were needed to obtain the 5 used in the study for each animal/side.  How were the final trials selected?  It is well known that speed of gait influences human gait, but that doesn't appear to have been controlled in this study.

Further details on the walkway used and its sensitivity would be helpful.  The authors state that they calibrated with a 3 kg weight, but not how large that was and thus what pressures were measured during calibration.  Based on the information available about that walkway, it would seem that typical pressures were quite low relative the the range of the sensors.
Validity of the findings: The results of the study seem sound in general, although my calculations with the provided data do not seem to identify the same mean maximum ground reaction force.  Perhaps I'm not understanding how the data are pooled between rabbits or by side?  Clarification of the statistical methods may be needed.

Of some concern to this reviewer is the wide variability in the duration of the stance phase.  Some trials are much slower than others, and those generally have lower ground reaction forces, as would be consistent with studies of human gait.  The authors note this skewed distribution of the stance duration, but not the potential consequence on the final results.  At a minimum, this should be discussed as a potential limitation.  The patterns of the gait cycle seem different for these long duration trials, while in contrast, those that are very short have much higher peak forces.  It isn't clear which of the two are more "typical" of rabbit gait that might be anticipated during a future experimental study.  It may be appropriate to consider if any of the trials should be considered as "outliers".  There does seem to be a modest correlation between the stance duration and the peak force.
It appears that in the figures, the data are converted to a %stance so that they can be depicted on a common graph.  The variable patterns seen for trials at different speeds are likely contributing a great deal to the shaded region, but it almost seems like you are trying to show both walking and running on one plot.

Another area of concern is the potential for skin movement as an artifact for calculation of joint kinematics.  This is a reasonable approach that is indeed more practical than other methods like x-ray or pins, however the skin motion could be significant.  Again, the authors clearly state this potential limitation, but not the expected magnitude of the impact on the accuracy of the reported results.  Would it be possible to calculate the lengths of the limb segments over the gait cycle and report on the degree of variability, which could reflect inaccuracies in identification of the joint centers.
Additional comments: It might be helpful to have scale bars in figure 3, both for the dimensions of the box and for the magnitude of the pressures measured.


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: RABBIT HINDLIMB KINEMATICS AND GROUND CONTACT KINETICS DURING THE STANCE PHASE OF GAIT
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors use clear, professional language, and have written an appropriate article in style and format, that is self-contained with relevant results and hypotheses.
Experimental design: This experiment is appropriate for PeerJ in scope and aims and fills a gap in the literature of normal single limb lab rabbit kinematics. This revised manuscript addresses reviewer concerns and is now sufficient for publication.
Validity of the findings: The findings are valid and the changes made to this manuscript make the interpretation of findings and discussion appropriate.
Additional comments: My apologies for not noting that you did indeed calculate time of contact (in this case, stance duration). I think in this case stride length is not nearly important as variables which measure loading, so this isn’t necessary.

Additionally, I would like to note that I do think that some form of velocity is an important variable to report. I would argue that with sufficient frame rates, using a marker (like the hip) can give reasonable measures of velocity and acceleration. Even if the distance/time measure is only when the rabbit is in view of the camera. In particular, it can give important information like whether rabbit trials were in steady state or accelerating. However, stance duration is certainly going to be correlated with speed.

Specific comments:

[Pg 3, Line 57] “Clinical” is spelled incorrectly

[Pg 3, Line 68] A minor quibble, but the phrasing in this paragraph initially sounds like you are reporting on your own data in the introduction. I think it would be helpful to say something like “In Gushue et al. (2005), a net vertical GRF curve showed XYZ...However these data only come from a sample of n=1, which makes it unclear if this is a typical gait pattern”, or something along those lines. This paragraph could also benefit from another sentence or two as it is a little terse. Can you elaborate just a little bit on the contrast you setup between rabbits having a “plantigrade type” foot morphology and a digitigrade gait pattern? Because it immediately jumps to a description of results from a paper that wasn’t cited in the previous sentence. I think this will help setup your study to make it clear what is known, unknown, and how your study addresses a gap in the literature. I think this adjustment will make for a stronger introduction and setup for the rest of the paper

[Pg 9, Line 216-218] In what ways were rabbit kinematics similar to rats during walking?