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: SOCIAL MOTOR PRIMING: WHEN OFFLINE INTERFERENCE FACILITATES MOTOR EXECUTION
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors report the results of one experiment in which they ask participants to perform either precision or whole-hand grips, after observing video of an actor performing precision grips in an interactive and non-interactive context, or without observing any action before (baseline). This experimental design allowed the authors to investigate: visuomotor interference, visuomotor priming, social motor priming and social motor interference. In the Interactive condition the socially appropriate response was a complementary action (performing a whole-hand grip). Results show that while visuo-motor interference effect  was only present in the Non-Interactive condition, in the Interactive condition, incongruent (yet socially complementary and appropriate) actions were facilitated with respect to congruent ones.
Although not so novel, in light of previous studies investigating similar processes, the results of the present study are interesting and the study is well designed. Moreover,  the article is well written and the images are very well done. I would suggest to better explain the novelty of the present findings and to clarify the hypotheses tested in the present study. Moreover I suggest to expand the introduction and better explain some methodological aspects as highlighted in the following points.

In the introduction, lines 89 – 94, the authors claim that “Another possibility is that a simple effect of co-representation might explain the interference effect. In that case, motor impairment would not last in a postponed task.” Could you please explain why the motor impairment should not last in a postponed task? This seems an important issue, given that this statement apparently sets the ground for your hypothesis. In fact, in lines 96-97, you write that “the present study has been designed to specifically test whether social compatibility effects are long-lasting and can modulate the perception-action coupling even after action observation.” I find this connection not very straightforward. It seems that you are implying that finding a delayed social compatibility effect would support the “continuum” hypothesis and disprove the “co-representation” one? If so, why and how? Please explain.

In the introduction the authors refer to studies reporting reduced visuo-motor interference when participants are acting towards reaching a joint goal. Please also discuss other studies indicating that during joint-actions performing complementary actions is not more difficult than performing imitative ones (Sacheli et al., 2015 Nat Commun, Sci Rep; 2018 JCN; Era et al., 2018 SCAN, Psych Res; Gandolfo et al., 2019 Acta) and suggesting that when interacting with a partner the need to predict the other’s actions and to program one’s own action accordingly might abolish visuo-motor interference, that is instead present when acting at the same time without needing to predict what the other is doing.
Experimental design: If the authors are interested in creating an interactive scenario, why was the action participants needed to perform (precision or whole-hand grip) pre-instructed? Participants could have been asked to complement or to imitate the action of the actor, without knowing in advance what action to perform (maybe adding more actions to possibly perform, to create more variability) and thus creating a more naturalistic scenario. Please comment.

In the data handling and results section Accuracy is not reported (number of correct grasping). Please report this measure.
Validity of the findings: Please highlight the novelty of your findings with respect to previous research. Are they new because they demonstrate that social interference also occur in a postponed task? Please elaborate more on this point.

In the Introduction (lines 88-90) you suggest that motor interference might be described as a continuum.
In Conclusion (lines 298-299) you write that “It seems therefore that joint action goals reduce visuomotor interference effects along a continuum”. However, it is not clear which of the reported results would support this conclusion. Please explain.

Also, in lines 296-298, you write that “interference due to observing a precision grip and subsequently perform a whole-hand grasp was partially reduced for the interactive compared to the non-interactive context (i.e., quicker RT and movement time)”. However, from the results it appears that  interference effect is abolished in the interactive condition and not just partially reduced. Please explain.

Looking at the plot in Fig. 5, it seems that the mean MGA value for PG in Interactive condition is almost comparable to the MGA values for WHG. This seems quite odd (and potentially interesting), as it would mean that participant’s grip aperture in Interactive-PG was as large as when performing a WHG. Also related to the previous point about the Accuracy, can you confirm that only correct trials (trials in which participants were performing PG are required by the instruction) were entered in the analysis? Please comment.
Additional comments: No additional comments


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

Input:
Title: SOCIAL MOTOR PRIMING: WHEN OFFLINE INTERFERENCE FACILITATES MOTOR EXECUTION
Review round: 2
Reviewer: 1

Output:
Basic reporting: The literature review and theoretical context is now much improved
Experimental design: While there are still limitations to the original design, the authors have addressed and discussed these adequately.
Validity of the findings: no comment
Additional comments: I would recommend another editing pass for the new sections by a native English speaker.


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: SOCIAL MOTOR PRIMING: WHEN OFFLINE INTERFERENCE FACILITATES MOTOR EXECUTION
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: I am happy with the work made by the authors.


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: VALIDATION OF COI METABARCODING PRIMERS FOR TERRESTRIAL ARTHROPODS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is well written and clear to understand. There is suffcient literature cited.
Experimental design: The research question was well defined and performed to a high standard.

One question was if the authors sequenced negatives and if so how these were dealt with in the bioinformatics.
Validity of the findings: The findings were valid and the conclusions reached backed up by the results presented.
Additional comments: L62: While this is true COI databases are also limited in taxonomic groups outside of insects.

Figure 1: Braukman 2018 or 2019?

L113: Mock community extracts were ….

L127: (( not needed

L172: Were sequencing negatives run?

L192: Figure 3 overviews the results of the processing steps……

L229: Amplicon concentrations (ng/ul) reached …..


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: VALIDATION OF COI METABARCODING PRIMERS FOR TERRESTRIAL ARTHROPODS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I have reviewed the manuscript entitled “Validation of COI metabarcoding primers for terrestrial arthropods” by Elbrecht and colleagues. In this work the authors evaluated a large number of potential primer sets for the amplification and identification of bulk arthropod samples using DNA metabarcoding. Their systematic approach to evaluation involved gradient PCR to determine amplification efficiency at differing TA, sequencing to determine detection of full species richness, and annealing temperature optimization for a subset of primer sets. They go on to recommend several sets of primers that performed well and advocate for the use of single primer sets for arthropod identification as opposed to the use of multiple primer sets to amplify all species.

Overall, I found this work to be impressive in its thoroughness, both in terms of the study design and the level of supporting documentation provided.  This is a valuable piece of work for the molecular ecology community and I commend the authors for its undertaking. My comments are mostly minor with one major suggestion.

Major comment:

The authors have gone to great lengths to evaluate a large number of primer sets for arthropod identification, but the concluding statements do not seem definitive. “Our study demonstrates that the fwh2, BF1/2/3 + BR2 and mlCOIintF based primer sets all perform well when metabarcoding terrestrial arthropod samples. For most of these primer sets, annealing temperatures of 46-50°C are ideal. When data is analyzed for Exact Sequence Variants(ESVs), the BF3 + BR2 primer set is recommended as it is not affected by primer slippage.”

I think the impact of this paper would be improved with more direct statements about which primer sets are recommended and for which applications. An abbreviated version of these statements should appear in abstract and in the conclusions. This paper is likely to be cited specifically for these recommendations and they should be easily accessible to the reader.

For example: “We recommend fwhF2 + fwhR2n for amplification of degraded DNA samples such as gut contents, and BF3 + BR2 when high taxonomic resolution is required”.

Minor comments:

Line 52 – Change “on” to “of”

Line 55 – change “targets” to “target”

Line 79 – Check journal style, but there is a missing “)” or citation brackets should be removed.

Line 84 – Suggest adding… given the increased cost and time “associated with amplifying and sequencing additional markers”…

Line 113 – Change “These” to “The”

Line 122 - Insert brief description of the utility of gradient PCR for this study. How is this tool being used to identify best primer sets? Describe that the heat block itself provides a gradient of temps so a full plate provides a range of TA across replicate reactions.

Line 129 – No replication? A statement here about why replication was not performed would be useful. I know this is addressed in the discussion, but you can also briefly justify the decision here.

Line 143 – Please clarify what the exact selection criteria were.

Line 149 – I found the structure of this section confusing. I suggest clearly breaking this into paragraphs for each topic, e.g., fusion primer design, PCR 1, PCR 2, library prep, sequencing.

Line 150 – change “where” to “were”

Line 154 – What is meant by “sequenced half the samples reverse orientation”? Meaning the primer tags were reversed, or adapter sequences? Please clarify.

Line 173 – Why was less DNA used for the Malaise trap sample?

Line 184 – Please translate code functions. What is fastq_pctid?

Line 206 – Again, please specify the exact selection criteria that were used.

Line 218 – The subsampling/bootstrapping process in Figures 4 and 5 is not described in this section. Please explain.

Line 263 – Fig S11 rarefaction curves. This may be worth moving to the main MS as it is a good visual representation of the main findings. I understand there is some conceptual overlap with Fig 4 but the shape of these curves clearly conveys the advantages of certain primer sets.

Line 319 – This statement seems overly bold. Sample replication is clearly very important, but PCR/Sequencing replication can also be important for generating consensus taxonomic assignment at the sample level and for identifying contamination.

Line 331 – Does this reflect sequencing bias or DNA degradation? The same pattern has been observed in DNA quantity by fragment length using qPCR of degraded samples.

Lines 352 – 354 – Is it also possible that primers for shorter fragments pick up more rare species because trace DNA is more amplifiable at short lengths? This could be an argument for the use of fwhF2 + fwhR2n or short barcoding regions with high taxonomic resolution.

Line 371 – Change “predicted” to predict

Line 423 – Change “,” to “;”

Line 440 – It is not clear from my reading of this MS why one would choose a primer set with a longer fragment over fwhF2 + fwhR2n. Short fragments have many advantages and if this provides similar taxonomic resolution to the longer fragments then why choose a longer fragment?

Lines 441 – Change “fwh2” to “fwhF2”

Line 451 – I commend the authors for providing extensive supplemental information. However, in my quick scan of the R code I did not find it very well annotated. If the objective is simply to document what was done this may be sufficient, but if the goal is for others to use this code then addition annotation would be useful.


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: VALIDATION OF COI METABARCODING PRIMERS FOR TERRESTRIAL ARTHROPODS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is written in excellent English. The structure is standard for a scientific article. The literature background is enough and all cited references are relevant.
Experimental design: The research question is well introduced. The experimental design is impeccable. The investigation is performed with the highest technical standards, rigorous scientific interpretation of the results, and transparency.
Validity of the findings: The recommendations derived from this paper will significantly improve the area in the future. The interpretation of the results are rigorous and absent of any speculation.
Additional comments: This manuscript by Elbrecht et al. is a long-expected, exhaustive and impeccably designed comparison of many primer sets previously proposed for metabarcoding of terrestrial arthropod communities. This information is invaluable to clarify the current chaos with a huge variety of primer sets for arthropod metabarcoding, which were proposed by different authors, many of them without any previous critical evaluation. The conclusions and recommendations presented in this paper will significantly improve the consistency of metabarcoding projects of arthropod communities in the future and will facilitate the comparison of results obtained from different workflows in the past. The authors are clearly committed to transparency in their results, so they made an effort to provide an exhaustive set of supplementary information files, which can be very helpful for many researchers working in metabarcoding primer design and evaluation. I strongly recommend the publication of this work in PeerJ. I include some minor comments and recommendations (mainly to correct some typos) below.

Minor comments:

L34: Correct “demonstrated” to “demonstrates”, to keep congruence with the present tense forms.

L 80-87: An important additional caveat of using multiple primer sets could be discussed here. Namely, the difficulty to merge read abundances obtained using multiple primer sets in order to extract some quantitative value, which basically makes multi-primer metabarcoding techniques a presence/absence detector, without any interpretable quantitative value.

L127: Remove double parentheses in  ((Vamos et al. 2017)

L132: What particular materials were used as positive and negative controls? This should be explained further here.

L150: Correct “where selected” to “were selected”

L176: Add the “n” to “an Illumina MiSeq”

L229-230: Correct “Amplicon concentrations reached (ng/ul)” to “Amplicon concentrations (ng/µl) reached”

L233: Correct “Amplification success for newly designed primers was mixed” to “Amplification success for newly designed primers showed mixed results”

L270: “The primer combinations fwhF2 + fwhR2n, BF2/BF3 + BR2, ArF5 + Fol-degen-rev and mlCOIintF”. The mlCOIintF primer (and its derivatives) is used in actually 3 “good” combinations. Also BF1 (which has a good BF1+BR2 combination and is targetting almost the same “Leray fragment”) is missing from the sentence. So I would rephrase this sentence to: “The primer combinations fwhF2 + fwhR2n, BF1/BF2/BF3 + BR2, ArF5 + Fol-degen-rev and those based on mlCOIintF and its derivatives”.

L271: Correct “recovery =< 95%” to “recovery ≥ 95%”.

L349: “Although these longer fragments improve taxonomic resolution”. This improvement is probably not significant in practice at the species level, since the analysed COI fragment is practically saturated after 200 bp. I propose to change the sentence to “Although these longer fragments might improve taxonomic resolution”.

L371: Change  “These complexities are difficult to predicted”  to “These complexities are difficult to predict”.

L372 and L388: “in vivo”. You mean “in vitro”. An “in vivo” metabarcoding procedure would imply all the flies, bees and bettles flying around the lab, while we sequence them.

L375. A recent publication by Collins et al. (2019) is very relevant to illustrate this non-specific amplification issue and could be added to the reference list:
Collins, R.A., Bakker, J., Wangensteen, O.S., Soto, A.Z., Corrigan, L., Sims, D.W., Genner, M.J. and Mariani, S., 2019. Non‐specific amplification compromises environmental DNA metabarcoding with COI. Methods in Ecology and Evolution, in press. Doi: 10.1111/2041-210X.13276

L392: Correct “PCR is also biased” to “PCR can be also biased”

L409: Correct “metabarcoding primer” to “metabarcoding primers”.

L409: Correct “given that of most” to “given that most”

L447-448. “As a general rule, the use of multiple primer sets seems rarely justified as it increases laboratory effort without substantially improving taxon recovery” I suggest to add: “As a general rule, the use of multiple primer sets seems rarely justified as it increases laboratory effort and adds complexity to the interpretation of read abundance, without substantially improving taxon recovery”

L458: Don’t forget to add the DOI to access the PrimerMiner results in Dryad.

Figure 1 (caption):
Delete “the” in “four primer sets showing the good performance”

Figure 1: Correct “Asses” to “Assess”. Correct “tempratures” to “temperatures” (2 times).

Figure 4: I think that the red cross symbol used to indicate “primer slippage” in this figure can be misleading to the readers, who could erroneously think that these primer sets are not recommendable to be used at all. I suggest changing this red cross to a symbol with less negative connotations, such as “*” or “PS”, and using a different, more neutral, color for this symbol.
I think that primer slippage is not really so big an issue as the authors are implying in this figure. Primer sets affected with primer slippage can be readily used in practice and the recovered sequences differing in just a leading base (or in 2 leading bases) are never a big problem, since they can be easily dealt with by most (if not all) taxonomy assignment algorithms, specially those involving a final taxonomic clustering step. Even if an ESV approach is used, the right ESVs can be recovered and assigned to the right taxon in the reference database, so that an easy and quick final step of taxonomic clustering would fix this minor issue and would produce a right taxon list.


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: VALIDATION OF COI METABARCODING PRIMERS FOR TERRESTRIAL ARTHROPODS
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 done an excellent job of integrating the feedback from all reviewers. I have no additional comments or concerns. I look forward to seeing the paper in print.


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: VALIDATION OF COI METABARCODING PRIMERS FOR TERRESTRIAL ARTHROPODS
Review round: 2
Reviewer: 2

Output:
Basic reporting: Nothing to add.
Experimental design: Nothing to add.
Validity of the findings: Nothing to add.
Additional comments: I am happy with the changes introduced by the authors. Well done and congratulations.


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: ANALYZING THE INTERPRETATIVE ABILITY OF LANDSCAPE PATTERN TO EXPLAIN THERMAL ENVIRONMENTAL EFFECTS IN THE BEIJING-TIANJIN-HEBEI URBAN AGGLOMERATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: From my point of view, the English language used in all text is good (I am not a native speaker).
The topic of the paper is more relevant for scientific debate and the selected study area is quite an important example. However, I think that the introduction section does not contain all relevant literature. In my opinion there are many studies about the topic covered that the authors should insert for improve this section. The following references could be considered:
-	Bonafoni, S., Baldinelli, G., Verducci, P., Presciutti, A., 2017. Remote sensing techniques for urban heating analysis: a case study of sustainable construction at district level. Sustain. For. 9 (8).https://doi.org/10.3390/su9081308 26 July 2017, Article number 1308. (ISSN: 20711050).
-	De Munck, C., Lemonsu, A., Masson, V., Le Bras, J., Bonhomme, M., 2018. Evaluating the impacts of greening scenarios on thermal comfort and energy and water consumptions for adapting Paris city to climate change. Urban Clim. 23, 260–286. https://doi.org/10.1016/j.uclim.2017.01.003.
-	Zullo F., Fazio G., Romano B., Marucci A., Fiorini L., 2019. Effects of Urban Growth Spatial Pattern (UGSP) on the Land Surface Temperature (LST): a study in the Po Valley (Italy). Sci Total Environ. 2019 Feb 10;650(Pt 2):1740-1751. doi: 10.1016/j.scitotenv.2018.09.331.
-	Huang, M., Cui, P., He, X., 2018. Study of the cooling effects of urban green space in Harbin in terms of reducing the heat island effect. Sustain. For. 10 (4). https://doi.org/10.3390/su10041101.
Furthermore, the literature about both the urbanization phenomena does not updated. I suggest to the authors some important studies on this important topic:
-	The Worldwatch Institute, 2007. State of theWorld, Our Urban Future. Norton, NY;
-	Barrington-Leigh, C., Millard-Ballb, A., 2015. A century of sprawl in the United States. PNAS https://doi.org/10.1073/pnas.1504033112.
-	Angel, S., Parent, J., Civco, D., 2012. The fragmentation of urban landscapes: global evidence of a key attribute of the spatial structure of cities, 1990–2000. Environ.Urban. 24, 249–283. http://dx.doi.org/10.1177/0956247811433536.
-	European Commission, 2006. Urban Sprawl in Europe: The Ignored Challenge, EEA Report 10. p.60.
Furthermore, the authors should be explain the acronyms OLS and GWR (line 92). The study area could be completed by inserting both the urban (in %) and the population density (in inhab/km2).
Regarding the figures, I think that some of them could be improved. Here are some suggestions:
Figure 1: The authors should insert a hillshaded relief such as basemap layer for the study area region. Furthermore, the used label must be reorganized.
Figure 2: The used legend is not useful. The authors should insert a graduated color legend where they show the used altitude intervals for distinguished the plain, hill and plateau belts.
Experimental design: In my opinion, the research question does not clearly defined. For this reason, I think that the authors should rewrite the abstract part, also because they insert their findings through various acronyms that are described later in the paper. Therefore, the abstract should be rephrased describing only a brief introduction to the topic, the main aims of this work and how their findings can be used in the sustainable and integrated development in Beijing-Tianjin-Hebei urban agglomeration.
The paragraph Methods is quite articulated. I suggest to the authors to include a flowchart which can facilitate the reader in understanding this part. The flowchart can increase the clarity of the various phases that compose the methodology.
Validity of the findings: The applied methods are functional to the analysis carried out and they are supported both by a significant amount of data and statistical tests. However, I believe that the conclusions section must be improved. That is because the conclusion is not really a conclusion but a summary - first part just repeats some facts stated already below. State only, what is new, how you contributed to science and if there can anything else evolve from your findings (i.e. lines 383-388 and lines 488-496).
Additional comments: In general, I think that the manuscript is a nice study and it fit the aims of the journal. I consider this a solid case study paper and my suggestions could improve the manuscript. These are minor revisions that, once made, will allow the paper ready for to be published in the journal.


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: ANALYZING THE INTERPRETATIVE ABILITY OF LANDSCAPE PATTERN TO EXPLAIN THERMAL ENVIRONMENTAL EFFECTS IN THE BEIJING-TIANJIN-HEBEI URBAN AGGLOMERATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript "Analyzing the interpretative ability of landscape pattern to explain thermal environmental effects in the Beijing-Tianjin-Hebei urban agglomeration" studied the relationship between thermal environment and landscape pattern over the Beijing-Tianjin-Hebei Area. It's an interesting study but has a lot of flaws. It also suffers from a poor writing style. I think this manuscript is not appropriate for publication until significant improvements are made.
Experimental design: 1) The authors used hot-spot analysis to study the clustering of land surface temperature (Ts). Ts is directly derived by the solar radiance and highly influenced by the land surface material, 3-dimension structure and other environmental factors. Not as socia-economical variables, What can be indicated from the the clustering of Ts? What's the meaning of the clustering of Ts?

2) How to determinate the landcape type for the window granularity. For example, for the window of 7*7, there were several landscapes in this window. How to determinate the lanscape of this window?
Validity of the findings: 1) For Section 3.3.1 and 3.3.2, some results seem to be unreasonable. For example, the result indicated the negative relationship between the water body and Ts. However, it's well known that water body has obvious Cooling effect. This may be attributed to the simple method.

2) The discussion section mostly describes the results and lacks in-depth analysis. In addition, the intellectual merits of the result of this paper should also be discussed.

3) The 1st paragraph of the Section 4.3 looks like a literature review. The authors need to compare their results with other studies and discuss on it.
Additional comments: 1) The literature review needs to be strengthened. A much better summary of the methods and variables employed in UHI pattern studies should be given.

2) The data section is too weak. The authors need to provide a much detailed description on data, not only in the Table. For example, what's the Ts data used in this paper? The method of the Ts product (MODIS) should be described. And the accuracy or reliability of this Ts product is better to be explained based on other references.

3) Sort the references.

4) L37, should be "between atmosphere and land surface"

5) L66-70, It's not true. There are a lot of UHI studies which introduced several environmental factors.

6) L73-75. It's also not true. There are a lot of UHI studies on metropolitan areas.

7) L220-221, Not clear

8) L223-226. Too detailed. Suggest to delete this description

9) L249, Please give the original or classic reference

10) L273-285, have little relationship with the topic of this paper.


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: ANALYZING THE INTERPRETATIVE ABILITY OF LANDSCAPE PATTERN TO EXPLAIN THERMAL ENVIRONMENTAL EFFECTS IN THE BEIJING-TIANJIN-HEBEI URBAN AGGLOMERATION
Review round: 1
Reviewer: 3

Output:
Basic reporting: The English language throughout the manuscript could use considerable improvement to allow for increased readability among the journal’s international audience. Below are a few examples of where improvements could be made, though the entire manuscript would benefit from additional proofreading and review by a native English speaker.

1. Certain statements could use significant rewording to increase clarity, as their current phrasing makes comprehension difficult (e.g. Lines 12, 95, 478-481).

2. Other statements, while comprehensible, are vague and could use additional detail. For example, on Lines 55-56: the correlation was the “most significant” compared to what? On Line 95, what sort of “other aspects”?

3. “This study” generally does not perform any of the actions that carried out by the authors (e.g. Lines 85, 163, 226, 229). These instances could be replaced with “we”.

4. Sentences generally should not begin with conjunctions, such as “and” (e.g. Line 26, 77, 94). These could be removed.
Experimental design: 1. While the main research gap appears to be laid out in Lines 66-70, your Introduction needs more detail leading up to your specific research questions. I suggest adding further justification for the objectives of the study that are outlined on Lines 84-97.

2. Lines 84-97: To increase the clarity and replicability of the study’s methods, methodological and analytical details mentioned in the Introduction would be more relevantly included in the Methodology section. For example, it could be stated that Pearson correlation coefficients were used to compare landscape pattern and surface temperature on Line 155, instead of the vague “relationship analysis”.

3. Lines 152-153: Additional clarity on methods would be helpful. How were the different landscapes integrated to produce EcoLSP and NDVI? Was NDVI not derived from the MODIS data, as described in Lines 127-129?

4. Lines 220-221: The claim that the different resolutions of basic data did not affect your research’s results does not have clear support. I would suggest providing some validation for this claim and/or reducing the certainty of the statement
Validity of the findings: 1. Lines 304-306: How was it determined that 5 km x 5 km was a turning point at which r2 became strong? This claim was not clearly supported by Figure 5, which shows only slight peak at the 5 km x 5 km granularity for 2 of the 5 landscapes.

2. Lines 383-388: These statements make normative claims that appear to overstate the author’s findings, which are entirely correlative. Additionally, these statements would be more appropriately placed in the Discussion/Conclusion, if they are to be included in the paper at all.

3. Lines 508-510: As it is currently stated, this conclusion does not appear to be fully supported by the results that were shown (see above comment for Lines 304-306). If this conclusion specified that the 5 km x 5 km optimal granularity was observed for only the ForLS and EcoLSP landscape patterns, then that would greatly increase the validity of this finding.
Additional comments: 1. Overall, results presented in this manuscript and methods used to produce them appear to be scientifically sound. The primary issues arise in the writing and in the interpretation of the results, both of which should be revised before acceptance. Care should particularly be taken to avoid attributing the relationships observed in the results to factors that were not explicitly explored in the study, as is done in the Discussion (e.g. Lines 444, 458)

2. The repeated use of “landscape” to refer to the different land cover types (ArtLS, ForLS, FarmLS) could lead to confusion among readers, as it does not align with many widely used definitions of a “landscape” (e.g. Forman & Godron 1981, Turner et al. 2001). Instead, I would suggest “land cover types” as a consistent substitute, as it is already used to refer to these metrics on Line 119.


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: ANALYZING THE INTERPRETATIVE ABILITY OF LANDSCAPE PATTERN TO EXPLAIN THERMAL ENVIRONMENTAL EFFECTS IN THE BEIJING-TIANJIN-HEBEI URBAN AGGLOMERATION
Review round: 2
Reviewer: 1

Output:
Basic reporting: In my opinion, the literature references are now complete and the manuscript represent a good contribution to science.
Experimental design: I think that the authors have clear addressed all my suggestions. The paragraph Methods are now clear and  the flowchart increase the clarity for the readers. This expedient allows the possibility to replicate.The authors have better specify their research question.
Validity of the findings: In my opinion, the conclusion section has been improved and it  supported by the results. Furthermore, it links to research question.
Additional comments: Dear Authors,
thanks for the revision of article.
I found you have clearly addressed all points I have suggested in my previous review.
The only small suggestion is to control some typos (i.e. line 152) and puntuaction errors present in the text.
I would like to recommend the paper 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: ANALYZING THE INTERPRETATIVE ABILITY OF LANDSCAPE PATTERN TO EXPLAIN THERMAL ENVIRONMENTAL EFFECTS IN THE BEIJING-TIANJIN-HEBEI URBAN AGGLOMERATION
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment.
Experimental design: no comment.
Validity of the findings: no comment.
Additional comments: The revision made significant improvement and I'm satisfied with it.


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

Input:
Title: DIFFERENT EXPRESSION PATTERN OF FLOWERING PATHWAY GENES CONTRIBUTE TO MALE OR FEMALE ORGAN DEVELOPMENT DURING FLORAL TRANSITION IN THE MONOECIOUS WEED AMBROSIA ARTEMISIIFOLIA L. (ASTERACEAE)
Review round: 1
Reviewer: 1

Output:
Basic reporting: The English is clear.
The references need a minor changes.
The structure of the article is appropriate but figures needs a revision
Experimental design: The research question is well defined and is relevant and the investigation was rigorously conducted but needs to be more detailed in some aspects referred in the general comments
Validity of the findings: no comment
Additional comments: The paper proposed by Mátyás et al, provides an overview of the genes expression patters along flower development of Ambrosia artemisiifolia. To accomplish this, authors refer to libraries prepared and published on a previous data report (doi: 10.3389/fpls.2016.01506), where RNA-Seq was performed to Male and Female flowers as well as leaves. The work proposed provides the follow up to the data report. Here, two more female samples were obtained and sequenced. The five data sets were analyzed together, providing an extensive, detailed, focused and clear overview of patter expression. Also, authors have carefully detailed the introduction, which represents one of the most interesting, complete and easy to read state-of-the-art, in flower development field. It should also be noted that the detailed Results & Discussion section is very clear and well structured, providing a very easy access to the information stored under this work.

Along this manuscript, major and minor questions have come to surface, that should be clarified or corrected by the authors, before final publication.

Major questions
1.	Under this study, five libraries are compared and, under Results and Discussion, there are segments where Female (and Male) exclusive transcripts are accessed, although, there are 3 Female libraries and only one Male library. I am concerned relating to the statistical significance of the Male library under this context. Is one single library relevant to provide a complete Male Knowledge, whereas it was need 3 females to provide that? I wonder if the addiction of more male libraries does not change the results.
2.	The number of individuals used along sampling is not detailed. How many individuals were used along sampling? Are 1F and 2F from the same individual(s)? How many buds were collected from each one of the nine developmental stages? If multiple buds were used, was it equally distributed? Also, according to repositories, there are 2 runs for 1F and 2 for 2F. How each one of these runs differ from each other? Are they biological or technical replicates?
3.	Authors used both Bowtie and Bowtie2 on this analysis, each one, were used with different instruments. These programs have different standards, scopes, defaults, etc. I wonder why did authors decided to use both that increment the entropy of the analysis. Also, as standard and defaults are different, authors need to clearly detail which parameters were used and the respective values. Considering that all samples have more than 50bp reads, bowtie2 should had been used on all samples. Authors should justify the option made.
4.	Along Methods, Line 258, under «Construction of A. artemisiifolia transcript datasets (...)» authors state that «The resulting differentially expressed transcripts were further clustered according to their expression patterns by applying SQL Database Manager». This statement should be more detailed: (a) latter, on results and discussion, authors clarify that Microsoft SQL Server was used as Database Management System. This information should be provided early in Methods. (b) There is no information of which kind of data was stored under this database. As far as I can understand, this database only contained the expression for each gene, but that is not stated. (c) The database construction should be detailed as supplement material. A different choose of fields along table creation may lead to different results, as computers will interpret the data diferentially. (d) The interaction mechanism with this database needs to be clarified. Did authors executed SQL queries directly on the database using a SQL client (in that case, which one?) or it was used any programming language interface, as R, Python? (e) Is this database publicly available? If so, provide instructions to access.
5.	The usage of RPKM/FPKM is broadly spread along RNA-Seq studies and the usage of this method to access gene abundance is well established. Although, bioinformatics’ communities are discussing the application of this calculation methodologies when comparing different data sets and studies. In order to better compare the expression along different studies, it is more acceptable the usage of TPM (Transcripts Per Kilobase Million). As the authors are using reads obtained from different instruments, I wonder if the calculation method is the best one.
6.	The proposed article is very extensive and detailed as already stated on this review introduction. Nevertheless, as authors have five libraries to work on, I wonder, if a cis/trans or co-expression analysis could be performed. Authors may compare the expression patterns of all the identified genes and check which groups are co-expressed/co-repressed, which may indicate co-regulation of those genes. I do not think that this analysis is fundamental to this paper, but it could contribute to the overall knowledge of A. artemisiifolia regulatory networks.
7.	Pp 304. Authors state that for RT-qPCR analysis only one reference gene was used. Usually for more robust results two or three reference genes are used when analyzing relative expression.

Minor questions
1.	Along Materials and Methods, on the «Plant materials and cultivation conditions», authors present the sampling first and the cultivation on the end. As this steps were taken in the opposite order, authors may want to adapt the text of this segment and start by describing the cultivation process and then the sampling methodology.
2.	The standard protocol of Illumina RNA-Sequencing states that cDNA is constructed using olido(dt) primers. Authors may include information on «RNA-seq library construction and sequencing» segment, to inform if cDNA for RNA-Sequencing was produced using oligo(dt)s or random hexamers.
3.	On titles, «A. artemisiifolia» is not in italics.
4.	RSEM package should be better cited (please, refer to https://doi.org/10.1186/1471-2105-12-323).
5.	Under «Digital gene expression analysis» it is refereed that RPKM was calculated. Although the information provided on the manuscript and on the repositories is that the authors performed paired-end sequencing. If both forward and reverse reads data sets were added to the programs, the authors did not calculated the RPKM but the FPKM instead (Fragments per kilobase per million mapped read). As the formula used is the same, this difference is only semantics but should be corrected in order to avoid confusing the reader. If the programs were not used with both Forward and Reverse reads at the same time, that should be clarified and justified as well.
6.	Primer3 should be better cited. This software is released with version control and so, the version used should be cited (check the top of the webpage). Also, articles should be cited instead of the webadress (please refer to 10.1093/nar/gks596 and 10.1093/bioinformatics/btm091).
7.	Authors used non-redundant protein database from NCBI and correctly provide the respective link, although, as this database is not curated and is not released with version control, I would recommend authors to provide the download date.
8.	Pp 344, 632 – Reference format. Should be Rocheta M et al., 2014b
9.	BLASTx is part of a package of BLAST programs and should be cited properly. Please, refer to blastx documentation to identify the correct paper to cite. Is none is specifies, authors should cite BLAST+ package, by referring to its proper article(s).
10.	On line 362: «(2*100 bp paired-end)». The information is redundant. Authors should state «(2*100 bp)» or «(100 bp on paired-end sequencing)». The first one is most commonly found.
11.	On «RNA sequencing and assembly of A. artemisiifolia floral libraries» segment, authors should include the reads length for the new libraries (1F and 2F). I found on supplementary material that those reads have 80 bp, but that information should be stated on the main manuscript as it the base of all the provided results.
12.	On lines 370-371: «Their de novo assembly resulted in 109,452 (1F) and 97,239 (2F)». Please add «contigs» to the end of this sentence as «Their de novo assembly resulted in 109,452 (1F) and 97,239 (2F) contigs». It is not clear if you are talking about contigs, scaffold, etc if we do not cross with supplementary material.
13.	I would like to propose to authors the utilization of «de novo» in italics, as that is a Latin expression and that is generally the standard. Nevertheless, I noted that authors were consistent by do not use italic on this expression.
14.	Figures numbering needs to be completely revised. The order of calling figures is: 1, 2, 7, … Also, figure 5 is never called on text.
15.	On line 400-401: «Representation of the significant sub-ontologies of cellular component, molecular function, and biological process are summarized in the Supplementary Figure 2». Do authors want to call Supplementary Figure 1 instead? I have only found one Supplementary Figure.
16.	Figure 4: The «A» letter used to identify panel A is on top of the plot yy scales.
17.	Pp 429-430. Authors use Q-RT-PCR in the subtitle and in text RT-qPCR. Please make it uniform.
18.	Pp 822 – garfical or graphical?
19.	Table 1. There are two Rocheta M references used in this manuscript, however, in table 1 it only shows Rocheta M et al., 2014. It is a or b?


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: DIFFERENT EXPRESSION PATTERN OF FLOWERING PATHWAY GENES CONTRIBUTE TO MALE OR FEMALE ORGAN DEVELOPMENT DURING FLORAL TRANSITION IN THE MONOECIOUS WEED AMBROSIA ARTEMISIIFOLIA L. (ASTERACEAE)
Review round: 1
Reviewer: 2

Output:
Basic reporting: The basic reporting style is adequate.
Experimental design: Based on the description of the experimental design, there is no biological replication. Certainly there was none for the RNAseq portion of the study, and it is unclear if there was biological replication for the qRT-PCR protion of the study since there was no mention of the number of biological replicates collected at each floral developmental stage in the first paragraph of the methods.  As such, no differential expression of genes should be reported.

Additionally, although there is a reference noting the use of the internal reference gene used for qRT-PCR, there is no solid evidence to suggest that that gene is not itself differentially-expressed through floral development. This could be easily remedied (assuming replication of the RNAseq libraries is done) by specifically showing the expression pattern of the GAPDH gene in the RNAseq studies (which are generally more accurate than qRT-PCR in any case).

However, this data does allow the presentation of the sequences of key flowering genes that were assembled from the sequenced libraries.
Validity of the findings: Sequences of floral genes are likely accurate. None of the differential expression is valid.
Additional comments: This reviewer understands the difficulty and cost of running RNAseq studies on multiple samples with adequate replication. However, the cost and time is not justification for failing to perform adequate replication.

If this paper was rewritten to just examine the sequences of floral genes (or indeed just the sequences of all genes expressed in the floral organs- GO ontology, any qualitative differences in sequences between floral genes in male and female organs, etc. then that would be acceptable for publication. Likewise, if there was true biological replication and subsequent qRT-PCR analysis of specific floral genes, then that too would be publishable- assuming there was proof provided that the internal control gene was stably expressed in the floral organs through development.


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: DIFFERENT EXPRESSION PATTERN OF FLOWERING PATHWAY GENES CONTRIBUTE TO MALE OR FEMALE ORGAN DEVELOPMENT DURING FLORAL TRANSITION IN THE MONOECIOUS WEED AMBROSIA ARTEMISIIFOLIA L. (ASTERACEAE)
Review round: 2
Reviewer: 1

Output:
Basic reporting: Minor questions
1.	On titles, «A. artemisiifolia» is not in italics. Still missing in line 244
2.	BLASTx reference is still missing in line 334.
3.	«Pp 429-430. Authors use Q-RT-PCR in the subtitle and in text RT-qPCR. Please make it uniform.» Now, I find 4 forms:
1.	qRT-PCR: Lines 415 and 507
2.	RT-QPCR: Lines 34, 414 and 496
3.	RT-qPCR: Lines 222, 298 and 615; Figure 6
Experimental design: No comments. It is ok.
Validity of the findings: No comments. It is ok.
Additional comments: Just revise the manuscript for minor corrections.


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

Input:
Title: THE CLINICAL SIGNIFICANCE OF COLLAGEN FAMILY GENE EXPRESSION IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Authors analyze clinical data and gene expression profiles of Esophageal squamous cell carcinoma (ESCC) patients from two databases, The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) to look for relevant collagen genes as potential biomarkers for the disease. The overall language used in the manuscript is satisfactory with minor rephrasing required in few sentences. The introduction provides a brief background however it should be expanded mostly on the need to look for biomarkers of ESCC and if these markers may be helpful in early detection. The discussion needs more expansion in a couple of directions, mostly related to results obtained from pathway analysis and co-expression network analysis. Also, throughout the manuscript authors are not elaborative with the specific terms used, these terms should be defined.
Experimental design: Based on the Clinicopathological information of the ESCC patients seems like, TNM and T-stage information is significantly correlated with survival in both databases, TCGA and GEO. Why have authors left out T-stage prediction model? The model should also be looked for T-stage, as is for TNM staging based model for both the databases.
Validity of the findings: Authors should elaborate on the basis for the selection of final 7 genes from the TCGA and 9 genes from the GEO and clarify in the text. Were individual AUC's for all genes selected for DEG based forecast model is above 0.6. Also, it’s not clear how the threshold was set.

In the TCGA database, there were 81 tumor samples and 11 normal samples. Authors should justify that such a difference between no’s will not put a bias in the study.
Additional comments: Major comments
Based on the Clinicopathological information of the ESCC patients seems like, TNM and T-stage information is significantly correlated with survival in both databases, TCGA and GEO. Why have authors left out T-stage prediction model? The model should also be looked for T-stage, as is for TNM staging based model for both the databases.

Please elaborate on the basis for the selection of final 7 genes from the TCGA and 9 genes from the GEO and clarify in the text. Was individual AUC for all genes selected for DEG based forecast model is above 0.6. Also, it’s not clear how the threshold was set.

Significantly enriched gene sets were obtained according to Oncogenic Signatures Gene Sets, Hallmark Gene Sets and KEGG Gene Sets. Authors should put information on NES, and how it is interpreted. Overall there doesn’t seem to be much consensus between 3 different gene sets, except for some overlap in p53 and oxidative phosphorylation. Authors touch the base here or some elaboration on possible discussion role of oxidative phosphorylation. However, authors should put forward a discussion on how functionally and mechanistically p53 possibly correlates to collagen gene expression in tumors. As there is evidence that p53 is implicated in connective tissue homeostasis.

Authors should clearly define and provide a brief background on some of the terms used in this manuscript, such as Hazard ration, GSEA, WCGNA, T-stage, N-stage, NES, etc, to make it clear to readers.

In TCGA database there were 81 tumor samples and 11 normal samples. Authors should justify that such a difference between no’s will not put a bias in the study.

For co-expression network analysis by WCGNA, authors should elaborate in discussion the findings from fig 5. Such as what key genes seemed to be co-expressing with low risk and high-risk gene sets. Was there any pattern observed?

Minor points

Lines, 53-54, “Thus, it is urgent to identify novel biomarkers and therapeutic targets to facilitate the development of treatments”. Biomarkers will also be helpful in early detection. Also, authors should shed some light on what’s the prognosis and chances of survival if these tumors are detected at an early stage.

Line 68, correct typo “patway”

Line 89-90, “All the gene expression levels 90 were normalized before studying”.  Normalized to what?

Lines 98-100, sentence not clear


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

Input:
Title: THE CLINICAL SIGNIFICANCE OF COLLAGEN FAMILY GENE EXPRESSION IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: No Comment
Experimental design: No Comment
Validity of the findings: No Comment
Additional comments: In this work to investigate the significance of collagen genes as potential biomarkers for the diagnosis and prognosis of Esophageal squamous cell carcinoma (ESCC), the authors carried out gene expression analysis of the 44 members of the collagen gene family in two datasets obtained from TCGA and GEO. They propose a DEG-based forecast model for predicting the survival of ESCC patients based on 7 collagen genes.

Line 89-90: All the gene expression levels were normalized before studying – How? Give details.

Line 92: using which statistical methods?

Line 101: Genes most related to overall survival of patients were shown?? Figure no?

Line 104: Multivariate Cox regression model was used to establish forecast models based on gene expression of the DEGs and overall survival of the patients via R software  - which R software?

Line 105: Formulas and risk score of each patient were obtained? What formulas – not clear.

Line no. 112-113: Gene sets enrichment analysis (GSEA) was performed to explore potential mechanism of collagen family genes by dividing the patient samples into high-risk and low-risk groups. Not clear. As I understand, 22 collagen genes were submitted to GSEA portal and important pathways identified.

Line no. 115: Some software javaGSEA is used. However no information is given about what is does, how it helps in identifying gene sets enriched in low and high risk groups, what are these gene sets (no legends provided for the supplementary tables). Not clear, please provide details.

Line no. 121-123: Risk-score-based DEGs were determined by dividing the patient samples into high-risk and low risk groups in TCGA and WGCNA performed to assess the relationships between collagen family genes and the risk-score-based DEGs as well as seven representatively enriched gene sets – what are these 7 enriched gene sets? How are risk-score based DEGS identified - not clear. Are these only collagen genes, or other genes as well – not clear. There are two sets of risk-scores, one based on 7 collagen genes for TCGA dataset, and another similar for GEO dataset – this is used to divide the patients in each dataset into low and high risk groups – right?

Line no. 139: what statistical methods – explicitly mention what analysis was done.

Line no. 166: How were the 7 genes in TCGA dataset and 9 genes in GEO dataset selected – specify. Any common genes between the two datasets? Why is this dataset dependent – discuss.

Line no. 168: In the formula of Risk scores, what are the multipliers (coefficients multiplied with the gene expression value of collagen genes) – please specify. Also give some justification of defining the risk score.

Line no. 184-185: No difference in the expression profile of the 7-genes in the low-risk and high-risk groups observed. Discuss.

Line no. 187-189: Fig 3G is not very conclusive, even patients with low risk score have low survival rates as for high risk score patients.

Line no. 192: Not clear what genes were submitted to GSEA? All Collagen genes? Only those collagen genes that were significant DEGs in high-risk group? Please provide details The whole GSEA analysis is very confusing. Provide details of how analysis was performed.

In the WGCNA analysis, how are the COL genes distributed across the modules? Discuss the functional enrichment analysis of genes co-expressed with COL genes from these modules. How the Fig S4 is obtained not clear. Provide details.

There is confusion in the analysis of the two datasets presented, one from TCGA and the other from GEO. It is not clear whether the DEGs observed in TCGA dataset are also DEGs in GEO dataset. Please clarify. Further, it is mentioned that TCGA data being based on RNASeq compared to microarray-based GEO data, the 7-gene forecast model is more accurate. How does it perform on the GEO dataset? That would provide a good validation of the proposed model. The difference in the ethnicity of the patients is being discussed as possible reason for no overlap between the two datasets. In that case it would have been more appropriate to consider datasets with similarity in the ethnic group of the patients considered.

In general, details of all the analyses performed are missing.

No Table legends provided for the supplementary tables. Similarly for supplementary figures, no legends provided.

English of the manuscript needs to be improved before publication. Also run a spellcheck before resubmission.

Significant amount of analysis is done but since no details are provided how it is done, it cannot be accepted in the current form.


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

Input:
Title: THE CLINICAL SIGNIFICANCE OF COLLAGEN FAMILY GENE EXPRESSION IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA
Review round: 1
Reviewer: 3

Output:
Basic reporting: The objective of the study is good as it focuses on the “The identification of novel biomarkers for early identification of the disease to facilitate the effective treatment for esophageal squamous cell carcinoma.” However, the manuscript has to be revised thoroughly so as to reach the standards of the journal.
Experimental design: Regarding the datasets used for the work, more details regarding the controls used and number of replicates used should be provided.
Validity of the findings: No comment
Additional comments: All over the manuscript, there are grammatical errors and the manuscript should be subjected to English language correction by a native speaker.


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

Input:
Title: THE CLINICAL SIGNIFICANCE OF COLLAGEN FAMILY GENE EXPRESSION IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA
Review round: 2
Reviewer: 1

Output:
Basic reporting: Satisfied with basic reporting
Experimental design: Scientifically sound
Validity of the findings: Findings well described with proper rationale.
Additional comments: Authors have revised the manuscript properly and have answered all queries raised.
I recommend accepting the manuscript.


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

Input:
Title: THE CLINICAL SIGNIFICANCE OF COLLAGEN FAMILY GENE EXPRESSION IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA
Review round: 2
Reviewer: 2

Output:
Basic reporting: OK
Experimental design: OK
Validity of the findings: OK
Additional comments: The authors have revised the paper and most of the comments have been addressed. However, the following two comments are still remaining.

1) Line 120: Prediction models
The justification for the use of risk-scores is still missing. Probably, a reference may be cited in the manuscript.

2) Line 229: Pathway analysis of Collagen genes
In reply20/21, the authors have mentioned that they have categorized samples into 'High' and 'Low' risk groups and then carried out GSEA across all the genes (not just collagen genes), while the title is misleading. Again, in the discussion, they focussed on explaining collagen genes and their involvement in pathways of cancer. For clarity, the authors may add what set of genes were used for gene enrichment analysis using GSEA, as the current heading indicates only collagen genes were submitted for enrichment analysis. Some details is required.


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: MULTIPLEXED ISSR GENOTYPING BY SEQUENCING DISTINGUISHES TWO PRECIOUS CORAL SPECIES (ANTHOZOA: OCTOCORALLIA: CORALLIIDAE) THAT SHARE A MITOCHONDRIAL HAPLOTYPE
Review round: 1
Reviewer: 1

Output:
Basic reporting: Although the text is for the most part clearly understandable, there are nonetheless a few places where it might benefit from the attention of an English-language editor. There are some minor grammatical errors, and in a few places missing words or non-standard grammatical constructs make a sentence difficult to understand. I have noted the most serious of these in the general comments section. There are also places where additional citations would be helpful.  In particular, citations are missing for some of the software or scripts used for data analysis.

 More seriously, the figures (as reproduced in the reviewer's copy) are far too small to read, and when enlarged their resolution is very poor - as a result I cannot read any of the labels, and individuals cannot be discerned in the STRUCTURE plot. In addition, the acronyms used for species seem to differ between the two figures and between the legends and figures (PK, PE vs. CK, CE).  It may be that the figures are adequate for publication (if acronyms are fixed), but I can't really evaluate them from the versions that have been provided to me.  Finally, it does not appear that the raw data files (sequence reads for the MIG-seq) have been provided.  There is a supplemental data file that includes "MIGseqDatafile"in its name, but it is a large spreadsheet of integers with no heading to indicate what the data represent (alleles? SNPs?).  Ideally, in order to be able to reproduce the results of the analyses, readers should have access to the original sequence read files (deposited to a public repository such as GenBank's Sequence Read Archive (SRA) or international equivalent).
Experimental design: This appears to be a well-designed study with results that are unambiguous and easily interpretable. This is (to my knowledge) only the second use of MIG-seq to delimit species in corals (by the same research group), and it seems to be a very useful tool to apply in cases where conventional barcode markers have failed to discriminate morphospecies.  Because this technique has not been used widely (compared to RADseq), it would be helpful if a bit more background were included for the reader who is unfamiliar with the definition of an ISSR and the basic concept of the MIGseq approach. Citations of some additional references (for instance the original Suyama paper) at first mention of the technique would be appropriate. The Richards et al. paper included a very clear, concise description of MIGseq (in both the Intro and Methods sections), and I would suggest including similar language in this manuscript.  The steps in data analysis seem to be described clearly, but as already mentioned above, citations are missing for some of the programs used.
Validity of the findings: The results are presented clearly and appear to show unambiguous genetic differentiation of the two Pleurocorallium morphospecies. There were, however, a number of different analyses that were run using different parameters (for example, different amounts of missing data and SNPs per locus), but the results of only one parameter set are shown. Instead of just stating "data not shown", it would be very appropriate to include the results of the analyses with other parameter sets as supplemental figures that could be viewed and assessed independently by the interested reader.  The conclusions are sound, but I suggest wording them a little differently: this study doesn't highlight the importance of conservation of these species per se, but it does suggest that separate conservation strategies will be required for the two different morphospecies.
Additional comments: 53: I think it would be clearer in this sentence to specify "family Corallidae" rather than "coraliid corals".

81-83: Your statement that no one has used high-throughput sequencing methods in precious corals is incorrect.  Herrera & Shank (2016, MPE 100: 70-79) used RADseq to delimit species in Paragorgia- the sister family to Corallidae - and they also included Corallium and Hemicorallium in their study as outgroup taxa.  It would be appropriate to acknowledge and cite their study.

96: This sentence is confusing.  Rather than "have at least two distinct morphological features" I think what you mean is "can be distinguished by at least two morphological features..."

100-101: This sentence is not clear and it seems like there might be one or more words missing.  Should it say that the two species *shared haplotypes* at eight mtDNA regions and EF-1?

104: The meaning of the phrase "whether they are the same species with the current gene flow" is unclear.  Perhaps this should read "whether they are the same species with gene flow occurring between them"?

120-121:  The formal name of mtMutS is the "mitochondrial mutS-like protein".  Note, however, that it has still not definitely been shown to be operating as a mutation-repairing gene in the octocoral mt genome. That function is still speculative.

190: The meaning of the statement "P. konojoi had only one haplotype in P. konojoi" is not clear.

Table 1:  Should "submatrine" be "submarine"?

201: The correct spelling is disequilibrium, not disequibilidium.


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: MULTIPLEXED ISSR GENOTYPING BY SEQUENCING DISTINGUISHES TWO PRECIOUS CORAL SPECIES (ANTHOZOA: OCTOCORALLIA: CORALLIIDAE) THAT SHARE A MITOCHONDRIAL HAPLOTYPE
Review round: 1
Reviewer: 2

Output:
Basic reporting: The mtDNA of anthozoans accumulates nucleotide substitutions at far slower rates than do bilaterally symmetrical animals.  These slow rates have made the inference of phylogeographic structure and distinguishing closely related taxa far more difficult for anthozoan taxa than most other animals.  The authors here employ a newly developed method to generate nuclear SNPs to tell apart two closely related octocorals that are commercially traded and may face threats to their populations.

The manuscript has two figures and both could be improved.  Figure 1 needs a major reworking because it does not convey the critical information that it should (that mtDNA cannot distinguish the two morphospecies).   Figure 1 should be a haplotype network instead of a rooted phylogenetic tree.  The long branch to the outgroup makes it impossible to see the limited mitochondrial variation that the figure should focus on.  In addition, the Fig. 1 legend should state the type of data (mtDNA) that the tree/network is based upon and the gene region as well.  Figure 2 is generally good, but could be improved by spelling out the two species' names in Fig 2a so the reader does not have to recall the abbreviations.
The background material presented in the Introduction is too narrow.  The slow mtDNA issue has been long-recognized for anthozoans (see Hellberg 2006 BMC Evol Biol 6:24, see also octocoral work by SC France) but focus here is not just octocorals, but even more specifically on coralliids.  The broader context of slow mtDNA in anthozoans should be provided by including some more of these references.
The English used is generally intelligible (other than in parts of the Discussion, see below), but awkward.  I note some suggested changes here, but the manuscript deserves a more thorough editing by a native speaker familiar with the subject.
Line 37: Indicate how many of each species here rather than the total for both combined.
37-38: "from the Ryukyu Islands and Shikoku Island..." ["to" implies that there are multiple sampling locations between the two named extremes, when there are actually just the two collecting sites].
53: "Precious, or coralliid, corals (Anthozoa: Octocorallia) play an ..."
64: Reference needed here
87-93: Any evidence that this approach identifies single-copy loci?
99-102: A critical sentence that need clarifying. What PROPORTION of haplotypes are shared between the two?  Are they the most common ones?
141: Says 87 in the abstract and elsewhere.  Why the difference?
183: Need to indicate how many of each species here, not just the total for the two combined.
189: There is no Fig. 1b
205: "either" unclear when there are three alternative data sets, not two
240: "composed" instead of "occupied approximately"
Experimental design: Meets standards - no comment
Validity of the findings: Their central conclusion, that the MIG-seq approach they have taken allows two potentially threatened precious corals to be distinguished even though mtDNA cannot do the same, is valid.  Some ancillary points made in the Discussion, however, confused me.  Generally, the writing in the Discussion was the hard to fathom.  Minimally, there appears to be confusion regarding what lineage sorting is and how it relates to the results they have obtained.  Lineage sorting depends on the effective population size, which is smaller for mtDNA than for nuclear DNA.  Thus, lineage sorting will be faster for mtDNA, contra what is stated in line 225.  If the two species diverged while sharing a single common mtDNA haplotype, then slow substitution rates would mean that it takes a long time for differences to accumulate between the genetically isolated lineages.  This is NOT lineage sorting, however, which refers to non-reciprocal monophyly due to the maintenance of shared variation present at the point of divergence.  Selection at nuclear loci, including the 12 diagnostic SNPs identified here, could speed them to reciprocal monophyly.
Additional comments: Nothing additional


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: MULTIPLEXED ISSR GENOTYPING BY SEQUENCING DISTINGUISHES TWO PRECIOUS CORAL SPECIES (ANTHOZOA: OCTOCORALLIA: CORALLIIDAE) THAT SHARE A MITOCHONDRIAL HAPLOTYPE
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have improved the English and addressed a majority of the reviewers' comments to clarify what were previously confusing statements. The figures have also been improved significantly in size, resolution and readability.  There are, however, still a number of places throughout the manuscript where minor grammatical errors or word choice make the meaning of a sentence unclear.  These include:

70: The scientific definition of "precious corals" is still not clear.  The best way to state this would be as in the paper's title, i.e. "The precious corals (Anthozoa: Octocorallia: Coralliidae), "

157:  This should read "using other techniques such as RAD-seq"

159: " the method can be performed on few or low-quality DNA" is unclear because "few" is not an appropriate modifier for "DNA".  Maybe this should read "the method can be performed with small amounts or low-quality DNA"?   or "with low concentration or low-quality DNA"?

162: The statement "Octocorallia, which is closely related to Heliopora spp," makes no sense (Octocorallia is the group to which Heliopora belongs).   In the context of the previous study, what would make sense to say here is "Closely related species of Heliopora were successfully delineated"

307: The statement "P. konojoi shared one haplotype in P. konojoi" has been changed in response to previous comments, but still makes no sense.  Should this say something like "All P. konojoi shared a single haplotype at both loci"?

332-333: To be consistent with the Introduction, Corallium (a specific genus) should be replaced with either "coralliid" or "precious" in both of these sentences.

396: "among different species or genus" should be "among different species or genera"

The Figure legends need to explain in more general terms what is shown, rather than just have telegraphic titles such as "Structure".  The figures should stand on their own and be understandable without the reader having to refer to the text to know, for example, what species or gene(s) have been analyzed.

The text states that samples were collected from the Ryukyus and Shikoku islands, but Table 1 does not indicate what collection sites are in the Ryukyus and which are at Shikoku.

Fig 1, a:  What is the red circle and the number 43 along the line connecting it to the pink circle?  Is that the outgroup?  If there were only two haplotypes found among P. konojoi and P. elatius a haplotype network is not very informative.  I recommend leaving it out and showing only the ML tree.
Experimental design: Reviewers' suggestions have been addressed, and appropriate references have been added.
Validity of the findings: Reviewers' comments have been addressed, and a supplementary file has been added to show the results of the Structure analyses run with different parameters.
Additional comments: Overall, the authors have done a thorough job of addressing the reviewers' comments and suggestions.  Just a few more minor corrections are necessary to make the text as clear as possible.


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: MULTIPLEXED ISSR GENOTYPING BY SEQUENCING DISTINGUISHES TWO PRECIOUS CORAL SPECIES (ANTHOZOA: OCTOCORALLIA: CORALLIIDAE) THAT SHARE A MITOCHONDRIAL HAPLOTYPE
Review round: 2
Reviewer: 2

Output:
Basic reporting: The English has improved.  Most notably, the figures are much improved.
Experimental design: Sound, as before
Validity of the findings: The previously confusing mentions of lineage sorting have been clarified.
Additional comments: I am happy with the revisions and recommend acceptance.


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

Input:
Title: THE CUTICLE INWARD BARRIER IN DROSOPHILA MELANOGASTER IS SHAPED BY MITOCHONDRIAL AND NUCLEAR GENOTYPES AND A SEX-SPECIFIC EFFECT OF DIET
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is well written, with introductory sections conveying to the main points. The literature is generally relevant, and I provide some additional suggestions to make it even more accurate. The Figures could be improved (see below).
Experimental design: The experimental design is clear and the research questions well defined. The methods have been described with enough details.
Validity of the findings: The findings are well supported by the data based on a consistent number of replicates, and the statistics, to my level of expertise, are correct. While the discussion is clear, it could be still a little bit more speculative given the few research reports in this specific field of investigation. The conclusion are well stated and linked to the original questions and limited to supporting results.
Additional comments: In this paper, Dong and collegues explored the relationship between mitochondrial/nuclear genotypes and the permeability of insect cuticle. Additionally, the authors also explored the interaction between the sexes and two types of diet (plant or yeast based) in Drosophila melanogaster. They compared three populations from three geographical distant origins and used repeated backrosses to each male strain to keep constant the mitochondrial DNA from the ancestral female line and progressively introgress most of the nuclear DNA from the male population.

This very fundamental and original question pertaining to insect cuticle has received very little attention probably due to the limitation of tools. The authors designed an original trick, based on the use of a red vital dye (EosinY),  to visualize and compare cuticule permeability in Drosophila wings. This simple, yet efficient trick, allowed them to semi-quantify the dye penetration into different wing areas, under controled conditions. To my point of view, this paper is an original and solid contribution which should be used in the near future as a landmark by entomologists and toxicologists.

My general impression after reading the paper is very positive and I have only few minor comments to improve the readability of the paper. The manuscript is well written, with introductory sections conveying to the main points. The literature is generally relevant, and I provide some additional suggestions to make it even more accurate. The Figures could be improved (see below). The findings are well supported by the data based on a consistent number of replicates, and the statistics, to my level of expertise, are correct. The experimental design is clear and the research questions well defined. The methods have been described with enough details. While the discussion is clear, it could be still a little bit more speculative given the few research reports in this specific field of investigation. The conclusion are well stated and linked to the original questions and limited to supporting results.

Minor comments:
L 118: is the average temperature in Benin 8.5°? It seems to be very low.
L129: I would write “...theoritical 99.99% removal..” since some regions of the chromosome are resistant to crossing over.
L180: you shoud add: “We used a semi-quantitative method to classify staining patterns as...”
LL 220-221: Why did the authors not expect to observe only one staining pattern? Explain.
L224: Table 1.
L263: The reference provided is uncorrect and should be replaced by “Antony and Jallon 1982 J.Insect Physiol” which is the first research paper showing sexual dimorphism in D.melanogaster cuticular pheromones.
LL 282-292: In this paragraph, I suggest you cite a recent PeerJ paper (Ferveur, JF., Cortot, J., Rihani, K., Cobb, M. Everaerts, C. (2018). Desiccation resistance : effect of cuticular hydrocarbons and water content in Drosophila melanogaster adults. PeerJ. 6:  e4318.) which explored different parameters related to desiccation resistance.
LL305-307: Could the authors provide a more precise information about the nuclear genes (or families of genes) which may be potentially involved?
LL319-320/LL 343-345: I agree with the authors that they must be cautious about their conclusions based on the small number of populations sampled.

Figures: I had several problems with the Figures.
1- All figures should be oriented in “horizon” format” so you do not need to twist your neck to read them.
2- Figure 3 is impossible to read given that it contains too many information compacted in a unfriendly series of graphs. The authors shoud find a more gentle way to show/share their data. Either they choose different colors to distinguish between diet, sexes, genotypes etc.. or they introduce a gap between groups of bars based on their categories. The legends below the bars should also be shown in a more didactic way, maybe on two rows, one for the genotype and the second one with the sex/food regime. Currently it is quite impossible to read them.


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

Input:
Title: THE CUTICLE INWARD BARRIER IN DROSOPHILA MELANOGASTER IS SHAPED BY MITOCHONDRIAL AND NUCLEAR GENOTYPES AND A SEX-SPECIFIC EFFECT OF DIET
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall the manuscript is clear and well written. Below are minor comments on the text and the English.

1- Lines 116-118. The Köppen-Geiger climate classification for Dundas and Benin appears to be inverted (the average temperature for Benin is not 8.5oC but closer to 26oC, etc).
2- Line 190. Shouldn’t it say “did NOT improve”?
3- There is a number of sentences with awkward or incorrect English that should be revised:
- Lines 23, 175: rather than “unravel” I would use “determine”
- Line 156-157 “To approximately control larvae density, we...”. I would recommend using “roughly”
- Line 205 should say “principal” not “principle”
- Sentence line 265-268 is awkward.
- Sentence starting at line 271 should directly follow sentence that ends line 270 (since they are logically linked by “Indeed...”)
- Lines 275-276 Should say “... results are interesting insofar as they suggest that...”
- Line 282 Should say “...it has previously been demonstrated that diet does...”
- Sentence line 284-286 is awkward.
- Line 288 I would suggest using “affect” instead of “concern”
- Line 289: “along with” should be deleted (and comma moved to the left of “including”)
- Line 294, commas after “we” and “therefore” are not needed.
- Line 311 Should say “.... cuticle permeability are either due to genetic adaptation...”
- Line 316, I would suggest using “similarly” instead of “comparably”
- Line 322 Should say “...would, in turn, allow the testing of whether...”
- Line 324 Should say “This hypothesis proposes that latitudinal...”
- Paragraph lines 356-358 should follow line ending line 355. As a free-standing paragraph is appears to “come out of nowhere”
- Line 729: I would suggest deleting “(take AA, AB, AC for example)” from legend
- Sentence lines 751-752 is not clear/difficult to read.
- Legend to Table S1 (starting Line 763) has some unnecessary information since it is obvious when reading the table: “The left half shows the numbers for males, the right half the numbers for females.” And “The top half shows numbers for flies on plant food, the lower half the numbers for flies on yeast food.” I would also label the columns “Nuclear genotypes” (to the left of the A, B, C row) and rows “Mitochondrial genotype” (above the A, B or C column), and eliminate explanatory text from legend.
- Line 779, please link “We distinguish three groups.” to the rest of the legend; as it stands it appears disjointed.
Experimental design: I am no expert in the statistical methods used for the analyses, so cannot comment on the appropriateness of the criteria and tests used. But the rest of the work is well done and documented, and addresses an important question in insect biology.
Validity of the findings: I am no expert in the statistical methods used for the analyses, so cannot comment on the appropriateness of the criteria and tests used. But the rest of the work is well done and documented, and addresses an important question in insect biology.
Additional comments: This manuscript reports the broad pattern of inheritance of factors that regulate cuticle inward barrier function in Drosophila. Through a careful crossing scheme between populations from 3 different climates the authors were able to isolate the contribution of nuclear and mitochondrial genotype, and determine the role of sex and nutrition and the interaction between these factors, in shaping the insect’s ability to repel dye penetration through its cuticle.

My only apprehension is that the work is quite preliminary: it would be nice to know the identity of some of the factors that are important regulators of cuticle inward barrier function. For instance, directly measuring the levels of the different classes of cuticular hydrocarbons (CH) and their pattern of inheritance might go a long way towards understanding how this barrier varies amongst different populations. Likewise, a number of possible candidate mitochondrial genes are mentioned (lines 63-71), and their level of expression could be measured in the various sublines. The expression of candidate nuclear genes could also be determined, including genes relevant to CH synthesis, cuticular proteins, etc. I recognize that these experiments might involve a significant amount of work. The current report is (to my mind) complete as it is but the results of the suggested experiments would qualitatively improve the relevance and impact of this research.


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

Input:
Title: THE CUTICLE INWARD BARRIER IN DROSOPHILA MELANOGASTER IS SHAPED BY MITOCHONDRIAL AND NUCLEAR GENOTYPES AND A SEX-SPECIFIC EFFECT OF DIET
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The manuscript by Dong and colleagues reports a study on the variations of cuticle inward barrier among Drosophila strains. The insect cuticle is mediating most interactions with the external milieu and this complex structure has certainly contributed to the spectacular evolutionary success of insects. Moussian is a renowned specialist of the cuticle and his work has provided many insights into genes and regulatory mechanisms underlying cuticle formation during Drosophila development. In this manuscript, the authors focus on the inward barrier properties of the Drosophila adult cuticle and the impact of genotypes and other factors. Cuticle composition regulates the penetration of external compounds and water, the latter being particularly important for the adaptation of Drosophila populations to varying ecological conditions, such as temperature and humidity. To address these issues, the authors used Drosophila strains from three source populations (Australia (A), Benin (B) and Canada (C)). The general strategy is based on a simple staining assay of adult wings, using patterns of Eosin Y penetration as a readout of cuticle permeability. Quantification was done by semi-automated image analysis, as described in the corresponding section, and staining patterns were classified in 4 discrete categories. Using this approach, the authors detected clear differences in Eosin staining patterns between A, B and C strains, without obvious correlation with climate classification. Since cuticle composition may relies on other parameters, such as mitochondria (that synthesize lipid-related metabolites), food and sex, the authors used a smart crossing scheme to exchange mitochondrial genotypes between strains, as well as a relevant sampling design to assay for any possible interactions (nuclear, mitochondria, sex and food). The main results of this studies are the influence of both nuclear and mitochondrial genotypes, and a sex/diet interaction, on the inward barrier efficiency.

The manuscript is well written, the material and method section is detailed, and interpretations seem appropriate to describe and discuss the data.
As detailed below, I only have some suggestions to improve the manuscript.

Main points
1)	Since the whole paper relies on a “new in situ method … based on the ability of the dye Eosin Y to penetrate the cuticle” (l99), some experimental validation of this assay and of its relevance to cuticle barrier would strongly strengthen the study. For example, Eosin patterns in a wild type strain versus a mutant background known to affect the barrier function should be very informative.

2)	As suggested by the PCA chart (Fig S2), the stronger effect observed is the nuclear genotype, as also supported by fig4. It should be important to highlight these findings, both in the text and also by reordering the discussion section.

Minor points
3)	I’m very surprised by climatic features of Benin (l118) and suspect some inversion between B and C. Also, I guess that the climate classification of Köppen is “Aw” instead of “Am”. The authors should carefully double check these points and correct them accordingly.

4)	The formulation “lower half at 55°C” or “upper half at 55°C” (l177-78) remains unclear for me. This should be rephrased.

5)	Also, the sentence “high frequencies of double stained areas patterns (female: 234, male: 252) and low frequencies of non-stained (female: 29, male: 34).”(l242-43) should be rephrased since it seems to refer to effectives instead of frequencies?

6)	The notion of “statistically significant” (l255) is vague. There is a vivid debate on its use and misuse in biology (eg PMID 30894741), and whether it was ever useful… The American Statistical Society is also advocating for an end to statistical significance and urge more thoughtfulness in interpreting data (see the special issue of The American Statistician (2019), 73).


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

Input:
Title: THE CUTICLE INWARD BARRIER IN DROSOPHILA MELANOGASTER IS SHAPED BY MITOCHONDRIAL AND NUCLEAR GENOTYPES AND A SEX-SPECIFIC EFFECT OF DIET
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have taken into consideration all my requirements, and therefore I think the paper can be accepted as it is.
Experimental design: valid (as in the first version)
Validity of the findings: valid (as in the first version)
Additional comments: The authors have taken into consideration all my requirements, and therefore I think the paper can be accepted as it is.


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

Input:
Title: THE CUTICLE INWARD BARRIER IN DROSOPHILA MELANOGASTER IS SHAPED BY MITOCHONDRIAL AND NUCLEAR GENOTYPES AND A SEX-SPECIFIC EFFECT OF DIET
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 revised the manuscript to my satisfaction. In particular the figures are much clearer and "reader friendly".
I only have a few suggestions for sentences that seem awkward to me:

Lines 23 “Testing different combinations of mito-nuclear genotypes, we unravel that the inward barrier efficiency...” Rather than “unravel” I would suggest using “show”.

Line 45 “...outward barrier and by consequence the desiccation resistance...” Rather than “by consequence” I would suggest using “as a result would affect”

Line 193 “... model because a high number of degrees of freedom is more penalised as with the AIC...” This sentence is not clear. Should it not say “...is more penalised THAN with the AIC...”?

Line 209 Sentence should start with “The” (“The aim...”).

Line 216 “We scored for the frequency of two Eosin Y staining...” I would suggest writing “We scored the frequency of occurrence of two Eosin Y staining...” (delete “for” and add “occurrence of”)


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

Input:
Title: THE CUTICLE INWARD BARRIER IN DROSOPHILA MELANOGASTER IS SHAPED BY MITOCHONDRIAL AND NUCLEAR GENOTYPES AND A SEX-SPECIFIC EFFECT OF DIET
Review round: 2
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors addressed the points raised and their paper 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: HIF INHIBITOR TOPOTECAN HAS A NEUROPROTECTIVE EFFECT IN A MURINE RETINAL ISCHEMIA-REPERFUSION MODEL
Review round: 1
Reviewer: 1

Output:
Basic reporting: Overall, the writing is clear and unambiguous.  However, some cosmetic changes to the English writing would improve the readability of the manuscript.

The background is sufficient, and references are pertinent.  Overall the basic reporting is good.

The top right panel in Figure 4A (I/R(+)) is too dark.  Please adjust the brightness to match the others (unless the authors have a good reason not to).
Experimental design: The research question is well defined, interesting, and timely.  It is true that neuroprotection of RGCs in various retinal diseases is an unmet need in the field.  The model employed is relevant, and the data are clean and presented clearly.  Methods are written clearly.
Validity of the findings: Data are robust, sound, and controlled.  Speculation is reasonable.
Additional comments: The authors show data that preventing HIF stabilization provides functional/anatomical rescue in I/R mice.
 This is a simple, straightforward study and the findings are clear.  The topic is timely, and interesting.

I have some specific comments:
1.  Can the authors comment on the revelance of the findings to human disease.  i.e. Relevance of animal model/contribution of ischemia to RGC degeneration in retinal disease.
2.  Can the authors comment more about what is known about HIF1a function in RGCs and supporting cells (astrocytes/microglia).  Can the authors rule out an involvement of topetecan in astrocytes/microglia/mueller cells?
3.  The authors should clarify that HIF1a is active in multiple other retinal cell-types.  Can the authors comment if there may be off target effects?