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

Input:
Title: EFFECTS OF THIOUREA ON THE SKULL OF TRITURUS NEWTS DURING ONTOGENY
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have thoroughly responded to the reviewers’ comments and recommendations. The additional details added to the text and supplementary materials improve the manuscript and interpretation of the results. However, some of the details provided in the response letter were not added to the text. I recommended adding a few of these details (listed below) in order to support the context and interpretation of these studies.

1.	The authors provided detailed responses and added text for much of the basic reporting. In the revised text, I suggest adding a sentence clarifying why hybrids were used. The descriptions of the reason are well described in the responses to both reviewers but still is not well described in the text. This would fit well added to the sentence on Lines 126-127 or added to the last paragraph of the Introduction.

2.	I greatly appreciate the additional information the authors added on the importance of TPO for biosynthesis of thyroid hormones. However, I suggest removing the sentence that was added on Lines 84-86 and keeping the focus on TPO. It is confusing to the reader to include inhibition of thyroidal iodine uptake (driven by the sodium-iodide symporter) and conversion of T4 to T3 (driven by iodothyronine deiodinases).
Experimental design: 3.	The authors addressed the questions regarding experimental unit and justified the minimal number of organisms used in these studies.
Validity of the findings: 4.	The responses clearly describe that there were no differences in the conditions in the containers (DO, pH, temperature) and no difference in mortality or growth across treatments. These provide confidence in the quality of the experiments, and, thus, I suggest including these observations in the text.

5.	In the response to Reviewer 2 Question 3.1, the authors describe histological analysis of the thyroid glands of this study. The results described in the response letter would provide great confidence in reduction of thyroid hormone synthesis in the thiourea treated organisms. If possible, I recommend added these qualitative results to the discussion to support the statements about thyroid hormone levels.
Additional comments: Minor suggestions:
a.	Line 73: Suggest moving source and purity of Thiourea back to the methods, but keeping the CAS Number in the Intro where Thiourea is first discussed.
b.	Line 118: Change ‘tread’ to ‘treated’
c.	Line 157: Remove the word ‘about’
d.	Line 170: Fix typo in ‘Supplementary’
e.	Line 174: Change ‘leading’ to ‘leads’


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: DYNAMIC RESIDUE INTERACTION NETWORK ANALYSIS OF THE OSELTAMIVIR BINDING SITE OF N1 NEURAMINIDASE AND ITS H274Y MUTATION SITE CONFERRING DRUG RESISTANCE IN INFLUENZA A VIRUS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article is well written.
Experimental design: I have issues with the originality of this work. This is essentially an incremental piece of work. It does not provide a substantial advance on the area described and neither does it have conceptual novelty
Validity of the findings: I have issues with the originality of this work. This is essentially an incremental piece of work. It does not provide a substantial advance on the area described and neither does it have conceptual novelty
Additional comments: The authors are recommended to have a thorough relook at this manuscript and significantly improve.


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: DYNAMIC RESIDUE INTERACTION NETWORK ANALYSIS OF THE OSELTAMIVIR BINDING SITE OF N1 NEURAMINIDASE AND ITS H274Y MUTATION SITE CONFERRING DRUG RESISTANCE IN INFLUENZA A VIRUS
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comments
Experimental design: No comments
Validity of the findings: 1. The 80 ns simulation time used in this study is too short of ensuring convergence. The authors have not provided the time evolution of RMSD also. Longer simulations and/or multiple copy simulations will be a better choice.
2. How the 150-loop dynamics affect the drug binding mechanisms of NA should be discussed clearly.
3. It would be helpful to show how the pocket cavity volume affects the OTV binding.
4. The disulfide bonds are essential for NA structure, and they must be careful about that, which is not clear from the methods section.
5. The binding free energies calculated with MMPBSA are shown to be very sensitive to many factors, such as the value of the dielectric constant chosen for the protein or the conformational entropy (which can be improved by running multiple replicates). The values calculated here must be interpreted very carefully.
6. The MMPBSA calculation provides the other energetic components (like vdW, Elect, Pol-sol, and non-pon-sol) in Table 1.
7. The role of molecular water in OTV recognition is missing in this text, as they play an important role in inhibitor binding.
8. A representative structure that summarizes the analysis in sections 2.1 and 2.2 would be beneficial.
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: DYNAMIC RESIDUE INTERACTION NETWORK ANALYSIS OF THE OSELTAMIVIR BINDING SITE OF N1 NEURAMINIDASE AND ITS H274Y MUTATION SITE CONFERRING DRUG RESISTANCE IN INFLUENZA A VIRUS
Review round: 2
Reviewer: 1

Output:
Basic reporting: This is a well-written manuscript. The conclusion is backed by simulation data and analyses.
Experimental design: The method section has been revised by the authors following the suggestion. The result can be reproduced following the protocol.
Validity of the findings: No comments
Additional comments: The authors have addressed all my queries and modified the manuscript accordingly. However, I still believe that a multiple copy simulation would be beneficial for dynamic study.


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

Input:
Title: EARLY MATERNAL SEPARATION IS NOT ASSOCIATED WITH CHANGES IN TELOMERE LENGTH IN DOMESTIC KITTENS (FELIS CATUS)
Review round: 1
Reviewer: 1

Output:
Basic reporting: The use of language is for the most part clear and professional.

There is adequate literature cited and sufficient background. I would like to see some references supporting the statements L37-38. It may help the reader if the authors noted that most of the literature in the introduction concerning early life adversity and telomeres is from research in wild animals, while the current MS studies domestic cats. This is an important contrast that could be explored (possibly in the discussion where human rearing of kittens is detailed).

The current study also deals with a particular type of early life adversity: loss of the mother. While to my knowledge there are no studies (wild or domestic) of mother-loss and telomeres, there may be studies of mother loss and other metrics of stress (stress hormones for example) and I would urge the authors to briefly research this and cite any relevant work. This will help build a case that mother-loss specifically (rather than adversity in general) has health consequences. Stress is also known to accelerate telomere loss.

The article structure is good. A section is missing: statistical analysis details in the methods. In my view this is vital for the reader to know the details of the analyses that have been carried out. For example, L105 reports a correlation but there is no info about how this p-value was generated and the reader therefore cannot know if it is legitimate. The authors must add a methods section detailing ALL statistical tests and analyses. In each case, the dataset and sample size, the analysis performed and info about the response and predictor terms and random terms etc.

The figures look good. The y-axis in both figures should be labelled "telomere length (T/S ratio)". The black regression line in fig2 should not extend beyond the bounds of the black datapoints. It would be helpful to see the sample sizes for each box on fig1. The raw data is present and looks straightforward to interpret.

The MS is self-contained and has well-conceived hypotheses and relevant results.
Experimental design: I am satisfied that this is primary research within the journal scope. The research is well defined and fills a knowledge gap.

I am satisfied that ethical standards were high.

As stated above, I am not satisfied that adequate description of the statistical methods is provided, and without the specific statistical info the analyses could not be replicated.

I would say the sample sizes are at the limit for rigorous tests, and this should be openly stated and the implications discussed.
Validity of the findings: I have two concerns about the sample sizes.

1) The number of samples for mother-reared offspring is very small, and quite imbalanced compared to OR kittens. The authors should state this and discuss possible implications in the discussion. The sensitivity analysis is a good addition.

2) The precise breakdown blood sample availability from the two groups is too confusing. L59 states that 10 mother-reared (MR) kittens were used. Line 76 states that 9 MR kittens had blood sampled at both timepoints, but figure 2 only shows 5 MR points, and the datafile appears to show 6 MR kittens sampled at both timepoints. L77 states there were an additional 4 MR kittens with blood samples at only one time point. To me this suggests 13 MR kittens were used, not 10 as reported L59. The data appears to contain 11 unique MR kittens. This confusion leaves the reader unclear about what the sample sizes were for different tests, and gives the impression of somewhat sloppy data handling. It is crucial this is corrected.

I would like to see the analyses repeated excluding the kittens that died, or with a predictor added for "survived to 8weeks Y/N". It is possible these kittens were already ill, biasing the results. It is also possible that their telomeres shortened so much that they died, thus masking the true effect because of the absence of their data at the second timepoint. I would like to see discussion of this and urge the authors to research 'selective disappearance'.

It is unclear whether analyses contain samples from the same kittens at the two time points. As stated above, this must be much clearer. If this is the case, kitten ID must be included as a random term, and should be nested within litter ID as a random term.
Additional comments: Overall I commend the authors on an interesting topic. While it appears adversity does shorten telomeres in wild animals, careful rearing by humans can remove these ill-effects. This is a useful and interesting finding. It's clear a lot of work has been conducted. While I am not an expert on telomere laboratory analysis, this appears to have been done carefully and correctly. There is a weakness in the data handling and analysis and I urge the authors to be careful and transparent to ensure the MS is ready for publication.


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

Input:
Title: EARLY MATERNAL SEPARATION IS NOT ASSOCIATED WITH CHANGES IN TELOMERE LENGTH IN DOMESTIC KITTENS (FELIS CATUS)
Review round: 1
Reviewer: 2

Output:
Basic reporting: This study is investigating the effects of maternal separation on early-life telomere length and telomere dynamics in kittens, which has not been investigated so far. It is therefore filling a gap in the literature. However, a lack of context in the abstract and the introduction, together with a not sufficiently detailed state-of-the-art and a too few number of studies cited, are masking the importance of this study. I provide detailed suggestions along with references (with 10 examples, but the authors are of course free to extend this suggestion and to choose other papers than those I suggested) that should be included to improve the background and context of this study.
In addition, my ability to assess the strength and validity of the findings at that stage is hampered by a lack of various key information in the methods. I provide some suggestions and have several questions that should help the authors advancing this issue. I hope the authors will find my comments helpful. I remain available for further exchanges or review of this article, would it be needed.
With best regards,
Mathilde Tissier

Clear, unambiguous, professional English language used throughout.
Yes

Intro & background to show context.
1.	Abstract: although your objective and results are clear, I think that the abstract is lacking context. You should add 1-2 lines of background before your objective and 1-2 lines after your results to replace your study into a broader context, so the reader might understand rapidly the extent of your work. In addition, you should state on which cell you measured telomere length (L21): white blood cells? For the results, it seems to me that your sentences L25-29 are redundant with what you said L24-26. I would keep only the sentence L25-29 that seem more adapted for an abstract. This would provide space for a sentence replacing your results into a broader context.

2.	Introduction: I find the introduction well written. Perhaps consider starting with a broader context, not directly and solely on telomeres. For instance, you could broaden your sentence L33-34 by stating that “Studies in multiple species have found that adverse early life experiences can result in modifications of adults’ behavioral, biochemical or molecular phenotypes. For instance, studies have pinpointed that telomeres shortening is increased in individuals facing harsh early life conditions”. In addition, you do not mention the potential relationship nor the existing knowledge/literature on the link between maternal effects, maternal separation and telomere length/telomere dynamics in other species, which is at the core of your study. You should include a paragraph on this (L42-43, between the state-of-the art on early life experiences and your specific hypothesis on the effect of maternal separation on kittens’ telomere length).

Below, I suggest some minor edits regarding the introduction.

Minor comments on the introduction
L35-37- consider revising. Here is a suggestion: Cells have a limited reproductive capacity, referred as the “Hayflick Limit”5. Telomeres shorten during mitosis and once they reach a threshold, the cell stops reproducing and enters a state of senescence or dies 6.

L38-39 – Telomeres are also

L41 – you mention health and well-being but you only cited studies referring to lifespan and mortality. Consider replacing well-being by “life-history” and to include studies connecting telomeres to health to support your statement.

L43-53 – references on other taxa are critically lacking to support your hypothesis and predictions in this section (see my suggestions).

L47 – are subsequently hand-reared by humans

L48-49 – no, you cannot say that this is the first study on telomeres in kittens; see Brümmendorf, T. H., Mak, J., Sabo, K. M., Baerlocher, G. M., Dietz, K., Abkowitz, J. L., & Lansdorp, P. M. (2002). Longitudinal studies of telomere length in feline blood cells: implications for hematopoietic stem cell turnover in vivo. Experimental hematology, 30(10), 1147-1152 > this is just one example.
You should rephrase this sentence, and make it more specific to the exact objective of your study, which is investigating the effects of maternal separation on early-life telomere length and telomere dynamics in kittens, which has not been investigated so far.

L49 – telomere length

L54 – Please include a prediction on the dynamics of telomeres, in addition to your predictions on telomere length (since you mention telomere shortening in your abstract and introduction + results)

Literature well referenced & relevant.
The literature on telomeres is extremely vast and I don’t expect you to cite all the articles published, but your state-of-the art should be a bit more referenced, including key papers, especially for the relationships between telomere length and life-history traits and health, on how telomere shortening in early-life may have long-term consequences or on the relationship between maternal effects and telomeres. You only have 22 references, which is very few, and offers room to cite more papers in your introduction.

Some examples (among which reviews or meta-analyses):

a)	Relationship between telomeres and health or life-history traits

Monaghan P, Haussmann MF. 2006 Do telomere dynamics link lifestyle and lifespan? Trends Ecol. Evol. 21, 47–53. (doi:10.1016/j.tree.2005.11.007)

Bize P, Criscuolo F, Metcalfe NB, Nasir L, Monaghan P. 2009 Telomere dynamics rather than age predict life expectancy in the wild. Proc. Biol. Sci. 276, 1679–83. (doi:10.1098/rspb.2008.1817)

Seeker, L. A., Ilska, J. J., Psifidi, A., Wilbourn, R. V., Underwood, S. L., Fairlie, J., ... & Whitelaw, B. (2018). Bovine telomere dynamics and the association between telomere length and productive lifespan. Scientific reports, 8(1), 1-12.

b)	Relationship between early life experiences, stress and telomeres

Price, L. H., Kao, H. T., Burgers, D. E., Carpenter, L. L., & Tyrka, A. R. (2013). Telomeres and early-life stress: an overview. Biological psychiatry, 73(1), 15-23.

Chatelain M, Drobniak SM, Szulkin M. 2020 The association between stressors and telomeres in non-human vertebrates: a meta-analysis. Ecol. Lett. 23, 381–398. (doi:10.1111/ele.13426)

Ilska-Warner, Joanna J., et al. "The genetic architecture of bovine telomere length in early-life and association with animal fitness." Frontiers in genetics 10 (2019): 1048.

c)	Relationship between maternal effects, maternal separation, telomeres in early life and phenotypes

Haussmann MF, Longenecker AS, Marchetto NM, Juliano SA, Bowden RM (2011) Embryonic exposure to corticosterone modifies the juvenile stress response, oxidative stress and telomere length. Proc R Soc Lond B Biol Sci 279:

Tissier, M. L., Williams, T. D., & Criscuolo, F. (2014). Maternal effects underlie ageing costs of growth in the zebra finch (Taeniopygia guttata). PloS one, 9(5), e97705

Chen, X., Zeng, C., Gong, C., Zhang, L., Wan, Y., Tao, F., & Sun, Y. (2019). Associations between early life parent-child separation and shortened telomere length and psychopathological outcomes during adolescence. Psychoneuroendocrinology, 103, 195-202.

BOTHA, Martmari, GRACE, Laurian, BUGARITH, Kishor, et al. The impact of voluntary exercise on relative telomere length in a rat model of developmental stress. BMC research notes, 2012, vol. 5, no 1, p. 697.

Structure conforms to PeerJ standards, discipline norm, or improved for clarity.
Ok

Figures are relevant, high quality, well labelled & described.
More information should be reported on the caption of Figures 1 and 2, namely the sample size, basic statistical information, whether raw data are presented or results from your model) on Fig 1 and the R2 and p-value on Figure 2.

Raw data supplied (see PeerJ policy).
I thank you for sharing your raw data.
Experimental design: Original primary research within Scope of the journal.
Yes

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

Yes, although a lack of context is hampering the importance of this study. Please see my comments above.

Rigorous investigation performed to a high technical & ethical standard & Methods described with sufficient detail & information to replicate.

I have four main issues resulting in my incapacity to assess the strength and validity of the findings, mainly arising from a lack of information in the methods section. These information are also crucial to assess the repeatability of your study.

1.	You did not mention how you determined age of OR kittens, for which date of birth is likely not known (you say line 62 “if known”: how did you proceed for the “not known”?). This could create a strong bias in your analyses, since date of birth is likely much more precise for MR and OR kittens. Therefore, you should address this in details in your Methods.

2.	On which tissue did you measure telomere length? Did you isolate white blood cells from your blood samples? Or did you measure it on total blood? If so, please justify by witing relevant references.

3.	Please provide information on whether you did the qPCR measurements in duplicates or triplicates. On one or two plates (did you use 96-plates or 384-plates)? Please also detail the composition of the Mix that you used to carry-out your qPCR, which primers and what were the cycling conditions. Please also include intra and inter-plate (if any) coefficients of variation. These are extremely important to assess the strength and the repeatability of your measurements.

4.	There is no section detailing the data nor statistical analyses that you conducted. Please add one. L95-98 of the results should be included in this section but is not sufficient to assess what you did. For instance, you say that you used a generalized linear model, but I assume that you mean generalized linear mixed model, since you included a random factor? In addition, which law did you use (normal)? If so, did your residuals follow normality? I am also concerned about conducing a Linear Mixed Model on these data given the (apparent) difference in the variance and the large difference of sample size between your two groups.

General comments and questions

L70: you should indicate at which age the kittens died (to understand whether this had an influence on your experimental design and analyses or not).
L74: approximately 1 and 8 weeks > please be more specific (give the information in days±SE)

L73-82: consider providing a table of how many viable samples with extracted DNA you managed to obtain at 1 and 8 weeks, for OR and MR kittens, respectively, as it is hard to have a clear idea when reading the text.

L81-82 – that’s a lot regarding your already low sample size for MR kittens (9 at both points and 6 at one point). This is raising the question on “how many samples did you have at each point at the end?’

General question: do you have any information on whether the 42 kittens made it to adulthood? If yes, you could include an analysis with a binary response (yes/no), and looked at whether telomere length or telomere dynamics influenced survival in kittens (while comparing OR and MR kittens).
Validity of the findings: 3.	VALIDITY OF THE FINDINGS

Unfortunately, the lack of information on the analyses conducted, the methodology (especially on the qPCR approach), the sample size of each category does not allow me to assess the strength, validity and repeatability of the findings. I could use the supplementary datasets to conduct analyses myself but my numerous questions on qPCR analyses would in any cases do not allow me to have sufficient information to assess the validity of the findings. In addition, I think that this information should be specified in the article, so that, by reading the methods, results and looking at the figure, the reader may be able to assess whether the analyses were performed correctly. You seem to have a much greater variation on your OR groups (Fig 1) than on your MR group (Figure 1). Nonetheless, I cannot assess if this is due to a difference in sample size, an unsuitable statistical/analytical approach, to a real difference in the variance between the two groups or to measurement errors.

I appreciate your supplementary sensitivity analysis but this should be developed in the methods section and not in the results.
Additional comments: No additional comments


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

Input:
Title: EARLY MATERNAL SEPARATION IS NOT ASSOCIATED WITH CHANGES IN TELOMERE LENGTH IN DOMESTIC KITTENS (FELIS CATUS)
Review round: 2
Reviewer: 1

Output:
Basic reporting: The clarity of the MS is much-improved. I appreciated the additional background information and cited literature in the introduction. The statistical methods are much more clear and easy to assess and understand. They seem appropriate to me.

The sample sizes are much more clear, although the table does not detail the number of kittens that had samples at both time points. This info would be useful.

I do not have the expertise to assess the validity of the qPCR techniques and interpretation.

Some minor comments:

L68 "other children" should refer specifically to "children raised by their mothers".

L175 refers to an interaction but it isn't explicitly clear what the interaction is. It is detailed in the methods but should be stated again here.

L237 "influenced" is too vague - there should be specific details about such effects, including direction and magnitude if possible.
Experimental design: No further comments.
Validity of the findings: The novelty is better discussed in the context of other studies.

The conclusions are discussed in the light of limitations of the study.

There is still some weakness in contextualising the findings: I see only 4-5 citations of other studies investigating similar research questions (in the discussion). Some discussion could be provided, for example, regarding why mother loss has substantial effects in some contexts and outcomes, but this is not evident in telomere lengths.
Additional comments: Overall this is a large improvement. I see only very minor issues that could easily be resolved.


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

Input:
Title: EARLY MATERNAL SEPARATION IS NOT ASSOCIATED WITH CHANGES IN TELOMERE LENGTH IN DOMESTIC KITTENS (FELIS CATUS)
Review round: 2
Reviewer: 2

Output:
Basic reporting: I have reviewed the documents received. I am overall happy with the response to the reviewer comments in the letter. However, I believe that some work is still needed before the work can be published. I have done my best to provide constructive and detailed comments to the authors.

Major comments

Introduction- while I appreciate the efforts of the authors in replacing their work in a broader body of work, I am a bit disturbed by the mix of ecological literature together with medical/psychological literature. The authors could perhaps just re-organize their sections to have two different paragraphs. Or highlight that both evidence from the evolutionary ecology and psychological/medical literature demonstrate the effects of early experiences on telomere length (L55). Then the reader will be less disturbed by the integration of both studies on European starlings and children in China. I do also believe that the transition to kittens should be improved (see suggestion below).

I appreciate your conclusion, which gives a good take-home message to the reader. But your whole paper, including your discussion, should bring anyone reading the paper to the same conclusion than yours. As it is, I believe that reader would end-up being a bit confused by statistical imprecisions, differences of samples sizes (which per se can be understood and could not be a major issue, but you have to be firmer on this matter), and limits of your study as discussed in details L197-237. I provide some details to help you work on those potential issues.
Experimental design: I still have questions and concerns on the methods, especially the statistical section. This is greatly related with your goal, to compare two groups that have different experiences, but with unbalanced sample sizes. Please see my comments below.
Validity of the findings: As stated in sections 1 and 2, there is still, to my opinion, some work to be done to support the validity of your findings. Please find my detailed comments in the general comments for the author below.
Additional comments: Detailed comments:

To improve readability, you could perhaps put your degrees of freedom as indices rather than in brackets (in the abstract and the result section).
L41- leads

L44 – delete also to avoid redundancy with L45

L66 – perhaps indicate the species of horse (to be consistent with the previous section in which you provide the information)

L69-70 – perhaps before mentioning kittens you could highlight the gap in the literature > effects of maternal separation on telomere length and telomere shortening in a variety of taxa. Then you mention the goal of your study. It will smooth the transition from the state of the art, including studies on children, to your study on kittens.

L81-83 – this should go with your predictions in the following paragraph (L83-88)
Methods

L112 – a space is missing after EDTA.

L116-118 – why?

From Table 1, if you compile numbers from OR kittens at Time 1, you don’t find the n=42 mentioned L93 of your methods (either n=40 if not counting the 4 samples used to develop the protocol or n=44). Please consider adding the number of kittens sampled (in addition to the number of samples); eg: n=19 samples from 10 kittens (1 kitten for which sample is only available at time 1)

L123 – Cawthon (not Cawthorn)

L133 – please provide the brand/company and details on the model of the machine used. Was it an automated machine?

L138-144 – this should be mentioned before description of qPCR measurements
Overall, I really appreciate the supplementary section provided for telomere measurements which seems complete/detailed enough to allow repeatability.

L161 – I don’t understand, you say that you used a GLMM but that your response variable followed a normal distribution. Do you mean that you used a LMM (Linear Mixed Model) and not a GLMM? This also assumes equality of variances, and since you have an imbalance in your sample sizes, this may be an issue. Did you ran any test to investigate this (Levene or else)? In addition, how did you confirm for normality (which test) to confirm your statement 165? You respond to part of these questions in the result section, but this should be stated here.

Results

L177-188 – did you look at the interaction between sample time and kittens group here (you mentioned it for the GLMM but what about this analysis)? It is a pre-requisit if you want to then separate the analysis by group. Also, why did you add this analysis? At this stage it is not clear. Perhaps you should indicate in the methodology what it brings to your questions/predictions.

L178-179 - This apparently stronger relation could solely be explained by a smaller sample size in the MR group. No?

Discussion

Consider replacing T/S ratio by relative telomere length to facilitate the reading.

L236-237 – in which way? By increasing the variance between individuals? Within individuals but at different time points? Yes it is a possibility, but if only stated like this, you give the impression to the reader that your results are not trustworthy. You really should develop further this idea and express whether – and if so, to which extent – this can be an issue for your conclusions.

I would expect you to discuss a bit more your results:
-	To which extent your study brings information to the field; eg: you can make comparisons with studies mentioned in your introduction, such as the study of Lansade et al 2018 on horses/foals. What could be the main difference? These foals were “abruptly separated” from their mother (L65). In your case kittens are indeed hand-reared and cared by humans, which could compensate in a way > although I agree with you, literature is needed to support this claim (L202).
-	Provide the reader with a stronger statement that your results (Fig 1 and 2) are not caused by selective disappearance of kittens with short telomeres (because they died), or by the fact that you don’t have the same kittens measured at the two sample times.  It is kind of mentioned L224-231 but perhaps to vaguely to be clear enough for the reader.
-	An extra suggestion: perhaps providing in supplementary material an analysis with only data on kittens from which you have a measurement at Time 1 and Time 2 could be helpful?

In addition, consider regrouping the “limits” of your study in a single paragraph? As it is, it gives the impression than each one of your argument is counteracted by a “limit” of your study, which makes the whole discussion not very convincing. I do appreciate the transparency and I believe that these limits must be discussed, but perhaps with more implications and in a short paragraph.


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

Input:
Title: EARLY MATERNAL SEPARATION IS NOT ASSOCIATED WITH CHANGES IN TELOMERE LENGTH IN DOMESTIC KITTENS (FELIS CATUS)
Review round: 3
Reviewer: 1

Output:
Basic reporting: Your revised manuscript entitled “Early maternal separation is not associated with changes in telomere length in domestic kittens (Felis catus). “ (Manuscript ID 52366) deals with the effect of one stressor (early maternal separation) on one ageing parameter (telomere length TL) in kittens.

General comments: This is an interesting study, however, it has limited data and narrow approach towards studying the telomere length that may be associated with aging. They conclude that there is no effect of early maternal separation on telomere length in kittens and did not had influence between week one and week eight.

Overall the revision brings a large improvement. I see only one big issue missing to assess the validity of the qPCR (see my comments in the methods section) and some minor issues that could be easily resolved.

Introduction
With the changes made the introduction provides an adequate background information.

Methods
I have several major comments regarding the technical part: see experimental design section

Discussion
Line 251
Not well formulated. We cannot be certain that the rate of attrition in this species of bird and in the cat is comparable, in particular because the metabolism is not comparable. Furthermore, telomeres are not measured in the same cells (Lymphocyte/erythrocyte) and the rate of renewal of these cells may not be comparable.

Figures
Figure 1: Please add the mean for each category on the figure.
Figure 2: Please use the same notation as in the text, OR and MR instead of orphan Y/N.

All other comments have been addressed.
Experimental design: I still have questions and concerns on the quantification of telomere length. Please see my comments below.

Methods
I have several major comments regarding the technical part:

Line 130 – viable DNA
What do you exactly mean? Is it not rather valid DNA or to be precise good quality DNA. Could you please rewrite these sentence? How do you control the DNA quality?

Line 135 – a column that retained DNA
Could you please just add the name of the manufacturer, please?

Line 138 and line 157
You write that you dilute your DNA at 10 ng/µL in your study. Then, with the help of a liquid handler you dispatch this DNA (10 ng/µL) in a 384 plates in order to do the amplification. And line 157 you write that the concentration of DNA in all experimental reactions was 2.5 ng/µL. So did you do a dilution in between?

Line 139 to 141
What is T and S? You have to explain the abbreviation the first time you used it, please.

Line 144
Please rewrite the sentence.
“Telomere lengths were assessed by qPCR (quantitative polymerase chain reaction) and expressed as a ration (T/S) of telomere repeat copy number PCR (T) relative to a single copy gene PCR (S).” Could you also just add the formula of the calculation or at least add references about how to calculate the T/S ratio?
May be it would be more judicious to place this sentence further upstream of the chapter on the quantification of telomere length? Perhaps just after the extraction….

Line 148
What are the concentration of both primers for the single copy gene, 1mM? Did you control if the chosen “single copy gene” is non variable among your population? Did you control the size of the amplicon?

Line 155 and 156
Can you please delete it to avoid redundancy with line 148 to line 150.

Line 164
You did a standard curve. Could you please add the starting concentration of the curve and the efficiencies of the telomere and single copy gene reactions, and also the r2

Line 166
What do you mean by positive controls? What are the function of them? Is it the same as the golden sample and what are the use of a golden sample?

Line 169 – “the ratios from all sets were averaged”
I don’t understand your sentence……could you please explain.
The inter-plate CV was 3%. For what: telomeres? Single copy genes?  Did you also calculate intra-plate variations? Or ICC (intraclass correlation coefficient)?

General remarks on the methods:
Can you specify the number of litters in each category in the material and method or in Table 1 and the average size of these litters (and the standard deviation). If all 6 kittens in the MR group come from the same litter this may explain the lower variance observed despite the smaller litter size.
Why there is such a large variation of age at T2? Are there differences in average age between OR and MR kittens. Please provide these information in the text or in table 1.
Rather than a regression between T1 and T2 why not do the telomere attrition between these two times and test the breeding effect of this attrition?
Validity of the findings: The conclusions are discussed in the light of limitations of the study
Additional comments: Overall the revision brings a large improvement. I see only one big issue missing to assess the validity of the qPCR (see my comments in the methods section) and some minor issues that could be easily resolved.


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

Input:
Title: EARLY MATERNAL SEPARATION IS NOT ASSOCIATED WITH CHANGES IN TELOMERE LENGTH IN DOMESTIC KITTENS (FELIS CATUS)
Review round: 4
Reviewer: 1

Output:
Basic reporting: All the comments have been addressed.
Experimental design: I have a question regarding the primers you used and the reply you did concerning the single copy gene primers.

Telc and Telg are telomeric primers normally used for monochrome multiplex qpcr (telomere and single copy gene assay are done in the same well, see Cawthon 2009). Also the sequences for the single copy gene given in your reply (feline GAPDH forward primer 775f and feline GAPDH reverse primer 859r) have a GC clamp. These clamps are used to artificially increase the Tm of the primers so that the multiplexing is possible (normally in multiplexing around 88°C = The 74°C acquisition signal or reads provided the Ct values for the amplification of the telomere template (in early cycles when the scg signal is still at baseline); the 88°C reads provided the Ct values for the amplification of the scg template (at this temperature there is no signal from the telomere PCR product, because it is fully melted). So the primers sequences you give in your reply are in favor of doing a multiplexing but in your paper you didn’t mention the GC clamps for your single copy primers. So did your single copy gene primers have a GC clamp or not? In your paper you didn’t do multiplex, why ?
Validity of the findings: All comments have been addressed
Additional comments: I really appreciate all the detailed answers given by the authors in order to assess the validity of the qPCR method. I am overall happy with all the response to the reviewer comments in the letter.

I have just one comment on the assessment of the DNA quality for the author’s future publications.
To do a telomere length analysis it is crucial to have good gDNA quality in term of quantity, lack of contaminants and integrity. Most of the time, especially if it’s the first time you used an extraction technique or if it’s the first time you extract gDNA from a new species, it is not sufficient to measure the gDNA quantity by a spectrophotometer like a Nanodrop and to have a look at the 260 nm/280 nm ratio and the 260 nm /230 nm ratio for assessing the gDNA to be of good quality. The ratios will inform you about the absence of contaminants but are of no clue about the integrity of the DNA. In order to check the integrity you need to do a gel (see figure 1 : DNA integrity gel in Seeker et al, 2016, Method Specific Calibration Corrects for DNA Extraction Method Effects on Relative Telomere Length Measurements by Quantitative PCR). DNA quality can be assessed using agarose gel-based separation confirming the absence of any degraded DNA in the form of a smear on the gel.

There are also some typo errors:
In line 148 : it’s 260/280 nm ratio and not 260/280 nM
In line 149 : it’s 260/230 nm ratio and not 260/230 nM
In line 161 : it’s 1µM and not 1mm

All other comments have been addressed.


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

Input:
Title: EARLY MATERNAL SEPARATION IS NOT ASSOCIATED WITH CHANGES IN TELOMERE LENGTH IN DOMESTIC KITTENS (FELIS CATUS)
Review round: 5
Reviewer: 1

Output:
Basic reporting: All comments have been addressed.
Experimental design: All comments have been addressed.
Validity of the findings: All comments have been addressed.
Additional comments: Your revised manuscript entitled “Early maternal separation is not associated with changes in telomere length in domestic kittens (Felis catus). “ (Manuscript ID 52366 4th revision) is now much clearer and better streamlined than the previous versions and it is obvious that the authors improve the manuscript. The authors have done a good job and properly addressed all my previous comments, so I have no more comments to add for the manuscript.

Sorry not to have emphasized these old typos before (pdf version):
1	Could you please be consistent throughout the manuscript in your notations? For example could you either write 0.98 or .98 as line 228 (0.98 and .97 for Time 1; 0.98 and 0.93 in Time 2). Please check throughout your manuscript (line 150, 181, 184, 228 etc… decimal numbers without zero).
2	Paragraphe 172-175: annotation for degree celsius is °C, check line 174, C is missing and in line 175 there is an inversion between the 2 letters 95C°.
3	In figure 2, scatter plot of correlations between telomere lengths at two time points.
Line 3, 28 orphaned kittens (r = 450 0.28, p = 0.14), remove 450.


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: GUICLUPEA SUPERSTES, GEN. ET SP. NOV., THE YOUNGEST ELLIMMICHTHYIFORM (CLUPEOMORPH) FISH TO DATE FROM THE OLIGOCENE OF SOUTH CHINA
Review round: 1
Reviewer: 1

Output:
Basic reporting: some grammatical suggestions made on uploaded pdf
Experimental design: fine
Validity of the findings: fine
Additional comments: Overall, well written and only minor grammatical suggestions are on the uploaded pdf.
I think the discussion needs a bit more work. Diplomystus dentatus is from Eocene Green River deposits of Wyoming, USA, and Horseshoeichthys is from Maastrictian deposits of southern Alberta, Canada. Both are from freshwater deposits. These fish would not have been able to reach the Pacific Ocean, as the uplift of the Rocky Mountains would have been well under way, and the palaeolake (Green River) and palaeostream (Horseshoeichthys) would both have been on the east side of the mountains which therefore would block them from reaching the Pacific Ocean. Current drainage of the area is the Hudson Bay or the Mississippi River, and likely palaeodrainage would have been similar.
These fish may still have been amphidromous, but perhaps leaving the lakes to spawn in connected rivers, as some fish do today. There are a number of northern Canadian lakes that contain salmonids that have two morphotypes – one that is amphidromous and the other that spends all its time in the lakes. Perhaps the diplomystines were doing something similar.

I think it is interesting that the larger ellimmichthyiforms are grouping together, but I’m not convinced by your suggesting of anadromy. Perhaps there is some other reason causing the increased size. Do you think it might instead by associated with climate differences between the Cretaceous and Eo-Oligocene?

The last paragraph of the palaeobiogeography section also needs to be reworked. You say that all ellimmichthyiform fossil localities (Fig. 1) are near to the recent coast. This is not correct. The freshwater Horseshoeichthys locality is about 600 km in a straight line from the Pacific Ocean and D. dentatus is about 1000 km from the Pacific coast, and both are on the wrong side of the Rocky Mountains. Other ellimmichthyiformes are from marine deposits, so it would be better to describe them as being found in shallow marine deposits (not ‘near the coast’). Additionally, these fossil deposits are not near the recent coast, with the Moroccan (Armigatus) and Albertan (Tycheroichthys) localities not being anywhere near the sea. This might just need a bit of rephrasing, but also a bit of rethinking in terms of the freshwater forms.


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: GUICLUPEA SUPERSTES, GEN. ET SP. NOV., THE YOUNGEST ELLIMMICHTHYIFORM (CLUPEOMORPH) FISH TO DATE FROM THE OLIGOCENE OF SOUTH CHINA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The text is clear and well written, as far as I can tell. I corrected some spelling mistakes in the pdf and suggested some English corrections.I have suggested three references that might be helpful in the discussion. The structure of the paper is good.
Experimental design: no comment, see general comments below.
Validity of the findings: no comment, see general comments below.
Additional comments: This is an important article describing a new Oligocene ellimmichthyiform fish from southeast China, representing the youngest representative of its clade. The description is detailed and I only have a few comments / remarks noted directly on the pdf. The phylogenetic analyzes are convincing and well done.
The biogeographical discussion is useful and well done.

In addition to the comments written directly in the pdf, I have the following remarks:

- Why did you choose NHMG 005532 as your holotype? From the photograph, it appears that very little detail of the skull is visible. NHMG 033659 shows many more cranial characters and several postcranial characters.

- In phylogenetic analyzes, what is the theoretical reason for using Elops in one matrix and Chanos in another matrix? Why not use both genera as outgroups in the same matrix.

- The characters supporting the nodes are detailed for each major clade. I would suggest that the authors indicate which of these characters are uniquely derived (ci = 1) and which are known in most terminal taxa for the most important clades. This information is important because the clade can be supported by very homoplastic character states and by character states unknown in most terminal taxa, which makes these nodes very weak. I realize that this information is available in Figs 1S and 2S (and I notice that there are very few uniquely derived character states), but it would be good to mention these character states in the main text.

- Lines 527-528: "It should be noted that the Diplomystus order or clade appears to show a tendency to enlarge their body size." This trend has been observed in many clades of ray-finned fish (Guinot and Cavin, 2018) over a long time interval (but strangely enough for clupeiformes but not for ellimmichthyiformes). This is a complementary explanation to the explanation linked to migratory vs non-migratory behavior proposed by the authors. This article also discusses some general paleogeographic and paleoecological patterns of clupeomorphs.

- Regarding the last paragraph of the discussion dealing with the Pacific distribution of ellimmichthyiformes and their potential close link with the sea to explain this distribution could benefit from a comparison with the distribution of Clupeiformes, and their numerous marine / freshwater transitions, studied by Lavoué et al. (2013, 2014).

- Figures 6. Why is Gasteroclupea always included in the Sorbinichthyidae when the analysis did not resolve this group as monophyletic? Maybe this is a detail, but I wonder why the authors chose long ghost lines for the genera rather than making them as short as possible? For example, the radiation of Diplomystus and the separation with Guiclupea may have occurred during the early Upper Cretaceous, with a single ghost line reaching the basal Cretaceous, rather than 5 ghost lineages reaching the basal Cretaceous.
‘Rhombichthys’ rather than ‘Rhomichthys’ in figures 8 and 9.

References
Guinot, G., & Cavin, L. (2018). Body size evolution and habitat colonization across 100 million years (Late Jurassic–Paleocene) of the actinopterygian evolutionary history. Fish and Fisheries, 19(4), 577-597.

Lavoué, S., Miya, M., Musikasinthorn, P., Chen, W.-J., & Nishida, M. (2013). Mitogenomic evidence for an indo-west pacific origin of the clupeoidei (Teleostei: Clupeiformes). PLoS One, 8(2), e56485. https://doi.org/10.1371/journal.pone.0056485

Lavoué, S., Konstantinidis, P., & Chen, W.-J. (2014). Progress in clupeiform systematics. In K. Ganias (Ed.), Biology and ecology of sardines and anchovies (Vols. 1–0, pp. 3–42). Boca Raton, FL: CRC Press. http://www.crcnetbase.com/doi/abs/10.1201/b16682-3

Lionel Cavin


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: GUICLUPEA SUPERSTES, GEN. ET SP. NOV., THE YOUNGEST ELLIMMICHTHYIFORM (CLUPEOMORPH) FISH TO DATE FROM THE OLIGOCENE OF SOUTH CHINA
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have responded to all of the comments I suggested in the first review and I consider the article to be acceptable now in its current form.
Best regards,

Lionel Cavin


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: POPULATION STRUCTURE, ADAPTATION AND DIVERGENCE OF THE MEADOW SPITTLEBUG, PHILAENUS SPUMARIUS (HEMIPTERA, APHROPHORIDAE), REVEALED BY GENOMIC AND MORPHOLOGICAL DATA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The language is understandable and professional throughout, but there are occasional grammatical errors or oddities and a number of long sentences. The long sentences in particular can make the point of a section a little hard to grasp. I have commented on some of these items in the annotated PDF, but a close reading for maximum clarity would be beneficial prior to full acceptance.

The figures are illustrative of the major findings of the work and have text that is of a readable size. I will note however that the color scheme used is not color-blind friendly, and that the resolution of the images in my copy of the PDF were not very high, and so some items were a bit pixelated.

I checked and was able to access the raw data and scripts associated with analyses in this study.
Experimental design: The methods as outlined are generally sufficient to allow replication, barring a handful of concerns/suggestions.

First, it is not clear how the insect specimens were collected, nor how they were stored between the collection date and use in the various experiments. I think it would be appropriate to add in these details (~L185).

Second, why does the sampling strategy for P. tesselatus from Morocco (7 individuals from multiple sites) differ so much from the sampling strategy employed for P. spumaris (L192)? Is it a protected or harder to locate species? I don't think this impacted the results of the 9 population work (the differentiation in the RAD data is strong), and the authors do exclude that population from another set of analyses and discuss how this difference in sampling may have impacted summary statistics, but I do think a justification of the sampling difference would be good to include.

Are the sampling locations selected for each country representative of the variation expected (especially for areas where the spittlebug is introduced)?

What type of PE sequencing was used? 2x100? (L248)

Next (L306), although I think 10 runs of STRUCTURE are probably sufficient to get a sense of the general pattern, I would have liked to see more replicate runs performed (perhaps 50-100). The authors could consider using the program ADMIXTURE (http://dalexander.github.io/admixture/), which uses the same statistical model but performs faster, if computational resources were a concern limiting replicate run number.

Related (L308), it is not explicitly stated if the deltaK method is used for selecting the best K (although the citations within the harvester program utilized by the authors suggest it was).
Validity of the findings: Although I think the authors treat their results with appropriate caution in the discussion and when drawing ultimate conclusions of their findings, I was struck by two results in particular.

First, the relatively small number of SNPs is surprising given A) the large number of reads recovered per individual, and B) the fairly permissive (p = 0.5) missing data filter allowed in calling SNPs from STACKS. Although the authors do note that the genome size is large, given that the average coverage is ~100x for the recovered loci and the proportion of heterozygous sites observed, it seems like there should be more SNPs. As their results were the same after removal of ~10% of their dataset (see second point), I don't necessarily think their results were strongly impacted in this case, but will note that population structuring methods can be influenced by relatively slight changes in allele frequency in smaller datasets.

Secondly, I am also a little surprised by how many Fst outliers were detected in this dataset (almost 10% of markers), although this may be a reflection of the wide range of locations and limited sampling sites that the organisms were sampled from, as the authors note.
Additional comments: I think these results build nicely on previous work in the system by providing a genomic-scale glimpse at differentiation, as well as by integrating some phenotypic data from the male genitalia. This work also contributes some explanations for both previously noted and newly discovered findings, which seem plausible when considered in the light of the insect's natural history information. As noted in several locations by the authors in the discussion, this work also provides a nice starting point for future work that can clarify the genetic landscape, degree of gene flow between populations of P. spumaris, and this species interactions with P. tesselatus.

I have attached an annotated PDF with suggestions for improving the clarity of the manuscript (see Basic Reporting section) as well as on some methodological questions (see experimental design).


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: POPULATION STRUCTURE, ADAPTATION AND DIVERGENCE OF THE MEADOW SPITTLEBUG, PHILAENUS SPUMARIUS (HEMIPTERA, APHROPHORIDAE), REVEALED BY GENOMIC AND MORPHOLOGICAL DATA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript reports relevant findings about Philaenus population structure. The experimental questions and the way the authors pursued them are thoroughly described.
Experimental design: The experimental design is solid
Validity of the findings: The results are well reported and clearly discussed
Additional comments: The article provides relevant insights about Philaenus genera.
I suggest minor changes that might improve the  draft:
1) I consider the difference between spumarius and tesselatus at nuclear genome level, while no differentiation is possible by just mitochondrial analysis, as the main finding of this paper. Try to better stress in the astract the importance of developing alternative and integrative approaches for the taxonomy of a group of species, instead of centering the discussion of the reflection of your findings on Xylella fastidiosa control.
2) I would remove any reference to Xylella fastidiosa transmission, since it is not clear how your findings could contribute to the development of a control strategy either directly or indirectly


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: POPULATION STRUCTURE, ADAPTATION AND DIVERGENCE OF THE MEADOW SPITTLEBUG, PHILAENUS SPUMARIUS (HEMIPTERA, APHROPHORIDAE), REVEALED BY GENOMIC AND MORPHOLOGICAL DATA
Review round: 1
Reviewer: 3

Output:
Basic reporting: In the manuscript “Population structure, adaptation and divergence of the meadow spittlebug, Philaenus spumarius (Hemiptera, Aphrophoridae), revealed by genomic and morphological data”, the authors use a combination of morphological, mtDNA, and RAD-sequencing methodologies to explore the evolutionary relationship between Philaenus spumarius and P. tesselatus. These species of spittlebugs are found throughout Europe, as well as in Northern Africa and the USA and they are of particular concern because they are a vector for a bacterial plant pathogen, X. fastidiosa. Previous work has identified P. tesselatus and P. spumarius as two distinct species using morphological data, but this is not reflected using mtDNA. The authors use collections from 9 sites in Europe, Africa, and North America to first, determine if genomic data can clarify the status of P. tesselatus and next, to look for evidence of selection on P. spumarius populations. The RAD-seq data supports previous claims that P. spumarius and P. tesselatus are two distinct species, despite a lack of evidence using mtDNA haplotypes. Further, they find evidence for positive selection on P. spumarius populations that is correlated with a handful of environmental variables as well as a signal of isolation by distance among the populations. They also find evidence to support the origin populations of several colonization events in the USA and AZO sites. The authors conclude with a discussion of the limitations of their work and the possibility that Wolbachia has had an impact on the mitochondrial DNA in P. spumarius, explaining the lack of differentiation in mtDNA compared to both morphological and RAD-seq data.

In general, I feel that this manuscript is an excellent example of the ways that genetic, genomic, and morphological data can be used together to generate convincing results about the evolutionary history of organisms. The authors utilize a widespread dataset with an appropriate number of specimens included at most sites. They analyzed their datasets with caution that is reflected in their methodology and further discussed in the discussion section. This work also contributes to the current understanding of drivers of evolution and speciation and offers new considerations as to the impacts of Wolbachia on mtDNA analyses. For these reasons, I feel that this manuscript would be of general interest to the readers of PeerJ. However, I have several recommendations that need to be addressed to improve the clarity of the manuscript, particularly to provide more explanation in the methods, broaden some of the ideas presented in the introduction, better explain the biology of P. spumarius, and improve the accessibility of the figures.

In the introduction, the authors could expand their explanation of the importance of understanding the evolutionary history of P. spumarius for models of risk assessment, as described in Lines 108-112. Addressing this more specifically would be helpful to understand why Xylella fastidiosa is relevant to the main topics of the manuscript when the methods/results are focused on the biology of P. spumarius.

All Figures: The combination of red and green is not an accessible combination of colors for people that are colorblind. Consider using alternative colors to benefit these readers, especially in the Structure plots that lack an alternative method to visualize the differences between groups.

Throughout the manuscript, some sentences could be restructured to improve clarity and ease of reading. For example, Lines 350-351 says “We did not exclude any variable at the start of the study based on their correlations, but we tested for correlation the ones that were significantly associated.” I think the second part of this sentence could be revised to make it easier for readers to understand when the authors tested for correlation. I have included a few more similar examples in the comments below.
Experimental design: Experimental design adequately tests the research questions and successfully pairs morphological and genetics data together.

The bioinformatic and statistical methods included an appropriate amount of caution throughout and I appreciated the authors' discussion of possible caveats as part of the discussion section.

Lines 185-193: It is unclear from the methods section how insects were collected. If P. spumarius is polyphagous, were insects collected off of a single host, multiple host plant species, or by using an alternative method? These collection methods could have an impact on the authors findings, as heterozygosity and population structure could also be influenced by host use or attraction to a pheromone bait or by variation in collection methods across sites. Further, has work been done to assess host preferences or host-associated differentiation within P. spumarius or is the species truly indiscriminate among the host plants listed? In general, a broader discussion of the biology of P. spumarius would be helpful – including mention of generation time and life cycle.

Lines 203-210: The description of aedeagus measurements does not include an explanation about how the measurement data was collected – manually or using a computer program for morphometric analysis? It would also be helpful to describe any data preprocessing that occurred before the PCAs described in the next paragraph.
Validity of the findings: All underlying data has been provided and is accessible on NCBI if not included in the supplemental materials. See general comments below for a couple links that failed to work in the methods section.

Discussion includes some speculation about the impact of Wolbachia on the mtDNA findings. The authors make it clear that this includes some speculation and I feel the commentary is an interesting addition to the end of the manuscript.
Additional comments: Minor Concerns
Line 124: Capitalize “new” in “New Hampshire”
Line 221: Revise “including RNase A treatment step” to say “including an RNase A treatment step”
Line 246: The link included in this line requires a university login and was inaccessible to me. Please consider revising this to a more publicly available source.
Line 250: The link in this line is out of date and no longer maintained. Please consider updating it to their new website.
Line 261: Change “ran” to “run”
Line 318: Change “correlation” to “correlations”
Line 386: Should the word “variables” be changed to “variable”? I’m not entirely sure what this sentence is trying to say. Please consider revising for clarity.
Lines 486-487: Consider revising this sentence for clarity – “However, there were a few individuals that were genetically more similar to others more distant geographically,…”
Lines 587-589: What is known about the phylogenetic relationship and/or divergence timing between P. spumarius, P. tesselatus, and other species found in the region? P. spumarius and P. tesselatus appear to be closely related, but I am curious about the remaining species in the region that the authors describe in the introduction as additional contributors to gene flow.
Line 632: Consider removing “however” from this sentence for clarity.
Line 762: Some references are in a smaller font than the rest of the manuscript.
Figure 2 Legend: Italicize Philaenus in the first line.
Figure 3: Is it possible to move the vector labels to make them easier to read without the overlapping data points?


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: POPULATION STRUCTURE, ADAPTATION AND DIVERGENCE OF THE MEADOW SPITTLEBUG, PHILAENUS SPUMARIUS (HEMIPTERA, APHROPHORIDAE), REVEALED BY GENOMIC AND MORPHOLOGICAL DATA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The language has been improved in the areas that were pointed out by myself and other reviewers of the previous version. In some newly-added sections, I have suggested a few additional corrections to minor grammatical errors, which I have marked with comments on the attached annotated PDF.

A few specific comments for continuity:
-Both "colonization" and "colonisation" are used in the manuscript. Either is acceptable, but I would stick with one or the other.
-Similarly, both "color" and "colour" are used in the manuscript.
-Both "Structure" and "STRUCTURE" are used in relationship to the model-based population structure analysis performed. I would stick with one or the other.

I also appreciate the authors switching to color-blind friendly colors in their figures, which I believe will improve accessibility for all readers.
Experimental design: I have no additional comments, as the added details provided in response to my and other reviewer's suggestions have addressed my previous concerns.
Validity of the findings: I have no additional comments.
Additional comments: I am satisfied by and appreciate the edits made by the authors. I would suggest one additional, very minor round of close reading to tighten up the flow, but am otherwise looking forward to seeing the final manuscript published.


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

Input:
Title: POPULATION STRUCTURE, ADAPTATION AND DIVERGENCE OF THE MEADOW SPITTLEBUG, PHILAENUS SPUMARIUS (HEMIPTERA, APHROPHORIDAE), REVEALED BY GENOMIC AND MORPHOLOGICAL DATA
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors of "Population structure, adaptation and divergence of the meadow spittlebug, Philaenus spumarius (Hemiptera, Aphrophoridae), revealed by genomic and morphological data" have adequately responded to all of the feedback from my initial review and have made all necessary revisions. At this point, I feel that the manuscript is prepared for publication in PeerJ.


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

Input:
Title: DEVELOPMENT OF LANDSCAPE CONSERVATION VALUE MAP OF JEJU ISLAND, KOREA FOR INTEGRATIVE LANDSCAPE MANAGEMENT AND PLANNING USING CONSERVATION VALUE OF LANDSCAPE TYPOLOGY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The study addresses the interesting question of how to evaluate landscapes for application in conservation planning and management. It provides an interesting case study and demonstrates how land-cover and landform data can be combined with expert evaluations. The story is mostly easy to follow and rooted in relevant literature within the field. The paper's strength lies in the ambitious attempt to build a consensus on landscape values from a descriptive and largely value-neutral framework of landscape type classification. I enjoyed reading the article. I had some issues with the structure of the text and justification for some of the methodology and the findings.
I had some problems with the structure of the article. It mostly follows the IMRAD-structure of scientific writing (introduction; materials and methods; results; discussion), but several deviations from this structure occur. I would recommend restructuring some of the material to avoid mixing results with methods and methods with discussion. This applies to the sections mentioned below, but the authors should go through the review systematically to check for inconsistencies in this regard.
Line 233–244, line 287–296, and line 308–318 summarise land cover and the cover of landforms and landscape types in the study area. Perhaps these sections could be moved to the results chapter, or alternatively to the description of the study area. Anyway, these summaries are not methods.
I would recommend starting the discussion chapter by building on the results. Line 434–445 is a somewhat confusing start of the discussion and could be moved or omitted. I would suggest starting the discussion with line 446.
Line 463–468 is a description of methods and should be moved to the methods section.
The section about limitations and shortcomings (line 562–572) is relevant and exciting, but should be moved to the discussion section, whereas the conclusion section could be shorter. I recommend that limitations and shortcoming is included as a separate section in chapter 4.

The tables, figures and maps are excellent, and they convey the information in an effective manner. An exception is Figure 3, whic is distorted in some way (stretched), and should be redesigned.
Experimental design: The introduction provides an exciting stage of the paper. It explains the dilemmas and conflicts in landscape management in the country, i.e., the different and often irreconcilable views on future development/trajectories for a landscape. Line 141–145 gives some good clues, in this respect.
The justification for the use of land-cover maps and landform maps is well justified in theory and well explained. However, more references should be added to provide examples of the value criteria used for landscape evaluation (e.g, the value criteria shown in Figure 7 part 2 (representativeness, specificity, etc.; see e.g, Solecka, I. 2018. The use of landscape value assessment in spatial planning and sustainable land management — a review. Landscape Research, 1-16. doi:10.1080/01426397.2018.1520206 or Erikstad, L., Lindblom, I., Jerpåsen, G., Hanssen, M.A., Bekkby, T., Stabbetorp, O. & Bakkestuen, V. 2008. Environmental value assessment in a multidisciplinary EIA setting. Environmental Impact Assessment Review, 28(2-3), 131-143. doi:10.1016/j.eiar.2007.03.005).
Several key concepts and terms are not clearly defined, and many of the terms are often used in very different meanings and settings in landscape research. Therefore, I would recommend adding a box, including key definitions of terms such as landscape, landscape type, landscape value, and the value criteria in box 7 par 2.
It is not entirely clear to me whether the landform map was created by Park 2004, or only the method. Please try to write this in a more pointed manner. If I understood the manuscript (line 263–296) you did this based on the methodology from Park 2004, adapted to the scale of the study area. Please write this section shorter and somewhat clearer.
Validity of the findings: The process with multiple assessments in a focus group, including documentation of the process in the supplementary material, is clearly described and easy to follow. However, the connections between sub-criteria such as representativeness, rarity, etc., and the numbers that the experts used in the evaluation is not fully transparent. This should be elaborated and rooted in theory. General problems with expert judgment should also be discussed in more detail. I would argue that putting numbers on values is a good idea only as far as the criteria for values such as "high value", "medium value", etc. is clearly described and defined. The lack of broader user participation except from expert should also be discussed further (this is briefly mentioned in the conclusion section).
I also think the difference between general landscape types (reoccurring units) and individual landscape areas should be discussed more. The conceptual idea of assigning landscapes to types is rooted in the tradition of systematic physical geography or 'landscape geography', the aim of which is to present and explain typologies of similar landscapes based on their material content. Grouping of similar landscapes into types is an effective way to communicate landscape information because affiliation to type alone will provide an extensive amount of information about any singular individual of that particular type. Since a type in a type system comprises a predictable, 'normal' amount of landscape variation, affiliation to type is also a useful reference and a good starting point for assessment of the unique character and properties of individual landscapes. However, variation within types and individual characteristics of singular areas is not discussed in detail, although the issue is raised by some experts in the supplementary material. I think this should be discussed further.
Additional comments: With these taken into consideration, I am happy to recommend the manuscript for a new review and possibly 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: DEVELOPMENT OF LANDSCAPE CONSERVATION VALUE MAP OF JEJU ISLAND, KOREA FOR INTEGRATIVE LANDSCAPE MANAGEMENT AND PLANNING USING CONSERVATION VALUE OF LANDSCAPE TYPOLOGY
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is an interesting study and the authors have developed the landscape conservation value for the Jeju Island using GIS and focus group interviews (FGIs). English is good but the paper should be well structured, especially in the introduction and discussion sections.
Experimental design: The article meet the standards.
Validity of the findings: I think all underlying data have been sufficiently provided,
Additional comments: As the paper currently stands, I have some questions and suggestions for making the paper more systematic.

Introduction:
It is necessary to restructure the introduction. It may contain some general content about Korea but too much local. I would recommend that the authors rewrite the paragraphs to be more concise and concrete in terms of presenting the core issues, research gap, and main questions.
In this regard, the front and back paragraphs should be connected, well structured. For example, the paragraph that starts with landform (i.e. Line 145) do not connect well with the preceding paragraph.

Materials and Methods:

2 of the Unit ‘km2’ should be superscripted.

What program was used to obtain the upslope contributing area (AS) and surface curvature (Cs)?

Line 288: spacing error, distinction(Fig. 4)

Where is Table B1?

Line 364: Relevant references should be added.
Line 372: is to have -> was to have

Results:

Line 413: In Figure 8 -> Figure 9?
Need to revise the legend of Figure 9 so readers can more easily check the high and low levels instead of quantile.

Table 3: In my understanding, the final LCV is standardized from 1 to 10. How do you get mean pixel LCV and Std. Deviation greater than 10? Please explain if there is anything I missed.

Discussion:
The discussion should be concise. For example, the starting paragraph is too common. In addition, it's a little unnatural with methodology and new results (i.e. susceptible areas).

Although the authors mentioned landscape terminology in terms of socio-ecological systems, human related cultural values will be omitted as the developed LCV deals with spatial content (i.e. land cover and landform).

Conclusion:
Line 553: In this context, land cover data…
That's what you might think, it is considered an extended interpretation that the land cover itself reflects cultural things.


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: DEVELOPMENT OF LANDSCAPE CONSERVATION VALUE MAP OF JEJU ISLAND, KOREA FOR INTEGRATIVE LANDSCAPE MANAGEMENT AND PLANNING USING CONSERVATION VALUE OF LANDSCAPE TYPOLOGY
Review round: 2
Reviewer: 1

Output:
Basic reporting: The issues with structure and clarity from the first submission is solved, and the revised manuscript is much clearer and more readable. The english is good, but some references appears to be at the beginnng of a sentence without furter introduction (at least in my tracked-changes-version). I have not proofread the manuscript, so pleas ensure high-quality proof-reading befor publication.
Experimental design: Comments from the first review are incorporated nicely. Problematic issues with the experimental design is now solved.
Validity of the findings: Comments from the first review are incorporated nicely, with a much better discussion of limitations.
Additional comments: Thank you for incorporating the comments from the peer-review in a wise and elegant manner. I think the manuscript is much improved, and shoul be ready for publication after proofreading.


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: DEVELOPMENT OF LANDSCAPE CONSERVATION VALUE MAP OF JEJU ISLAND, KOREA FOR INTEGRATIVE LANDSCAPE MANAGEMENT AND PLANNING USING CONSERVATION VALUE OF LANDSCAPE TYPOLOGY
Review round: 2
Reviewer: 2

Output:
Basic reporting: Overall, the manuscript has been improved according to the comments. But, the typos are still found (e.g. Line 116: n terms of). The authors are advised to correct the ms thoroughly.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Thank you for your hard work.


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

Input:
Title: MYOCARDIAL CONTRAST ECHOCARDIOGRAPHY ASSESSMENT OF MOUSE MYOCARDIAL INFARCTION: COMPARISON OF KINETIC PARAMETERS WITH CONVENTIONAL METHODS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The introduction was very long, and sometimes it is not easy to follow. A good introduction should identify the article topic and provide essential context. I suggest rewriting the introduction in brief.

The conclusion should be more specific and has conclusive statements. Please rewrite!

The figures should be including the (P values) between the MI and sham groups to become more understandable for the readers.
Experimental design: 'no comment'
Validity of the findings: It was the first time that compared the myocardial contrasts echocardiography (MCE) with the speckle tracking data in the small animal model. However, the evaluation of MCE by the gold standard (TTC stain) was previously reported in Bhan et al. 2013 (doi:10.1152/ajpheart.00553.2013)

Results. In fact, It could not understand why the data of myocardial infarction( MI)  mice and sham were mixed and presented as one group (correlations figures). The Author aimed to demonstrate the usefulness of the  MCE method with MI.  Thus, it could not plot the data as one group, even though the study sample was very small. Please comment.
Additional comments: Many limitations should be considered and mentioned.
The first and major limitation should be the groups' sample size, which can affect comparisons of small differences between methods. Therefore, the results should be confirmed in a larger series.
When assessing the left ventricular function, an anaesthetic regimen should be chosen carefully to minimize its effect on cardiovascular physiology. The adverse effects of anaesthesia on conventional and speckle tracking echocardiographic parameters have been previously reported.
Moreover, the apex foreshortening should be considered. The apex foreshortening could result from the chest anatomy of animal models, surgical operation, and more frequently in acquisitions obtained from less experienced sonographers. The effect of apex foreshortening on conventional and strain measurements should be stated as a limitation.


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: MYOCARDIAL CONTRAST ECHOCARDIOGRAPHY ASSESSMENT OF MOUSE MYOCARDIAL INFARCTION: COMPARISON OF KINETIC PARAMETERS WITH CONVENTIONAL METHODS
Review round: 1
Reviewer: 2

Output:
Basic reporting: - The English was overall clear and unambiguous.
- The literature was sufficiently referenced
- The raw data set was shared.
- Self-contained with relevant results to the hypothesis
Relevant comments are given in the general comments to the authors.
Experimental design: Original, rigorous, well-defined research using methods with sufficient detail.
Validity of the findings: Robust, statistically sound data.
Additional comments: The authors describe a readily available echocardiography tool to non-invasively measure blood volume and perfusion after myocardial infarction, and validated it with conventional methods.

Overall the authors have a well -written manuscript with solid scientific data using well-described methods.

There are a few issues that should be addressed:

Major issues:
The authors should clarify better the usefulness of measuring noninvasively blood volume and perfusion, and what it could be used for in research.

Minor comments:
Abstract:
-The first sentence in the abstract should be simplified/ split in two:
“This study explores the use of and provides proof of concept for a minimally invasive assessment of myocardial infarction in mice using myocardial contrast echocardiography (MCE) and existing equipment and software readily available to the average researcher. “
- the abbreviations that are not used more than two times can be taken out (this is also true for the manuscript)
Manuscript:
-p1 line 35-36: Also small animal MRI is being used. – please add
-	Table 1 is redundant if all the means and p values were added to the graphs.

 - Figures: now from only looking at the graph, I understand the method does not work, therefore: in addition to the arrows to the data point of the failed MI, please separate the failed MI point with a different color (but same shape) and an additional legend in the figure. For the statistical test state that this point was excluded. If possible, add the TTC for this datapoint.

Figure 1: Point out in graph 1C &1F, point out where wash in rate and peak enhancement were taken from.

 Line 357 An M-mode: should be: “A M-mode..”

Line 158: polyethylene PE 20 tubing : PE is confusing in this sentence, please erase – as later you use it for peak enhancement.
Line 156: “proof of concept for an innovative complementary technique to conventional TTC staining and echocardiography”: add noninvasive / perfusion measurements to the sentence
-Discussion: add noninvasive MRI (dynamic contrast enhanced/ late gadolinium enhanced) to the discussion. E.g. https://pubmed.ncbi.nlm.nih.gov/26282195/


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: MYOCARDIAL CONTRAST ECHOCARDIOGRAPHY ASSESSMENT OF MOUSE MYOCARDIAL INFARCTION: COMPARISON OF KINETIC PARAMETERS WITH CONVENTIONAL METHODS
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comments
Experimental design: No comments
Validity of the findings: No comments
Additional comments: No 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: MYOCARDIAL CONTRAST ECHOCARDIOGRAPHY ASSESSMENT OF MOUSE MYOCARDIAL INFARCTION: COMPARISON OF KINETIC PARAMETERS WITH CONVENTIONAL METHODS
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have addressed all issues raised.


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: NEUROMEDIN U AND NEUROTENSIN MAY PROMOTE THE DEVELOPMENT OF THE TUMOUR MICROENVIRONMENT IN NEUROBLASTOMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article must be written in English but the authors should improve it avoiding typing errors.
The authors included a sufficient introduction and background with explanation of own hypothesis. However not revelant literature on  Neuroblastoma  was referenced. I suggest to cite Luksch R et al. 2016.
The article should be improved in clarity and should be implemented in Methods and Results section with more detailed description of data.
The hypothesis was not fully sustained by overwhelming results. The authors should provide additional experiments to adequately support own hypothesis.
Experimental design: The authors define well the research question, however several past studies provided Neuroblastomas gene expression profiles resulting in a number of gene expression
signatures being used to evaluate the patient’s risk (De Preter et al., 2010; Oberthuer et al., 2010). Unfortunately, findings derived form these studies have never been translated into clinical practice.

The knowledge gap was identified and the study attempted to fill the gap but I suggest to tone down the wording on the functional consequences of authors' hypothesis.

The investigation seems to be conducted rigorously but not all experiments showed a high technical standard.

Methods were absolutely not described with sufficient information to be reproducible by another investigator.
Validity of the findings: The authors' hypothesis could have a good rationale, but the study needs to be improved in experimental design with adeguate Neruoblastoma risk stratification. It will add value to the literature providing novelty to the actual information on Neuroblastoma.

The data on which the conclusions are based are not robust and nedded to be improved.
The conclusions are appropriately stated and are connected to the original question investigated. However, the experiments provided do not support adequately the hypothesis.
Additional comments: In this study, Yang and co-workers analyzed the molecular differences in the tumour microenvironment between INSS stage 4S and stage 4 examining gene expression microarray data from two public datasets. Biological processes of differentially expressed genes (DEGs) were assessed by gene ontology enrichment analysis revealing that 216 DEGs were enriched in aggressive biological processes. The authors highlighted hub genes using STRING database and Cytoscape verifying  gene expression in Oncomine database and found Neuromedin U and neurotensin elevated in stage 4 . To functionally validate in silico findings the authors performed transwell and wound healing assay to explore the function of the genes in neuroblastoma cell line SK-N-BE(2). Moreover, they analyzed leukocyte population infiltrating tumour showing a high infiltration of regulatory T cells and type 2 tumour-associated macrophages in stage 4 but not in stage 4S. The authors conclude from their data that neuroblastoma aggressiveness may be associated with the action of neuromedin U and neurotensin in modulating tumour microenvironment and suggest that this information might be used for the development of targeted therapies.

Specific comments:
1. A major limitation of this study is the relatively low number of cases in the two groups of two datasets. Taking also into account that the study has as starting point a re-analysis of public microarray datasets there is a certain risk of over-fitting and over-interpretation of the results. I strongly suggest to validate the results in an independent cohort. This task might be achieved optimally by analyzing another microarray dataset. An example is E-MTAB-161 dataset containing information of 504 NB smaples. Validation of the data in an independent cohort would substantially strengthen the significance of the results.

2. In the gene expression microarray dataset examined, the authors extrapoled the gene expression data of stage 4S and stage 4. However, since the high NB tumor heterogeneity as well as clinical outcome, to compare NB patients stage 4 versus 4S could have high risk again in over-interpretation of the results. Do the authors take into account only NB patients stage 4S with disease regression? It is note that patients stage 4S have different outcome according also MYCN amplification.  It would be very interesting to repeat the analysis using stratification risk groups according to the High Risk Neuroblastoma Study 1.7 of SIOPEurope (SIOPEN) specifications considering HR group patients stages 2, 3, 4, and 4S with MYCN gene amplification or stage 4 samples with an age at diagnosis over 12 months of age; ‘low/intermediate- risk’ (LIR) group the remaining patients. The study would benefit from integrating the performed analysis by adding the Kaplan–Meier analysis on Neuromedin U and neurotensin and stage risk.

Minor revision
1. In general, the Methods section would be implemented with more detailed on procedure protocols. An example in “6.3 Western blot” control antibody used is lacking as well as information on antibodies incubation time, protein extraction and gel running. The authors would make substantial changes to the manuscript in writing and editing.
2. Same comment for Methods section. The Results section should be expanded and described more comprehensively.
3. In paragraph 2 of Results, the authors explored protein-protein interaction of 216  DEGs. However, the authors should provide the results of the analysis before showing the top 25 hub genes.
4. Paragraph 3 of Results. The study would benefit from sharpening the hypotheses on NB tumour progression. I suggest changing the last sentence since NB tumour progression don’t’ follow the roles of Vogelstein “multistep tumour model”.  It is not very clear as CIBERSORT works, please provide more detailed since from this analysis the authors based own speculations.
5. Paragraph 4 of Results. The authors showed western blot of two proteins of interest. However, the two proteins are both of 20 kDa. Did the authors incubate the antibodies on the same membrane? If, yes, they should run protein lysate so that they can incubate the full membrane with two antibodies in parallel, without stripping. Moreover, the authors should show the effects of silencing on RNA. I suggest to perform RT-PCR.

6. The authors should support own hypothesis on the role of Neuromedin U and neurotensin in tumour aggressiveness with additional functional tests, such as clonogenic assay.

7. Conclusion. I suggest to tone down the wording on the functional consequences of genes of interest, and even more on their potential significance as therapeutic targets.

8. Written English could be improved and typing error had to be corrected. Some examples where the language have to be improved include “wound healing assay to detect the funtion of the genes in neuroblastoma cell line SK-N-BE(2) in abstract section, change funtion in function; lines 134 add “t” to scrip!!


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: NEUROMEDIN U AND NEUROTENSIN MAY PROMOTE THE DEVELOPMENT OF THE TUMOUR MICROENVIRONMENT IN NEUROBLASTOMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this manuscript, Yang and coworkers describe the molecular differences in the tumour microenvironment (TME) between Neuroblastoma INSS stage 4S and stage 4. The work is presented quite well and shows that Neuromedin U (NMU) and neurotensin (NTS) were elevated in stage 4. The authors also demonstrate that NMU and NTS promoted the proliferation and invasion of SK-N-BE(2) cell. Lastly, the authors showed a high infiltration of regulatory T cells and type 2 tumour-associated macrophages in stage 4 but not in stage 4S. However, there is no direct link here to show a relationship between NMU /NTS and TME. Overall this is an interesting piece of work, but much work needs to be done to improve the robustness of the findings.
1.	Please could the authors check the manuscript throughout for grammatical errors as there are sentences which do not make sense.
2.	The introduction needs to be improved. The authors should describe better lines 93-97 to provide more justification for the study.
3.	The figures need to be improved:
-	Figure 1. Could the authors label A and B for the upper and lower panel?
-	Figure 2. The authors state that NMU and NTS were significantly elevated in stage 4 however no statistics are shown on the bar chart.
-	Figure 4. There is no correlation between figure legend and labelled panel, please add the western blot images in the figure. The wound healing assay is incomplete. Have the authors measured the migration distance? They need to report the statistics of this analysis.
Experimental design: In general, the Materials and Methods section is not well described the authors should provide more detailed on procedure protocols. Some examples are reported below.
1.	In 6. Validation of hub genes section, the author should describe better how they select NMU and NTS for further validation
2.	In 6.2 siRNA transfection section more detail is required on how this was performed and timing of experiments.
3.	In 6.3 Western blot section more detail is required in regard to how the proteins were obtained and how the western blot was performed.
4.	In 6.5 Wound healing assay section please clarify and provide more detail in regard to how the microscopy was conducted and how the wound healing was measured.
Validity of the findings: As for the Materials and Methods section, the Results need to be better described. The authors state that NMU and NTS may be implicated in the modulation of TME and neuroblastoma development, however, this hypothesis was not fully supported by results.
1.	In supplementary Table 2 the authors reported the 25 hub genes screened by CytoHubba ranked by Degree method, could they also report the genes obtained after combination with the Oncomine database? Furthermore, could the authors comment on why NMU and NTS were selected for further validation?
2.	In “3. Significant tumour-associated lymphocytes infiltration occurred in stage 4” section it is shown that tumour-associated macrophages type 2 (TAM-M2) and regulatory T cells (Tregs) were significantly infiltrated in stage 4, but there is no direct link here with NMU and NTS expression. The authors should discuss the possible causes this.
3.	The “4. NMU and NTS promote cell proliferation and invasion of SK-N-BE(2)” section needs to be improved. First, cell proliferation and migration are different concepts. Wound healing experiments can indicate the cell migration while the BrdU assay, MTT assay or Ki67 may be used to analyze cell proliferation. Furthermore, no statistical tests have been conducted to support the effect of downregulation of NMU and NTS on cell migration.
Additional comments: No additional comments