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: MOVE IT OR LOSE IT: INTERSPECIFIC VARIATION IN RISK RESPONSE OF POND-BREEDING ANURANS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors used professional language throughout

Literature is well referenced

Background and introduction: Much of the introduction is devoted to the impacts of land use change on declining and imperiled species, and the connection to the three common species and their behavioral responses is not clear from a conservation standpoint.
For improvement, the authors should remove unnecessary information (specific examples in general comments) and establish relevant background material that will set up the specific research question.

Figures are of journal quality
Experimental design: This article is within scope of journal

Research question: The research question itself is not clear, and authors provide no predictions, hypotheses, or relevancy for examining perceived risk at habitat edges, including potential species specific responses.

Investigation standard: Estimation of body size in southern leopard frogs not of high standard. Size was either measured or estimated, with no indication of the percentage that were estimated, nor if estimation was in line with actual measurement. This referee suggests removing this section of analysis.

Study design and field methods are sufficiently described, though a figure delineating “middle, edge, and boundary” categories would be helpful to the reader.

While the authors investigated a myriad of factors that might influence movement, there was not sufficient background material addressing the reason for some of the investigations.  For instance, change in behavioral choice by season/month was investigated, but there was no explanation or hypothesis laying the framework for this investigation.

Analyses are not clear, and need substantial revision, including reporting degrees of freedom, and some type of goodness of fit for the model. Reporting the GLM (with model fit) and breaking down results by parameter in the model may help reduce the confusion. If the model is informative, showing a test against an intercept only model would help convince readers.
Validity of the findings: Impact and novelty: This study offers an examination of behavioral responses to simulated predation at habitat edges, but does not expand upon the importance of the change in behavioral response to a sufficient degree. While this information may be useful, there needs to be a stronger rational provided.

Data: Beyond the uncertainty of estimating size, data seem adequate, though there is not a clear connection between analyses and results.

Figure one, while only reporting proportions, show a clear biological pattern worth reporting. However, significant revisions of the results are necessary for the validity of the study to be interpretable.

Conclusions: Relevant supporting research is provided, though the organization of the conclusions seems out of place with actual study. As it reads, the first and last paragraph of the discussion are about broad land use change patterns, but this study did not examine broader land use patterns, nor did this study indicate how knowing behavioral response will affect these populations.
Additional comments: By examining the predator response behavior of three anurans at habitat edges, this study offers insight into the potential effects of habitat fragmentation and land use change on three common species of anurans.

Introduction needs to both reduce unnecessary content, and introduce observational study better.

Lines 61-67: Unsure if this section is relevant.

Lines 74-77: Unsure if relevant.

Content setting up the observational study, including the relevance and ecological impact of alterations to predator response need to be added to the introduction. As written, there is little connection to the background and measured responses.

Lines 91-98: Site description unnecessary.

Line 99-101: Why does abundance of an organism make it a good model species?

Lines 99-109, given the scope of your study, it is important to give some background information on each species, especially as it relates to those species being relevant for study. However current background information, especially in lines 105-105 is not useful in the context of this study.

Lines 110-123: Much of this information is relevant to your study, and can be used to make predictions. As such, most of it would better serve the manuscript if moved to the introduction, thus providing the relevant background to set up your research question. Adding in a section on the relevancy and impact of different predator responses would also benefit the introduction.

126: Is a 1.5 meter path cut through a forest enough of a habitat alteration to warrant calling the boundary edge habitat?  If so, provide a reference.

129: How is an anthropogenically created habitat edge different from a mown path/forest edge?

145-150: This is more relevant background material that needs to be moved from the methods.

150-151: For what percentage of leopard frogs was SVL measured, and for what percentage was SVL estimated? A breakdown of estimated vs measured sizes needs to be included, in the supplementary material. Even so, it is unclear if these data are accurate enough to include.

157-160: Firstly, why measure month response? There is no relevant background material to support this analysis. Secondly, results should not be reported in the methods.

Analyses and results:  The authors state that they utilized a general linear model with logistic regression, though they did not show this model. The authors give specific results, i.e. frogs are more mobile at habitat edges, but do not provide overall responses of species and habitat section.

Discussion: Authors need to provide more ecological context for their results. Lines 215-218 are good, though more synthesis on why understanding behavioral responses to predators will improve future management/conservation work would be beneficial.


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: MOVE IT OR LOSE IT: INTERSPECIFIC VARIATION IN RISK RESPONSE OF POND-BREEDING ANURANS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors need to set up, in the abstract and introduction, that they are comparing different species due in part to their life history characteristics (i.e. arboreal vs semi-aquatic).

The manuscript contains many transitions, that seem to be in there for the sake of transitions. While not disqualifying from a scientific integrity perspective, it can be distracting when reading.
Experimental design: No comment.
Validity of the findings: No Comment.
Additional comments: The manuscript is a valuable addition to the field. It is well-executed. With minor improvements to the writing, it will be suitable 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: MOVE IT OR LOSE IT: INTERSPECIFIC VARIATION IN RISK RESPONSE OF POND-BREEDING ANURANS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This manuscript is much improved from the first draft and adequately addressed all comments.


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

Input:
Title: CHRONIC INTAKE OF HIGH-DOSE OF BLUEBERRY LEAF EXTRACT DOES NOT AUGMENT THE HARMFUL EFFECTS OF ETHANOL IN RATS
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In this manuscript, effect of high-dose BLEx (blueberry leaf extract) has been investigated on alcohol metabolism during chronic intake of ethanol.  Then, authors have concluded that chronic alcohol intake could increase the liver CYP2E1 and anti-oxidative protein expression, while the chronic high-dose BLEx did not augment (= modulate any) harmful effects of ethanol.  It is an important area for investigating whether functional foods (or so-called health foods) can improve the disease risks and also attenuate the harmful effect of disease settings.  Because the functional foods sometimes interfere with the medication, it may be necessary to demonstrate their non-toxic or no side effect in individual settings.  Before publication, please consider the issues raised as following.

1. As mentioned in Introduction in lines 77-78 with authors’ previous report (Yamasaki et al., 2016), this reviewer could understand why authors wanted to investigate the effect of high-dose BLEx on alcohol metabolism during chronic intake of ethanol, although the BLEx did not show any modulating effects in this paper.  To avoid concluding that results obtained here indicate just no effects, authors can emphasize the experimental differences in detail between previous work and this study in the Introduction and Discussion sections).

2. Line 85 in the Materials and Methods section: please describe how to prepare the hot aqueous extract of BLEx in detail.

3. Please describe the reason concerning the concentrations/amounts of BLEx and ethanol used in this study in text.  If you have any available data of pilot study(ies) or previous paper (as mentioned above), please discuss this point.

4. Please mention about HO-1 in the Introduction section, like as stated in the Discussion section.

5. In the Figures 1A and 1C, please annotate the experimental difference of ethanol 1% and 5%, for example, with additional lines and arrows, as stated in line 103.

Others
Line 42, in the Abstract section.  Typo?  ‘dody’
Line 138, in the Materials and Methods section. Typo?  ‘1,09,572’


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: CHRONIC INTAKE OF HIGH-DOSE OF BLUEBERRY LEAF EXTRACT DOES NOT AUGMENT THE HARMFUL EFFECTS OF ETHANOL IN RATS
Review round: 1
Reviewer: 2

Output:
Basic reporting: With respect to basic reporting, the manuscript by Yamasaki et al. is well written in professional English where appropriate. In addition, there are sufficient literature references used within the context of the manuscript. Moreover, the manuscript is structured in a traditional way such that it is easy to follow. The manuscript is also self-contained such that only results relevant to the hypothesis are presented. However, more detailed are needed within the statistical section such that readers can fully understand the authors’ choice in statistical methodology. Lastly, the authors should further develop a conceptual framework explaining why they think blueberry leave extract did not aid or reduce the harmful effects of ethanol intake. Could it be a time issue where the extract might have been given first, followed by ethanol? Could the null findings be related to a species phenomenon less relevant to humans? Might it have been the chronic dosing of ethanol vs an acute effect? These concepts should be addressed in a theoretical fashion within the discussion section.
Experimental design: With respect to the experimental design used by the authors, the manuscript represents original research and methodological approaches. The research question is well defined, and presented within a sociological context of drug use. Indeed, a relevant knowledge gap is attempted to be addressed with detailed research approaches. This reviewer believes that the authors of the manuscript attempted a rigorous investigative approach to answer their research question. In addition, the research provided by Yamasaki et al. was conducted within the conformity of ethical standards within the animal research field. While methods are described in detail, the use of table 1 should be moved to the methods section for further clarification. Additionally, the primer sequences table should be moved to a supplementary section.
Validity of the findings: With respect to the research findings, Yamasaki et al. present null data where the impact of blueberry leave extract on the deleterious bio-chemical effects of ethanol is found to be inconsequential. Despite not finding significant differences within the research data, this reviewer believes it is important to disclose non-significant findings. Indeed, the merits of the work is validated by clear data provided where across multiple assays, the research found similar results. Ethanol exposure does not differ when compared to ethanol plus blueberry leave extract. However, given the low number of animals per experimental group, this reviewer suggest the authors add more rats to each group in order to reach satisfactory conclusions. Regarding the statistical approach, much work is needed. Specifically, the authors should revisit the statistical section of the manuscript and fully describe their approach. More detail is required within this section.
Additional comments: The manuscript presented by Yamasaki et al. is well written and presented. It is interesting to find that blueberry leave extract does not protect against the effects of ethanol. In addition, blueberry leave extract does not further complicate the harmful effects of ethanol in rodents. The authors also conduct an array of assays examining food consumption, protein liver enzymatic levels, and mRNA expression of liver enzymatic levels. Comments that follow are intended to improve the manuscript. Within the abstract, on line 33, the word ethanol would be changed to alcohol. On line 36 the wording should be changed to “leaves have attracted attention as they are rich”.  On line, the last sentence of the abstract should be written as “Thus, chronic BLEx intake does not ameliorate the harmful effects of ethanol”.

Within the Introduction section, on line 61, the test “excessive ethanol consumption is a risk factor for liver disease (Gao et al) should be deleted. Within the methods section, on line 98, please state both the total number of animals, and rats per treatment group. On line 198, more distraction is required the statistics used. Did the researchers us an ANOVA? The results section requires much improvement. Clear statistical values are required per each assay conducted. Indeed, the text in the results section reads more like a figure legend. Furthermore, the western blot appearing in Figure 4 (A) should be redone or removed from the manuscript as the qualitative validity of the blot is poor. Lastly, similar to the paragraph on 302, the authors should continue to expand their conceptual notion explaining why blueberry leave extract did not impact the effects of ethanol at the behavioral, protein, nor mRNA level.


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: CHRONIC INTAKE OF HIGH-DOSE OF BLUEBERRY LEAF EXTRACT DOES NOT AUGMENT THE HARMFUL EFFECTS OF ETHANOL IN RATS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Issues raised by this reviewer in the previous manuscript have been accordingly improved in the current version.


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: DIGITAL DISSECTION OF THE PELVIS AND HINDLIMB OF THE RED-LEGGED RUNNING FROG, PHLYCTIMANTIS MACULATUS, USING DIFFUSIBLE IODINE CONTRAST ENHANCED COMPUTED MICROTOMOGRAPHY (DICE ΜCT)
Review round: 1
Reviewer: 1

Output:
Basic reporting: The study presents a detailed description of the lower back, pelvic girdle and hindlimb elements of the musculoskeletal system of Kassina maculata. Hypotheses and results are relevant and the manuscript is overall well written in clear and unambiguous English. I have a few suggestions to improve the manuscript which are detailed below.

Kassina maculata was transferred to Phlyctimantis in the phylogeny of Portik and Balckburn (2017 Evolution 70-9: 2017–2032). Please clarify why you choose not to adopt the latest systematic proposal.

In the introduction it would be nice to expand a little on the morphological adaptations of K. maculata which enabled the occupation of terrestrial and arboreal habbitats (lines 59-61).

The 3D digital models are very good, however most figures are lacking the scale bar. More importantly, figures showing gross anatomy can be improved. Most lack the scale reference, there is variation on background color (figure 8) and position of labels (lettering). Figure 7B has very poor quality. The spaces between each figure of fig 9 are visibly different, and it would be more elegant to use a scale bar on figures 9D and E. There is no need of adding a red arrow to highlight an element if it already has a legend (iliacus externus in fig 7 and plantaris longus in fig 9). Also, please check the order of reference of figures in the text.
Experimental design: The manuscript presents original research, with a well-defined and relevant research question, detailed methodological description and high technical standard.

Regarding the methods section, Table 1 is very informative, but I was unable to understand the difference in staining between rounds three and four, besides resolution. The authors recommend further staining on round 3, and there was no modification neither in stain concentration nor in time. Please clarify.
Validity of the findings: In the results, the anatomical description of muscles includes the tendinous elements, which is great! However, I noticed some important ones were missing. For example, the plantaris longus usually has two tendinous proximal attachments and inserts by the Achilles tendon, and those were not mentioned. The longissimus dorsi may have a tendinous insertion attaching to the urostyle, and the iliacus externus usually attaches to the proximal end of the femur by a strong tendon. Please check.

The discussion is well stated and linked to the research question. I suggest the authors take a look also into Fabrezi et al. (2014 Evol Biol DOI 10.1007/s11692-014-9270-y) and Fratani et al. (2018 Zoology, 126, 172-184) which include information on muscle and tendon variation and its relation to locomotion.
Additional comments: Minor comments:

62 – “robust to maintain” please rephrase that. Maybe add “in laboratory conditions”?

79 – While Xenopus is regularly used “as” a model…

145 – You should use “specimen” instead of species here. Please provide collection numbers for the individuals used in the study.

162-164 – This sentence should go in the materials & methods section.

176 – Does the Longissimus dorsi has a tendinous insertion in the urostyle? Not clear in the figures.

240 – (…) K. maculata “does” not deviate…

363 – (…) in “a” detail “level” that is challenging...

384-386 – Please include a reference paper for this sentence.

437 – (…) biomechanical significance of this “muscle pattern”...

488 – I don’t understand “exemplar specimen” here. Did you mean example specimen? Single specimen? Model specimen?

516 – (…) are critical for “the” creation of anatomical…

Table 2 - Extensor brevis superficialis (EBS) and flexor digitorum brevis superficialis (FDBS) appear to be missing.

Figure captions:

Figure 6 – There is no muscles in this fig so the sentence “See main text, or Table 2 for muscle
abbreviations” is not necessary.

Figure 9 – Please clarify in each caption if it is dorsal or ventral view of the specimen. There is a point where it is supposed to be a comma after semitendinosus.


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: DIGITAL DISSECTION OF THE PELVIS AND HINDLIMB OF THE RED-LEGGED RUNNING FROG, PHLYCTIMANTIS MACULATUS, USING DIFFUSIBLE IODINE CONTRAST ENHANCED COMPUTED MICROTOMOGRAPHY (DICE ΜCT)
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall the paper is well written in clear English, however, I think the writing still needs some polishing throughout the manuscript. I have pointed out at some examples below. Although the anatomical descriptions are clear and used terms are consistent with the nomenclature, they are too long so I would recommend summarizing the anatomy section of the Results. It might be important to consider how relevant these are, as muscle positions are summarized in Table 2.  Maybe adding an extra column to Table 2 with ‘Muscle description’ and giving a short description such as: ‘thin parallel muscle’ would help to reduce the descriptions in Results. Often, the muscles are compared to other frog species, which I consider should be part of the discussion instead (see below).
Scientific names are used throughout the manuscript, however, according to taxonomic nomenclature it is expected to abbreviate the scientific names after the first time a species name is mentioned and continue abbreviating from then on. So, Kassina maculata, should become K. maculata the second time it is mentioned. As well as the species genus, if the genus Rana is mentioned and later on the text a different species from the same genus is included it should be abbreviated as R. spp. The literature is well referenced, relevant and extensive. A few points are highlighted below, where I consider necessary to check the literature again and extend it. The segmentation and the 3D model are very well done, some comments for the figures are explained below that have to be addresed, a scale bar is needed in most of the images.
Experimental design: This paper investigates the gross anatomy of the musculoskeletal system of the spine, pelvis and hindlimb of K. maculata, as a generalist model (in locomotion modes) of a frog using diceμCT and gross dissection. The authors stated the aims clearly, although some re-writing should be done (see below), and present the first 3D model of the spine, pelvis and hindlimb anatomy of a frog with multiple locomotion modes that has potential use in a number of studies of functional morphology and biomechanics of jumping and walking. The methods are well defined but the writing needs some polishing. I would recommend to summarise Table 1, and include in Methods the staining and scanning details used for the final scan from where the model was generated (see below).
Validity of the findings: The research undertaken in this work is meaningful, the results are clear and the conclusion is relevant and well stated. The addition of a 3D PDF available is great and provides a useful tool for anatomical studies of anurans. The findings are interesting and deserve publication, once the comments included here are addressed.
Additional comments: Introduction
Line 59. It should read ‘red-legged running frog…’
Line 63. Generalist of what? Locomotion modes?
Line 74.  Literature is incomplete, a number of papers have provided tools to visualise soft tissue anatomy of different animal groups through diceμCT. See, for example, Bribiesca-Contreras and Sellers 2017 and Düring et al. 2013.
Lines 82-88. Aims of the work should be re-written as it is hard to follow in its current form. Capital letters do not follow after commas, it should be changed to lower case.
Line 87. Specify the source and location of the library and groups that are represented on it. It is not clear from the text.

Materials & Methods
 Line 91-92. Which one is the subheading? If both, one should be highlight some other way to distinguish between the two.
Line 95.  Is the removal of the heart part of the staining procedure? It should be explained why the heart was removed.
Line 96-97. Sentence is not clear: ‘to limit internal exposure to fixative and, initially, staining solution’. What is stained first and then fixed? The following sentence states that the sample was fixed first. The sentence should be re-written.
Line 99. Specify if this was done by moving the specimen to a PBS solution where if was left soaking overnight or by rising the specimen or so. It is not clear.
Line 100. ‘using 1 x phosphate buffered saline (PBS)…’ could be changed to ‘using a PBS solution’.
Line 101. ‘To enhance contrast’, delete ‘the’.
Line 102. What is the concentration of the solution? Include in the text or if different concentrations were used for each round of staining and scanning specify so or refer to table 1.
Line 103- 105. ‘The specimen underwent five rounds of staining…’ This sentence is too long and not too clear. It should be re-rephrased.
Line 138. Define acronym TMT in first use.
Line 146. Re-phrase sentence ‘This animal had been euthanized following experimental data collection not discussed here (euthanasia methods as above’. Perhaps something like ‘This animal had been euthanized as mentioned above’?

Results
It is not recommended to have references in results, although, is tempting in anatomical studies like this. Keep them to the minimum if you consider it extremely important to be mentioned here or, I recommend moving the comparisons with other species (lines 177, 179-180, for example) to the discussion.

Line158. A comma is missing after digits.
Line 162. Just saying ‘Figure 3-6’ is enough.
Line 162-163. ‘All muscles are named…’ This sentence is weird and should be re-phrased. Is there any nomenclature you used in particular? The muscle names are consistent in all these papers?
Line 210. Delete ‘for PY position’ in the brackets.
Line 214-216. This sentence can be moved to discussion and start the paragraph with something like: ‘In K. maculata the II originates…’.
Line 218-219. ‘Both the TFL and GL insert on…’.
Line 227. Add a comma before ‘however’.
Line 299. ‘…form the majority of the muscle tissue…’, do you mean mass? If so, it should be specified. The whole sentence should be re-phrased as is hard to follow.
Line 245-248. This sentence should go in the Discussion.
Line 263. ‘The smaller portion has similar origin and insertion…’.
Line 268. ‘Gemellus’ should start with lower case.
Line 268-269. The sentence within the brackets does not follow any previous idea, it should be deleted or moved as part of the discussion.
Line 325. ‘Origination’ should be ‘origin’.
Line 330-334. This sentence should be moved to the Introduction and, if necessary, mention a brief summary in the Results. The following sentence should be re-worded as it can’t be followed easily.

Discussion
Discussion is clear and consistent with the aims. The limitations of the method employed are well stated and clear. I would recommend however, including more references when talking about the limitations of using diceCT as multiple studies have discussed issues with staining, such as tissue shrinkage. It would be also important to add what you did to resolve those issues. Explaining why you decided to skin the sample during the staining period could help others to replicate your method. The anatomy discussion is good and clear, moving some parts from results could make it easier to understand (see above).

Line 349. Delete the comma after ‘first’.
Line 353-355. This sentence needs to be re-written.
Line 384. Reference is missing.
Line 400. Move reference to the end of the sentence in Line 402.
Line 466. A reference is missing at the end of the sentence, if the entire idea comes from Vickerton et al. 2013 this reference should be moved at the end of the sentence. Shrinkage of tissue has been studied repeatedly in similar studies using diceμCT, examples could help to make your point stronger as levels of shrinkage are not reported in this study. For studies where quantitative data of muscle is needed, knowing the shrinkage level would be very useful.

Conclusions
You do not mention anything about the use of diceCT in your conclusions. This is a crucial part of your study and such, it should be mentioned here. A brief conclusion stating in the technique was useful or not to visualise the soft tissue anatomy of the pelvis and hindlimb of K. maculata, and what is the bigger issue with this, such as the lack of tendons and mention that this is why it is important to combine traditional dissection.

Line 507. I don’t believe you are providing any quantitative data in your study.
Line 509. It is not common to include references in the conclusion. Re-phrasing the sentence could help to remove them or, at least, make the idea clearer. ‘According to previous morphological descriptions, K. maculata possess… ‘.

Figures and tables.
Figure 1. Add scale bar. The quality of the figure could be improved, traces of cellophane are evident in B.
Figure 2. Again, a scale bar is needed and quality could be improved. Adding a black background to the image would make it cleaner and the divisions of the different views won’t be evident.
Figure 3. I don’t understand what you mean by surface, it is superficial? ‘Superficial digital dissection’ could be best.  Re-phase second part: ‘See main text and Table 2 for muscle abbreviations. For interactive 3D PDF, see Supplemental Information’.
Figure 3-6. Figure legends must be improved, see above. The axis is barely visible in all the images, its size must be increased and a scale bar must be included.
Figure 7-9. The figure legends should be polished. For example, in Fig. 7., you can write something like this: The red arrow indicates the dorsal fascia in A, the fleshy iliacus externus […] in B and […] in C. The dashed lines show…’. In Fig. 8, you can write: ‘** show the presence of intersegmental septa and * denotes the separating septa’.
Table 1. I think this table is long and some parts should be better summarized, you don’t need two columns for the number of rounds and name of scan test type. Also, it could help the readers to include in Methods what parameters where changed during the scan test, such as solution concentration and skinning of the specimen, and mention why you decide this. As well as mentioning the details for the final scan used to produce the model.


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: DIGITAL DISSECTION OF THE PELVIS AND HINDLIMB OF THE RED-LEGGED RUNNING FROG, PHLYCTIMANTIS MACULATUS, USING DIFFUSIBLE IODINE CONTRAST ENHANCED COMPUTED MICROTOMOGRAPHY (DICE ΜCT)
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript presents a very interesting and useful methodology for the study of the musculoskeletal system in Kassina maculata, a specie with multiple locomotor modes and habitat uses. The technique is well explained and is useful as a guide for the detailed study of the musculature of this and other species. Although it is a non-destructive technique that allows keeping the material intact, it not allows visualizing tendons and ligaments. For that reason, the authors also carried out traditional dissections, which together allow a more detailed study of the musculature and the pelvic morphology.

The manuscript is clear, unambiguous, and there is a professional English language used throughout. Anyway, I suggest some changes for better reading and comprehension.

-In the first place, I suggest adding more information about the importance of studied the musculoskeletal system in anurans and the interspecific difference of the musculature.

-Secondly, the authors remark (Line 59) that K. maculata has multiple locomotor modes,[but they don’t add to the list that the specie can also climb (Porro et al., 2017)] and also mentioned that it is a terrestrial and arboreal frog. These make K. maculata a good “generalist frog model system”. Anyway, in line 81 the authors wrote: “We, therefore, present the first detailed digital dissection of a generalist (walking and jumping) terrestrial species”. I wonder why here the "generalist" definition is only limited to walking and jumping. Then, in the discussion section, the authors discuss that K. maculata is also an arboreal frog. I suggest writing a more clear definition of a “generalist frog model system”.

Literature is well referenced and relevant. Anyway, some of the quotes of the manuscript are not ordered chronologically and some of them have not the same format (for example &/and). Also, please check that all references have the same format (for example uppercase or lower case in the titles).

Figures and tables are relevant, well labeled and described. Nevertheless, I suggest some changes to improve them that are detailed below in general comments.
Supplementary material represents a very interactive way to study the muscle. If its possible I suggest editing the name of the viewpoints (for example: instead of back and front, ventral and dorsal views).
Experimental design: This is one of the first studies in anurans to use this very modern and not-destructive technique for the study of the musculoskeletal system. The selected specie has multiple locomotor modes and also is a terrestrial and arboreal specie. This makes the study very interesting not only from the field of comparative anatomy but also for biomechanical and morpho-functional studies
The methods are well detailed and are replicable.
The aims of the studied are well defined, anyway, the authors repeat this information in Line 62 and -a more complete idea- in line 83. I suggest reorganizing the paragraphs and emphasize the three principal goals of the work only once.
Validity of the findings: The data of digital dissection present here is a novelty and useful to extrapolate to other anurans species with similar locomotor modes and habitat use.
Also, the comparative analysis of the musculature with other species is adequate. Anyway, for better comprehension, I recommend checking the organization of the results. It is important that the description of the muscle follow the same sequence of the figures, which is also the sequence of a traditional dissection (from the most superficial layer to the internal layer).
The conclusion is well stated, linked to the research question. Anyway, in line 507 authors wrote that they presented a complete and quantitative assessment of the musculoskeletal anatomy. Instead, I think this research has a qualitative approach.
Additional comments: -Specific observations and recommendations.
*Abstract:
Line 17: I recommend adding the three goals of the work.
In Line 38, the authors remark the importance of the digital dissection of a terrestrial frog, nevertheless they mentioned that these frogs are not only terrestrial but also arboreal. This makes it more interesting for study. I think it would be important to remark this feature here and all over the manuscript.

*Introduction:
Line 46: the quote of Calow & Alexander is incomplete.
Line 47: Please check the paper year of Nauwelaerts & Aerts.

*Mat&Met:
Line 151: lack the quote of Duellman & Trueb 1986

*Results:
Line 154 to 264: I suggest reviewing the first paragraph; it seems that much of the information here is more adequate to material & methods section.
Line 170: It is not necessary to quoted Ecker 1889 in this section. You can add the quote in material & methods.
Line 190: please mention the muscle Coccygeosacralis before to the name abbreviation.
Line 191:  The CS and CI can be first seen in figure 3 (which shows the more superficial layer of muscle). The same for IE in line 202. For a better reading please mention since the first figure where the muscle appears. On the contrary, the reader could assume that this muscle appears in the Medial digital dissection.
Line 226, 243: It is better to start the sentence with the complete name of the specie.
Line 288: For a better reading of the text and figures, I suggest to inform that, both TiAL1 and TiAL2 can be already seen in figure 3.
Lines 295 to 300: neither the EBS nor ABP were mentioned in these lines.
Line 306: delete )
Line 322: check the word tarsometatarsal
Line 329: I recommend check the last part of the result section. Most of the information here could be incorporated into the Introduction or Discussion sections.
Line 336: Delete A-C from “Figure 10 A-C”
Line 342: check the word sacro.

*Discussion:
Line 373: Please review in this line and all over the manuscript that Lithobates catesbeianus is now Rana catesbeiana. Also Bufo guttatus is now Rhaebo guttatus. I recommend checking the current names of all the species in http://research.amnh.org/vz/herpetology/amphibia/

Line 375/376. It is not necessary to repeat this information due it has been written in Mat&met.
Line 378-384: I think that if you also discuss the locomotor modes that these species have (all together with the similarity and differences in the musculature) could improve this section. Moreover, it would be interesting to discuss the function of the muscles that are present in Ascaphus truei and Leiopelma hochstetteri. Can it be associated with the locomotor mode or use of habitat?
Line 393. Please mention all the locomotor modes for the specie.
Line 419. In the sentence: “Since lateral rotation of the IS joint is only freely permitted by the Type IIA morphotype (by definition)…” please write the corresponding quote.
Line 433. I think this section “GR and SM myology” can be incorporated into “comparative musculoskeletal anatomy” section. Please write the complete name of the muscle in the subheading.
Line 491 to 496: I suggest incorporate this paragraph to the previous section.
Line 503. I suggest mention GR and SM in this line.
Line 504. I don´t understand the authors when said: “augment anatomy…”
Line 569. The reference is incomplete.
*Figures
Figure 3 to 6: delete or increase the size of the x,y, z-axes in the bottom of the figures.
Figure 6 C. Please check this. Tarsals bones are between tibiale and fibulare and metatarsals.
Figure 7: (B) I suggest improving this photography. The quality is not good enough. (C) the photography is very shiny.
Figure 8: I recommend checking the size of white spaces between the photography. In D to F I suggest replacing the asterisk for head arrows, the septum will be more easy to identify.
Figure 9: I highly recommend edit this figure. The figure has many details that can be easily resolved. For example, a black bar under C label, the blue backgrounds between the photography is not equal, pinholes (F, G and I), and pinhead (H, F and I), different size of white spaces between the photography. The use of Photoshop or other software could be helpful to improve the figure.
*In Table 2:
 The origin and insertion of the tensor fascia latae (TFL) are not mentioned in the table. Neither the muscle of the foot: plantaris profundus (PP), flexor digitorum brevis superficialis (FDBS), transversus plantae proximalis and distalis (TPP and D), and intertarsalis (IN), extensor digitorum communis longus (EDCL), abductor brevis dorsalis (ABD), tarsometatarsal (TMT).
Please check the name of TiAL1 and TiAL2. Also, you wrote “tibialis anterior brevis” instead of “tibialis anticus brevis”.


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: DIGITAL DISSECTION OF THE PELVIS AND HINDLIMB OF THE RED-LEGGED RUNNING FROG, PHLYCTIMANTIS MACULATUS, USING DIFFUSIBLE IODINE CONTRAST ENHANCED COMPUTED MICROTOMOGRAPHY (DICE ΜCT)
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: In revision, you adequately addressed my suggestions. I believe the paper is of high quality and deserves publication. However, I just have a few more minor comments.

Intro
Line 93. Delete “(e. g. Through Morphosource)” at the end of the paragraph or include statement in previous parenthesis.

Results

Just use abbreviations in the text as you have already referred the reader to Table 2 where muscles acronyms are listed.

Line 175. Change comma for colon after ‘pelvic muscles group’ as you are listing muscles.

Discussion

Line 394-395. ‘May have’ is repeated twice, delete one.

Table 1. I recommend deleting ‘test round’ in the headers and changing ‘Total stain duration’ for something like ‘Cumulative stain duration’.

Figure 1-9. Why is the scale bar quoted as being ‘approximately 1 cm’? You should get precise measurements from CT images.


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: ANTIOXIDANT ENZYME CYCLING OVER REPRODUCTIVE LUNAR CYCLES IN POCILLOPORA DAMICORNIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: On the whole the manuscript is well written. I feel there needs to be more detail provided on the reproduction of P damicornis, as it is very interesting and relevant to the results here. The monthly planulation is not universal and varies a great deal geographically.
The captions for the figures could be improved. Figures should stand alone but there is not enough detail here for that to be the case. The titles of the figures should be removed from the figure with the details in the caption. It is unusual for results to be included in the figure caption. I think the figures may look better without the gridlines.
It is unusual to place report p values in an abstract.
The authors listed all the authors for many of the citations instead of using et al. This needs to be fixed. The authors also have not used standard unit abbreviations throughout the methods section.
The aims could be explained more clearly. What are the specific questions or hypotheses for the work?
Experimental design: Authors explained the relevance of this project well and I appreciate the argument that we must understand what occurs in normal conditions prior to understanding how environmental change might affect these factors.
Methods are described well with considerable detail.
Validity of the findings: I think this is important work. The authors need to discuss whether they can be sure that their findings are actually do to the reproductive cycle of the coral rather than occurring in response to the lunar cycle. I expect that they are due to reproduction, but this design does not preclude the lunar cycle being responsible, so this possibility needs to be addressed in the discussion. It would be good to repeat this work in a location where planulation is not talking place, so that changes due to the lunar cycle can be assessed.
I think the authors need to consider the possibility that P damicornis might be spawning gametes at this location. Spawning in this species can occur in corals that are also producing brooded larvae, but it is not obvious so can easily be missed.
Additional comments: This is a good contribution to an important area  where there are still considerable gaps in our understanding. I have made some editorial suggestions on the pdf.


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: ANTIOXIDANT ENZYME CYCLING OVER REPRODUCTIVE LUNAR CYCLES IN POCILLOPORA DAMICORNIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this manuscript the authors seek to characterize variability in antioxidant enzyme activity that is associated with monthly reproductive cycles in a brooding corals. Identifying the scale and timing of this variability could help investigators to separated induction of antioxidant enzymes by exogenous stressors from variation associated with endogenous cycles. In general, the language was clear, but I have detailed suggestions for improvement that will be provided at the end of the “general comments” section.
Additional references should be provided in two areas: (1) The authors have customized protocols for enzymatic activities; however, these analytical methods (e.g., choices of substrate, assay buffers, wavelengths for monitoring metabolite production or consumption) are certainly built upon previously developed methods which should be cited. (2) Related to multiple isoforms of SOD (see comment related to L464-7 in the last section).
The structure of the manuscript was appropriate. Checking for conformance to PeerJ requirements seems like it should be a job for the editorial staff.
I have several comments about figures: (1) The enzymatic activity figures are relevant (2-9, portions of 10-11); however, they are incompletely labeled. Asterisks are used to indicate statistical significance, but it should be clear from looking at the figure which specific contrasts are significant. Presently this information is only in the figure legend. A more informative presentation could be achieved by using distinct letters to indicate statistically different groups, or by marking the significant comparisons with brackets. (2) The manuscript frequently refers to reproductive and non-reproductive periods (e.g., Figure 4 caption), but these are not clearly defined or labeled on the figures. Are only the first quarter points considered reproductive, or (as suggested by Figure 4 caption) is new moon also included in the “reproductive” period? (3) I do not consider the western blots within figures 10 and 11 to be relevant. My specific concerns about the western blots will be included in comments on experimental design. In addition, the western blots are not described in the figure legends. (2) All the plots of enzymatic activity are necessary, but they could be more easily interpreted if they were combined into a smaller number of figures that each contain multiple sub-plots. One possibility would be to group Figures 2, 4, 6, 8 and 10 into one figure (because a reader would want to look at difference among enzymes in monthly timing) and Figures 3,5,7,9 and 11 into a second figure (for similar reasons).
Raw data are provided. I suggest that the Excel workbook containing raw enzyme values should be rearranged such that it is clear which values correspond to which colonies. This might be easiest if the columns corresponded to colonies and the rows corresponded to individual measurements (or the reverse). This is important given that the experimental design employs repeated sampling of the same colonies over time (as discussed below).
Experimental design: The manuscript contains original primary research that is within the scope of PeerJ (this also seems like it should be a job for the editor). The research question is well-defined and meaningful. While the rationale was clear within the main document, it was less clear within the abstract. A brief justification should be provided to indicate that there is reason to think antioxidant enzyme activity might vary over reproductive cycles. I do have some specific concerns about the methods and they ways that they are presented.
Individual colonies were sampled repeatedly over time, but the data were analyzed using one-way ANOVA. I believe that a repeated measures analysis is necessary given the experimental design used.
The methods used for waveform analysis are unclear. A frequency of 1 is equivalent to a perfect sinusoidal fit, but it is unclear how this parameter scales (i.e., 1.72 is considered a “not strong” fit in L353, but it is unclear what basis is used to designate a curve fit as strong or weak).
I recognize the potential value of western blot assays, the large amount of work the investigators devoted to this aspect of the project, and the frustration they likely felt when faced with variation in antibody batches. Still, I do not feel that the Western blot analysis makes a meaningful contribution to the manuscript. I have several concerns: (1) It is not clear what criteria the authors used in their preliminary assays to determine that “all antibodies…work in this coral species” [L271]. (2) It was later found that the authors could not reliably measure three of the four targeted enzymes, so no data are presented. There is no need to discuss these measurements that ultimately didn’t work. As is the description and discussion of failed measurements take up substantial space and result in substantial repetition within the discussion. I suggest that the failed assays not be mentioned in the methods or results. In addition, I suggest that these failed efforts will be briefly discussed (all three discussed together in one brief section). (3) The apparently even levels of SOD protein do not provide any additional insight and the data are relatively limited. No loading control is shown and there is no replication. How was the expected size of the protein calculated, or was this inferred from previous studies? There are multiple forms of SOD in most cnidarians (as well as their symbionts), so I wouldn’t necessarily expect only a single band. (4) I might have missed this but I’m not sure that the supplementary figure showing the full blot is referenced within the paper (or perhaps in the figure legend). If these data are retained, it’s important for the reader to know that they can look at the full blot.
Validity of the findings: Some of my concerns are reflected in the preceding section. I recommend that the authors incorporate repeated measures into their statistical analysis. Additional information is needed to enable full evaluation of the waveform analysis. Additional information would also be needed to ensure that the SOD western blot analyses are valid (although I have recommended removing these).
Temporal patterns in enzymatic activity are interpreted in the context of lunar reproductive cycles. There is no direct demonstration of causation. The interpretation is strengthened by the repeated sampling through two lunar cycles and the waveform analysis. No environmental parameters are measured, and the reproductive status of each colony is unknown. The authors do address some of this uncertainty late in the discussion (L478-481). Some qualification of the interpretations should be given earlier in the manuscript (perhaps around L400). As a suggestion, the authors point toward controlled laboratory studies as a natural future extension of the work. The authors might consider also adding monitoring of reproductive output. This would be particularly powerful within the context of a repeated measures design, such as the authors have used.
Additional comments: My most substantial comments have been incorporated into the preceding sections. I think data are valuable, but would particularly like to see revised statistical analyses and presentation of significance results on figures.
L54 language imprecise. Suggest something like “wide application in assessing responses to diverse stressors” or “their involvement in physiological responses to a variety of stressors”
L74 This is intended as a question and not a criticism. Regarding “prior to adopting these enzymes into our suite of diagnostic tools.” Arguably the authors have already done this to some extent (Downs et al. 2006 & 2012 and Rougee et al 2006 and 2012 include Richmond as a co-author). I have not cross-checked all of these references in detail, but at least some of them include measurements of some antioxidant enzymes (protein levels rather than activity, I think) in the context of ecotoxicology studies. The authors can make their own decision about this, but it might be useful to edit the statement. Possibly something like “Prior to continuing and expanding our use of these enzymes…” (?)
L78 This use of “defense enzyme activity” is vague. Suggest “cyclical variation in the activity of xenobiotic metabolizing enzymes…”
L88 I don’t think ROS has been defined yet (defined in L92).
L96-7 I don’t know what is meant by “systems other than those found in Cnidarians” (what “systems”?) I think the authors mean, “studies conducted in non-cnidarian species” or similar.
L111 Presumably antioxidant enzymes exist for many purposes, some of which are in included in this sentence.
L117 enzymatic activities are not necessarily “enzymatic stress levels”
L118 I’m not sure what is meant by “application”. Authors might simply mean “distribution” or they might mean both a broad distribution, and the frequency with which P. damicornis has been used in physiological or toxicological studies.
L119 As written, it might imply to a non-specialist that brooding is unique to P. damicornis. I suggest “unlike many other common reef-building corals…”
L125-126 I have no idea what the authors are referring to here. If previous studies have demonstrated monthly cycles in antioxidant enzymes, they should be cited and clearly described here and differentiated from the current study. Similarly, which studies have looked for and failed to find an annual cycle in antioxidant enzymes? Such a cycle might be expected in response to seasonal changes in light and temperature (as indicated by the authors in other parts of the manuscript). L473-474 suggest that monthly and/or annual cycles have already been characterized for at least some of the antioxidant enzymes. The manuscript needs to clearly indicate how the present study distinct from the work by Rougee et al. 2014.
L128 syntax doesn’t make sense here.
L131 I don’t understand this either. Internal cycling is a natural phenomenon. What stresses are the authors talking about? Presumably they are actually interested in anthropogenic stressors as well.
L143-145 I didn’t fully understand this. The authors discuss variation along the length of the branch, but it’s not clear which portion of the branch was used (presumably a fragment that incorporated both the tip and mid-branch polyps)? I don’t really know how the sampling design reduced variation. Samping branches from different parts of the colony is different from sampling tissue from different portions of the branch. (I know the authors know this, it just needs to be explained more clearly).
L139-153 In the comments to reviewers, the authors included a justification for the sample size based on preliminary power analyses and other considerations. This information could be included somewhere in this paragraph.
L154-6 Two very different ideas are lumped into this sentence. Please separate. The first part probably belongs in the previous paragraph.
L157-8 It’s stated twice that the samples were stored at -80. I think this could be streamlined (I think the authors mean that they were frozen immediately upon collection and then later after pulverization, but the description could still be simplified). I don’t think naming the freezer model is necessary.
L162-3 This largely repeats L157-8 and the two descriptions of pulverization should be combined.
L172 It is not necessary to describe the preservation of the zoox samples because they were not used in any analyses within this manuscript.
L178-9 I’m pretty sure this also includes buffers
L227 microtiter
243 substrate [comma] and buffer
L245 Since preceding sentence is plural and two classes of GPx are measured, I suggest “GPx enzymes use…” or “Both forms of GPx use…”
249 decreases are proportional
L250 96-well microtiter
L256, L261 the NaN and CHP (and maybe some other components) are only added to assays for one of the GPx forms
L266 I have no idea what is meant by “Materials…were sourced in-house” I think the sentence could just be deleted. (but I also think this section should be deleted).
L266-310 I’m not commenting in detail because I feel this section should be deleted.
L316 I don’t think the variance itself is significant. I think the differences in the means are significant in the context of the variance associated with each group (but as previously mentioned, I think a different analysis should be conducted).
L317-320 Were these analyses really conducted in GraphPad Prism as indicated in L313? I could not find a waveform analysis function, although it might be possible do perform one.
L320 I don’t think it’s necessarily true that values above or below these are caused by external factors, just as it’s not necessarily true that all this variation was caused by “internal” factors.
L338 suggest replacing “cycles” with “phases”
L358 suggest “across all moon phases and throughout the acute collections”
L361 Conversely to what? Several patterns have been described.
L363 please be more specific than “collection periods throughout the sampling cycle” Significantly higher than all other sampling times?
L395 is “potential” needed here?
L397 changes in activity of antioxidant enxymes is not necessarily “stress flux”
L399 suggest more precise wording than “reproductive baselines”
L402 write out species
L407 Not sure what is meant by “greater” More importantly?
L420-422 This is not necessarily true. For example, an increase to 66 during a “low” part of the cycle might be due to external stimuli. Reproduction varies on a lunar cycle, but also exhibits some annual periodicity. In the winter a smaller change might indicate a response to some external stimulus.
L422-425 This statement is relevant to all enzymes and not specific to CAT. Suggest it be moved to an introductory or concluding paragraph.
L444-6 Not sure exactly what is meant here. Are the authors suggesting that with some additional experimentation, wave fits would be useful, or that some other curve fitting could be done? If additional experiments are needed, how should they differ from what has already been done?
L455-457 Needs some edits. As is current syntax is confusing “Previous studies analyzing GPx activity…confirmed activity and presence”
L460 agreement problem: increases…that (plural/singular)
L463 not sure what is meant but “adaptive function of their cellular detoxification”
L464-467 Authors should cite some work from Allemand lab in discussion of multiple SOD isoforms. Although the work was conducted in anemones, it is in my mind the most thorough study of SOD activity conducted to date in cnidarians. I don’t have access to full internet right now, but I think the most appropriate reference is likely Richier et al. 2003 (which the authors have cited elsewhere).
L473-4 Within this sentence, it is unclear how the present study is different from what was already done by Rougee et al. (see similar comment L125)
L478-481 To do this, changes in environmental conditions should also be measured.
L489-490 I don’t agree that the blots confirm that the assays were effectively designed.
L500-501 The limitations of the western blots don’t necessarily impede proteomic assays. The probably are specific to antibody-based measurements. Proteomics more commonly refers to mass spec-based methods (such as those discussed in L502-3).
Figure 1 caption: replaced “of interest in “ with “sampled during”
Fig 2 caption (and perhaps other captions) “moon phase cycle” doesn’t make much sense. Suggest “moon phase” or “phase of the lunar cycle”


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: ANTIOXIDANT ENZYME CYCLING OVER REPRODUCTIVE LUNAR CYCLES IN POCILLOPORA DAMICORNIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: I am satisfied with the changes made by the authors and feel that the manuscript is much clearer in its intent, methods and results now.
Experimental design: I have no further comments. I am happy with the changes made.
Validity of the findings: The results are now more clearly presented and discussed than they were in the first version of the manuscript. I have no further comments.
Additional comments: The authors have made a thorough attempt to consider all comments made by both reviewers in their new version of this manuscript. I am pleased with this new version and feel that it is ready for publication. I have no further suggestions or criticisms of 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: ANTIOXIDANT ENZYME CYCLING OVER REPRODUCTIVE LUNAR CYCLES IN POCILLOPORA DAMICORNIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: I understand from their response that the authors were not able to conduct a repeated measures analysis due to analytical issues. This should be mentioned somewhere in the statistical section. It is not strictly correct to conduct one-way ANOVAs because independence is an assumption of the test.
Validity of the findings: No comment
Additional comments: The manuscript is much improved, but there are issues that remain to be addressed.
1.	I think there is at least one error in Figure 2. To really understand what is going on, it would be helpful (1) if authors referred explicitly to subfigures in text, and (2) if subfigures were arranged in the order that they are discussed. As is, SOD is 2b but is discussed last. The text does not match what is shown in the figure.
2.	The same is true for Figure 3. L318 says SOD activity is higher on day 5 than days 3 and 4. That is not what is shown in Figure 3b (day 5 is the lowest in the figure.
3.	Regarding waveform analysis, it is helpful that the authors explained to me in the comments how they did the analysis. This information needs to be included in the text such a reader could reproduce it.
L16 [,] and action
L68 suggest replacing “expression” with “activity” (since activity was measured in this study)
L126 suggest authors consider a different phrase than “inherent stress.” This is just my opinion. I would suggest a phrase like “inherent variation in redox state” or “inherent variation in cellular physiology” or “endogenous cycles in cellular physiology”
L223 suggest removing “Using the following protocol”
L394-408 Repeating a variation on my comments on the previous draft. I think it is very important to mention that SODs are a family of enzymes that even vary in their subcellular localization. It’s not particularly surprising that there could be cycles driven by multiple factors on multiple time scales. This is true of some of the other enzymes measured too. It should be clear to readers.
L424-430 The idea that proteomics (given sufficient analytical ability) would be useful is good, but the transition doesn’t make sense as written. I wonder if this text is left over from earlier version that included the westerns. There is nothing wrong with the overall text, it just needs to flow more smoothly. “This work” doesn’t particularly highlight deficiencies in capacity to do proteomics. It’s more that the proteomics would be a great follow-up and the abilities currently aren’t there.
L449 agreement problem. Data…help
Figures 2 and 3 Why do the captions say “CI = 95%” if confidence intervals aren’t shown? According to the caption, the error bars are standard deviation.


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 COMPLETE CHLOROPLAST GENOME OF FAGUS CRENATA (SUBGENUS FAGUS) AND COMPARISON WITH F. ENGLERIANA (SUBGENUS ENGLERIANA)
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is well written report of a chloroplast genome. The work is new and well performed. However there are now dozens of chloroplasts genomes in databases, they are very well conserved as it is in this case. In my opinion the results should be part of a more comprehensive genome analysis if Fagus species or it should be downloaded in a database. There is no reason for a specific publication
Experimental design: This is a well performed work, well described methods and no special comments
Validity of the findings: This is a report on a chloroplast genome from a new species that just confirms that in this case is also very well conserved. They found 0.197% of differences between the two species studied. This finding is not a reason for a full paper publication
Additional comments: The result should be put in a database or to be included in a more comprehensive analysis of Fagus species. AT this point there are dozens of chloroplast genomes and it is well known that they are well conserved in most of the plant species studied. The novelty is inexistent.


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 COMPLETE CHLOROPLAST GENOME OF FAGUS CRENATA (SUBGENUS FAGUS) AND COMPARISON WITH F. ENGLERIANA (SUBGENUS ENGLERIANA)
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is a very good study concerning the assembled chloroplast genome of Fagus crenata. The manuscript is generally very clear and well-written although there are minor text revisions that I think the authors can still perform. For instance, the authors repeat across the text that the “assembled genome was a total of XXX bp” I see this sentence published in many studies but I still think the correct sentence would be “has a total of XXX bp”. Other minor typos should also be checked (e.g, line 99” following by manual modification”).

Figures and tables are clear and very well explained. Congratulations to the authors for this!

The main limitation of this paper is that the Discussion is very poor. In fact, there is almost no discussion. This is really a disappointment because the authors have clear, solid results that could be used to construct a very good Discussion. References are very scarce and limited.
Experimental design: The experimental design is overall correct but more details are needed. For instance, why was a NJ tree constructed? This seems to be a poor analysis in comparison to other widely-used methods (e.g, maximum likelihood in RAxML, Bayesian analysis in MrBayes). I also assumed that you only used the coding genes to built the tree….is that correct? Please clarify.

Where is the list of microsatellite regions? This should be part of the main text. There are no other SSRs above tri-nucleotide repeats?
Validity of the findings: Data is robust and sound but there is almost no Discussion. No clear conclusions are given.
Additional comments: This is a very good paper that can potentially be published in PeerJ. It has no flaws and it is overall very clear. The only weakness is the lack of a solid discussion.


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

Input:
Title: THE COMPLETE CHLOROPLAST GENOME OF FAGUS CRENATA (SUBGENUS FAGUS) AND COMPARISON WITH F. ENGLERIANA (SUBGENUS ENGLERIANA)
Review round: 1
Reviewer: 3

Output:
Basic reporting: The authors report on the sequencing, assembly, and annotation of the chloroplast genome of Fagus crenata (subgenus Fagus), and its comparison with the only other chloroplast genome available for the Fagus genus (F. engleriana subgenus Engleriana).

This is a very concise manuscript describing in detail the organization of the chloroplast. It is well written in a clear manner and professional English language is used throughout.

To the best of my knowledge, the Introduction and background cover the relevant literature.

There are some oversights on part of the authors regarding the availability of their data that hinder the full reproducibility of their work.

1.	The authors should deposit the original short reads from their NGS experiment on an appropriate database (for example the SRA at NCBI).

2. The Multiple Sequence Alignments (MSA) of the full chloroplasts mentioned in line 102 and used to produce the phylogenetic tree shown on figure 2 should be included as supplementary data as a plain-text file in an appropriate format (FASTA aligned, Clustal, nexus, phylip, for example).
Experimental design: The methodology is, in general, of sufficient clarity and detail. There is only one point of their methodology that I consider should be carried out with more care: the phylogenetic inference presented on figure 2.
Firstly, the authors should be more precise when detailing the construction of the Multiple Sequence Alignment. Currently, it is only stated that MAFFT v7.308 was used (Line 102). From the previous line I assume that this was done inside Geneious v9.0.5 but no other information of the parameters used is stated. MAFFT v7.308 offers a range of multiple alignment methods (FFT-NS-i, E-INS-i, L-INS-i, G-INS-i and some others), each being a collection of parameters best suited for different situations. For the case of closely related chloroplasts either L-INS-i or G-INS-i could be the best option (this should be tested on their data and explicitly stated on the methodology section).
Secondly, the default parameters of the Neighbor Joining method under Geneious (or any other phylogenetic reconstruction software) should be adjusted for the Multiple Sequence Alignment used. The most important parameter being the DNA substitution model selected and its modifications (gamma parameter and/or invariable sites). These should be calculated through an appropriate estimator application (for example jModeltest2, prottest, or the one included in MEGA). It is probable that the structure or topology of their tree may not change but the resulting phylogeny will have a better estimate of individual branch lengths and total length of the tree (which also should be included on the description of the tree).
Lastly, although the tree on figure 2 shows bootstrap confidence values, no mention of how many repetitions were used to calculate those values. The information of how many total/informative positions were on the Multiple Sequence Alignment  should also be included.

For the sake of helping visualizing the conservation/divergence of the chloroplast data I would suggest the following changes be made:

On lines 121-122 the authors state: “The two Fagus chloroplast genomes were relatively conserved (Figure 3) with the IR region more conserved than the LSC and SSC regions.” The IR, LSC, and SSC regions should be marked on it. Using Morella rubra as the reference makes it difficult to assess the relative conservation between both Fagus species. I would suggest making Fagus crenata the reference for this figure.

These same data could be summarized on a table showing the percent identities in a pair-wise manner (F. crenata vs F. enlgeriana, F. crenata vs M. rubra, and F. enlgeriana vs M. rubra) for the whole chloroplasts and for each of the regions. Also, the total gaps on the alignments should be included.

Optional minor detail:

The “most variable regions” (Lines 127-128) could be ordered from highest to lowest.
Validity of the findings: Although this reviewer understands the importance of reporting the assembly and annotation of the chloroplast genome for future studies into the inter- and intra-specific genetic structure and diversity of the genus Fagus, I encourage the authors to expand on its discussions about the implications of the divergence found on coding and non-coding regions, and the relevance of the SNPs and SSRs  as possible markers.
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: THE COMPLETE CHLOROPLAST GENOME OF FAGUS CRENATA (SUBGENUS FAGUS) AND COMPARISON WITH F. ENGLERIANA (SUBGENUS ENGLERIANA)
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have addressed all of my stated concerns.
Experimental design: The authors have addressed all of my stated concerns.
Validity of the findings: The authors have addressed all of my stated concerns.
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 PHARMACOLOGICALLY-INDUCED AFFECTIVE STATES ON ATTENTION BIAS IN SHEEP
Review round: 1
Reviewer: 1

Output:
Basic reporting: Dear Jessica, dear Caroline, dear co-authors,

As you may guess my identity quite easily anyway (and the editors did not think that our past and current exchanges are too much of a conflict of interest), I make this a non-anonymous review.

As with your other recent work, I very much appreciate your approach, which is presented in clear language, refers to the relevant literature and is properly structured. In respect to the formalities, I am unsure only because it seems that raw data should be supplied which, at least, was not included in the materials available to me.

In my view, a balanced set of stimuli is necessary for an attention bias test such that, in a neutral state (your control group), both stimuli elicit a similar amount of attention. Only then, we can hope to see changes towards either the negative or the positive stimulus according to some experimental manipulation. I assume that the “memory” of the dog (that is no longer visible) and the picture of a real sheep is an attempt to achieve such a balance. Perhaps, you could be more explicit about this? Moreover, the implication of the fact that the stimuli to which attention is directed are different (live animal versus photograph; memory versus current presence) would strengthen the discussion in my opinion. Given Fig. 2, the balance in respect to attention in the control group was more or less though not ideally achieved (they directed more attention towards the dog-wall than the sheep-wall). Does that have consequences on what you try to find

Your conclusion mainly focuses on positive states (the inclusion of which was novel in this experiment; e.g. line 41). As far as I can see, you could not replicate the previous results (introduced on lines 54-49 and 67-73) as clearly, either. I think this merits some more space in the discussion. I think the notion that the previous experiments could have been spurious results needs to be considered as well. Even if, in the end, the arguments that you list and that indicate that the current experiment may have had its problem are the most likely explanation for the lack of differences in your current study.

Quite often, you talk about emotional “states” without specifying whether you mean short-term emotions or longer-term mood. I think, at least conceptually, this makes quite a difference because, e.g. emotions are much more specific than mood states. I think the manuscript could be improved if you were more specific in this respect or explain the complications about being more specific and what your results could imply for these different types of emotional states.

It seems that you have a clear notion (which is well argued for) what states you consider more or less negative, “neutral”, and positive. I think it would help the reader if you would list the different treatments in this order (from strongly negative to positive) throughout in the text and in the figures. One might expect some monotonous trend (or some [inverse-]u-shape) across these treatments and the existence of such a relationship would be much more easily visible if the treatments were accordingly sorted.

As far as I can see the internal body temperature is not really introduced. Therefore, it seems to come as a little surprise on lines 197ff. As you provide this data at some length, a more proper introduction with specific hypothesis may be useful.
Experimental design: As far as I can see, the experiment was well designed and executed. I have some minor to moderate comments on your statistical procedures, though, that you might want to reconsider in a revised version of the manuscript.

Overall, I find it quite hard to follow which statistical procedures were used for which outcome variable. Perhaps, this could be added to one of the tables. Moreover, in some instances, it is not clear to me, which fixed and random effects were included in which model. Some more detailed listing would be here as well in my view. In addition, it may be useful to know which specific procedures from which packages you used for the different models. A detail: if you used R 3.5.1 it should be cited as R Core Team 2018 (and not 2015).

I would very much appreciate if there was one single table for all the different outcomes that were evaluated. This provides an easy overview and the possibility for an assessment of multiple testing issues (the increasing probability for finding a random low p-value with each additional test conducted). This would mean to combine Table 2 (in which global tests are given) and Table 3 (only pair-wise comparisons, why no global test?) and move those evaluations only mentioned in the text to the table, too (lines 276-284).

In my view, a bias can best be explained as the concurrent focus of attention. Therefore, the first outcome variable that I would be interested in would be the proportion of time spent attentive on the dog wall divided by the total attention spent on the dog or sheep wall. (The overall attention to these walls could be used as a weight in the evaluation to account for the fact that this proportion can more exactly be calculated the longer the attentive period). This would directly show a shift from one wall to the other. It would also account for the fact that the sheep in the test could not be attentive to both walls at the same time (sum-one-constraint). I am not sure whether and how this pattern could be seen directly in the absolute time that was directed to one or the other wall. Similarly, I miss the amount of time standing near (or the relative proportion of dog-door versus sheep photo) or sniffing the dog door (or, again, the relative proportion).

I was unsure whether all of the outcome variables are really needed. E.g. is there really an expectation in respect to the attention to the door wall, the back wall or sniffing the environment? If yes, I think these would need to be stated. I would rather tend to drop these outcome variables.

Moreover, I think some outcome variables are evaluated several times in slightly different ways, e.g. the duration of vigilance, failure to be non-vigilant, and vigilant for 180 sec are basically the same outcome variable. In my view, the survival analysis does the proper job for censored durations here and should be the only model reported for vigilance. Similar cases exist for the categorised outcomes that are only mentioned in the text and for the other two survival models (sniffing photo and sniffing environment).

Some more statistical details:
•	Line 218: how was test order coded? Test order across treatments or within a test-day? You did not include any interactions? Was there a specific reason for this?
•	Line 220: Type of elimination approach? Based on which criterion?
•	Line 230: How can a Fisher-Test be applied to a contingency table larger than 2 x 2?
•	Lines 242ff: Did you include the same random effect (clustering) as in the mixed models? If not, why not?
•	Lines 247: Usually, such auto-correlations are only valid for equally-spaced time points. This is not the case here. Did you somehow adjust for that?
•	Lines 287ff: Main effects can only be interpreted in a model including an interaction if sum-contrasts are used. Did you do so?
•	Lines 296-297: I am lost here. I am not sure what you did nor why this (subset?) was evaluated.
•	Fig. 3: Obviously, many survival analyses use the terminology of “failure-time”. Here, I find this term confusing. Could you use another Y-axes description?
•	Fig. 4: This data was not right-skewed, either? I am not sure what the full and what the post-hoc models were here (specifically for the comparisons within a time-point, there is no repeated measurements of the individuals, right?). In my view, the random effect here might have included the sheep identity nested in the test day. Was that the case?

In respect to statements as on lines 215-216, I advise the reading of the ASA statement on the use of p-values (https://doi.org/10.1080/00031305.2016.1154108), but overall this has no major implication here. But e.g. in Table 2, the close-mouthed bleats reach a large p-value only even though the mean differences between the groups are quite large.
Validity of the findings: I always admired your pharmacological approach such that I was almost comforted a little that this approach does not automatically lead to a straightforward interpretation of results. You discuss this issue well and at length. I was wondering whether there are some data of e.g. place preference tests with sheep receiving one or the other drug that you could cite or whether such tests could be recommended as possible next steps to solve some of the issues that you raise. Alternatively, do you have other recommendations how the effect of the different drugs should be evaluated in sheep?

As stated above, I think a somewhat more critical general discussion (lack of replication of previous results) would be good.
Additional comments: I have only some minor additional comments:
•	The abstract may need some revising in respect to the results if some of the issues mentioned are changed.
•	Line 46: only before? It could also be for longer within a certain time window, right?
•	Lines 119-120: Could you elaborate a bit more why this was your expectation?
•	Line 163: I am aware that you previously define what you mean, e.g. by “happy”. Nevertheless, this is one place where just using “happy” struck me as a bit too simplistic (and anthropocentric). Could you re-evaluate your terminology in this respect? E.g. using the drug name instead of the presumed state?
•	Line 175: Could this process of removal be perceived (heard) by the sheep? Do you have any (qualitative) indication that the sheep knew whether the dog was still there or not?
•	Lines 188-190: Should/could you state which treatments were involved for the 6 occasions of score 3?
•	Results: As you summarise some of the results in the text, I am often unsure which specific outcome variables you address. Could you be more specific in the text?
•	Lines 342ff: But you did find difference between the treatments. Therefore, this ceiling effect would not have been a worry for all of the treatments, right? Also, the fact that censoring took place mainly in one of your groups, actually makes the groups more distinct and should therefore not be a problem (as it is accounted for in the survival analysis).
•	Line 396: “less clear” and then the difference between happy and other sheep: is this not a contradiction?
•	Figures: I would welcome if some more of the outcome variables were shown graphically. Also, I very much like to see some of the raw data as well (e.g. by using boxplots).
•	Table 2: Could you include the degrees of freedom in this table, too?
•	Conclusion: I would like to see a mention of the “negative” treatments as well.
•	Line 447: Are dose-response studies the only necessary studies or could alternative approaches be useful (see above)?

I hope to see a revised version of your manuscript soon and I think given my comments above, this should not be too hard to achieve. Best wishes, Lorenz (Gygax, HU Berlin)


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 PHARMACOLOGICALLY-INDUCED AFFECTIVE STATES ON ATTENTION BIAS IN SHEEP
Review round: 1
Reviewer: 2

Output:
Basic reporting: The literature is appropriate and well referenced, except for R version 3.5.1 (it is more probably the R Core Team 2018 than 2015 and the full reference should be “R Core Team. 2018. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL http://www.R-project.org/”. Everything else is fine.
Experimental design: No comment, but see (minor) comments on the Materials & Methods section.
Validity of the findings: No comment, but I would have liked to see more discussion about future studies going towards a more on-farm setting of the attention bias test, and critics about the stimuli used (see my comments).
Additional comments: The authors present a modified attention bias test to assess emotionally induced positive and negative states in sheep. The approach is interesting and overall well explained. The study is well performed and in general well reported but see my detailed suggestions thereafter:

Abstract
Lines 20 and 63: add a dash between “pharmacologically” and “induced”.
Lines 23-24 and 35: for consistency with the manuscript put a capital letter for each treatment group: “Eighty 7-year-old Merino ewes were allocated to one of four treatment groups: Anxious (m-chlorophenylpiperazine), Calm (diazepam), Happy (morphine) and Control (saline)” and “Anxious group”.
Lines 25, 30, 153, 163, 204, 208 and 378: use the SI units, i.e. “min” instead of “mins”
Line 25: You say there that the test arena was “4 x 4 m” while line 179 it is “4 x 4.2 m”, so please be consistent.

Introduction
Lines 45-46: please precise that “An attention bias occurs when an animal attends to certain types of information before or for longer than others”, as you explain it after.
Lines 46-48: could you please reformulate this sentence that is currently unclear.
Line 51: “principle” instead of “principal”
Line 56 and 64: I suggest reformulating “Anxious sheep” to “anxious-induced sheep” as you use the formulations anxiety-like states, induced calm and controls in this paragraph referring to previous studies.
Lines 79-80: include the “e.g.” in the references’ parenthesis.
Lines 90-92: I understand that pharmacological agents can be used in a more standardized and repeatable manner that environmental manipulations, however I think you should discuss in the discussion section) the advantage of using various environmental manipulations to induce emotional states for future applications in more on-farm settings. Animals that are not experimental animals have their emotional states influenced by the environment, and not by drugs.
Line 98: add “e.g.” in the parenthesis, and if possible give examples of other contexts where diazepam was used to reduce anxiety-like behaviours.
Line 105: remove the coma after euphoric-like.
Line 118: add that the Calm and Happy groups “would both also pay more attention […]”.
Line 122: replace “morphine treated group” by “Happy group”.

Materials & Methods
Lines 142-143: could you please rephrase. It is weird to say at what time the first injection was (without specifying that it was always 30 min prior to testing) and when the last test was performed.
Lines 147-149: could you precise whether the sheep were used to be handle? I know the sheep were 7 years old but they were only handle for (negative) handling procedures it could have an impact on how they reacted to the test, e.g. being highly vigilant. Also, please precise whether the sheep were bred, raised and tested on the same farm and how they were housed, specifically before the attention bias test.
Line 151: can you provide the peak effect of all the drugs used: it is always 30 min after i.m. injection?
Line 153: remove the capital letter of “Anxious” state.
Line 161: define “i.v.” used for the first time in the manuscript.
Lines 164-166: precise the mode of administration. Was it also i.m. for the morphine and saline?
Line 169: in Figure 1, please precise in the legend that two video cameras were used: a first camera was position to obtain the view angle presented in this picture while a second camera was positioned above the arena to the right of the photograph.
Line 172: “life-size” instead of “life-sized”
Line 173: precise that the photograph was from an unfamiliar female conspecific. Also, you should discuss the fact you used the same dog and photograph of sheep for all the test. This can be seen as problematic for generalisation of the test because you observe responses to a specific dog/photograph. Additionally, I would like to see discussed the difference between the two stimuli used: one is a live animal whereas the other one is a life-size 2D colour photograph. Concerning the experimental set-up, after 3 s you hide the dog by covering the window: don’t you think that this movement might attract vigilance towards the window?
Lines 176-177: add the detail about the camera right after you talk about it such as “[…] behaviors were recorded using a Sony Handicam video camera (Sony, Australia, model number HDR-XR550) positioned above the test arena in an adjacent building”.
Line 182: in Table 1, could you precise whether you assess the positions of the sheep ears to define its vigilance behaviour. A sheep with passive ears for instance cannot be defined as vigilant. Also, did you measure the attention and vigilance behaviours continuously, or e.g. every 1 s? Moreover, see my comments below on the elimination definition (as you present the urination behaviour only).
Lines 184-188: explain the hypotheses behind the behavioural measures (i.e. activity, arousal)
Line 199: remove the double parenthesis next to each other such as “(Model number DS1922L-F5, accuracy 0.5°C, resolution 0.063°C, weight 3.3g; Embedded Data Systems, Lawrenceburg, USA)”.
Line 206: It seems, according to the data files, that data from 4 temperature loggers were missing (sheep No. 20/40/41/70). Please correct it or explain the missing data.
Line 215: according to the R version used, it is more probably the R Core Team 2018 than 2015. Also, please provide the full reference detail: R Core Team. 2018. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL http://www.R-project.org/.
Lines 236-238: give the reference about the pscl package right after you talk about it such as “Vocalization data were analyzed using a negative binomial hurdle model using the package pscl (Zeileis, Kleiber & Jackman, 2008), to account for the presence of excess zeros in the dataset”.
Line 239: you analysed the eliminations that is according to your definition the number of urinations and defecations, however you give the data and present the results only for urinations. I guess there were not enough defecations for the analysis, but please precise it or change the elimination definition.
Lines 242-243: give the reference of the analysis right after you talk about it such as “All latency data were analyzed with Cox’s proportional hazards model using survival analysis (Therneau & Grambsch, 2000; Therneau, 2015), as described by Monk et al. (2018b)”.

Results
Line 266: “Control group” instead of “control group”
Lines 276-278: please precise that it is on 20 sheep for each group and add the missing comas such as “More sheep in the Happy group entered the zone closest to the dog wall (14/20) compared to the Control, Calm and Anxious groups (6/20, 7/20 and 4/20 sheep, respectively; Fishers Exact Test, P=0.009)”.
Lines 280-281: I suggest changing the sentence to “Seven, 4, 3 and 2 sheep from the Anxious, Happy, Control and Calm groups, respectively, urinated during testing”. Also, see above my comments about sheep’s defecation behaviour.
Line 286: This paragraph, with all its statistical results, is difficult to read. Maybe you could make a table to synthesize them. Be consistent in the way you present the P-values (P in capital letter and no space before and after the equal sign).
Line 289: add the missing “=” in “(X2 (8) = 870.6, P<0.001)”
Line 291: add “min” such as “at times -30 or -20 min (P>0.1) (Fig 4). At time -10 min, […]”. In the Figure 4’s title replace “Mean (± se)” with “Mean ± s.e.m.”

Discussion
Lines 307-308: I suggest to replace “displayed a higher, albeit not significantly higher” with “displayed a higher, albeit not strongly supported statistically”
Line 308: add a capital letter to “Control animals”
Line 317: are you sure you want to introduce the background noise during test as the first explanation for your results, though I agree the background noise is important when testing attention and you clearly and sincerely discuss the issue.
Line 323: add a coma after “However”
Line 326: “there was a high number of […]” instead of “there were a high number of [...]”
Line 329: remove the double parenthesis such as “(unpublished data from Monk et al., 2018a)”
Line 337: put “respectively” between comas
Line 341: Here would be the good place to discuss future application of the attention bias test in more on-farm settings, in which the emotional states are induced by the animals’ environment. Also, I would like to see somewhere in the discussion some critics about the stimuli used (live vs. 2D image and no variability in the stimuli) and therefore how generalizable the approach is.
Lines 348, 352 and 360: precise “anxious-like” response/state
Lines 381-384: please rephrase the sentence in two sentences. Also, position the references after introducing the studies such as “previous studies in cattle (Lee et al., 2017) and rodents (Olivier et al., 2003; Bouwknecht, Olivier & Paylor, 2007) […]”.
Line 391: remove “in sheep” that is redundant in this sentence.
Lines 432-433: I understand that lengthening the test duration could potentially overcome the high number of censored data points observed for vigilance behaviour, however I would not recommend this because then test sheep might not be focus for a long enough time and you take the risk of not being able to explain why they (not) react in such a way.


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

Input:
Title: IMPACT OF BODY WEIGHT GAIN ON HEPATIC METABOLISM AND HEPATIC INFLAMMATORY CYTOKINES IN COMPARISON OF SHETLAND PONY GELDINGS AND WARMBLOOD HORSE GELDINGS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is well written in all its parts and literature is well covered and referenced.
Experimental design: The topic of the manuscript is appropriate for the Journal and the information is of significant interest to the Journal\'s readers.
Some clarifications that should be added to the material and methods should be added to the manuscript as described in the general comments for the author
Validity of the findings: The data  is well presented and reflected the novelty
The conclusions are appropriately stated and connected with the research question.
Additional comments: The title accurately reflects the major findings of the work, however, I suggest to add “geldings to Shetland ponies and Warmblood horses.
It is useful for the authors to mention the reason for the selection of only geldings animals in their study.
Line 92-93: have age differences between the ponies and the horses been considered when comparing the results between two groups?

Line 119-121: Serum amyloid A (SAA), plasma glucose, serum insulin, serum triglycerides (TG) and serum non-esterified fatty acids (NEFA) concentrations were analyzed only in t0 (October 2015), t2 (October 2016) and t5 (December 2017). Have these parameters been considered in winter only?

Line 114: Did the authors used a 14-gauge-catheter for blood sampling from ponies?

Line 155-169: Could the authors clarify why they used surgical method rather than classical liver biopsy?
Could the surgical operation influnce animal appetite, weight gain and the results of studied blood parameters?

Line 231: Did the animals suffer from additional health problems due to excess nutrition such as colic?
Were there any observations regarding the consumption of drinking water during the study? kindly focus on these observations.
Line 237-238: Could the administration of non-steroidal anti-inflammatory drugs 20 days before sample collection in one animal have any impact on studied liver parameters?

Line 417-418: When discussing the results it is important to consider the time of sample collection (t0,2, 5). Also the impact of season on the results should be discussed.


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

Input:
Title: IMPACT OF BODY WEIGHT GAIN ON HEPATIC METABOLISM AND HEPATIC INFLAMMATORY CYTOKINES IN COMPARISON OF SHETLAND PONY GELDINGS AND WARMBLOOD HORSE GELDINGS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: line 109: please explain basal state more clearly (e.g. basal serum levels etc.)
line 125-143: please explain more clearly at what timepoint which blood parameter was measured
Validity of the findings: Results: Pain scores from the LPS challenge are not given in the text or tables
line 252: basal insulin? levels during CGIT??
Additional comments: No comments, nice article with interesting results and discussion


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

Input:
Title: IMPACT OF BODY WEIGHT GAIN ON HEPATIC METABOLISM AND HEPATIC INFLAMMATORY CYTOKINES IN COMPARISON OF SHETLAND PONY GELDINGS AND WARMBLOOD HORSE GELDINGS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The topic of the manuscript is appropriate for the Journal. Clear, unambiguous, professional English language used throughout.
Experimental design: Materials and methods section don't needs any changes. Literature well referenced & relevant.
Validity of the findings: No comments
Additional comments: No comments