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: INVESTIGATION OF INHIBITION EFFECT OF DAIDZEIN ON OSTEOSARCOMA CELLS BASED ON EXPERIMENTAL VALIDATION AND SYSTEMATIC PHARMACOLOGY ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment. Please see below
Experimental design: No comment. Please see below
Validity of the findings: No comment. Please see below
Additional comments: The manuscript entitled “Investigation of inhibition effect of daidzein on osteosarcoma cells based on experimental validation and systematic pharmacology analysis” by Zhu explored the effect of daidzein on osteosarcoma and the potential molecular mechanism. The paper has been well-designed and evaluated; but some points should be improved before publication:

1.	Some grammatical/formatting errors must to be corrected in the entire text.
2.	Regarding the quality control of the cell line, the presence of mycoplasma was investigated in cell culture? Was cell culture negative for mycoplasma?
3.	The IC50 values of daidzein on 143B and U2OS cells are 63.59 μmol/L and 125 μmol/L. This compound showed low potency. What is the clinical importance of this? A positive control should be include.
4.	Normal cell line (or primary cell culture) should be used to assess the selectively of daidzein.
5.	In vitro invasion and migration assays must be carried out using non-cytotoxic concentrations. The interpretation of these assays using ½IC50, IC50 and 2xIC50 is not appropriated.
6.	In figure 1, use “IN VIVO experiments” instead “IN VITRO experiments” for xenograft model.
7.	For all photograph from microscopy, include more details, e.g. bar scale, total amplification, etc. Add more details in all figure’s legend.
8.	Add statistical analysis in figures 2, 3 and 6. Add statistical at the legend of figure 4.
9.	The quantification of WB figures is stimulated. Include the MW in each protein of WB.
10.	In the experiments with overexpression of Src, the reversion is not clear. The negative control also presented more cells that are viable. I suggest you using a more sensitive assay than MTT (like annexin-V/PI staining by flow cytometry).


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: INVESTIGATION OF INHIBITION EFFECT OF DAIDZEIN ON OSTEOSARCOMA CELLS BASED ON EXPERIMENTAL VALIDATION AND SYSTEMATIC PHARMACOLOGY ANALYSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The current version of the manuscript has been improved in grammar compared to the previous version. Some errors in the description of the methods were also remedied. The literature review, the article structure and the presentation of results are adequate.
Experimental design: The experimental design is also adequate. The methodological detailing allows the replication of experiments by other researchers.
Validity of the findings: The results presented are interesting and allow for advances in the field of study in which the study is inserted.
Additional comments: In general, the corrections made by the authors substantially improved the current version of the manuscript.


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

Input:
Title: INVESTIGATION OF INHIBITION EFFECT OF DAIDZEIN ON OSTEOSARCOMA CELLS BASED ON EXPERIMENTAL VALIDATION AND SYSTEMATIC PHARMACOLOGY ANALYSIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have answered all the questions I concerned, and provided abundant data to support their conclusions.Thus, the manuscript can be accepted for publication.
Experimental design: Above
Validity of the findings: Above
Additional comments: Above


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

Input:
Title: HALLMARKS OF GLYCOGENE EXPRESSION AND GLYCOSYLATION PATHWAYS IN SQUAMOUS AND ADENOCARCINOMA CERVICAL CANCER
Review round: 1
Reviewer: 1

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: Re: #60970
This manuscript claims that a comprehensive analysis of glycogene expression in cervical cancer tissues by the microarray expression analysis provides a possible prognostic biomarker for diagnosis of cervical carcinoma, i.e. metastatic potential, immune evasion and drug resistance.  Although further reverification on the number of cases is necessary, the data presented are useful, and the manuscript is acceptable for publication.
  As indicated by authors, glycogene expression is not directly correlated with final expression of glycoconjugates, and accordingly with cell biological properties of cervical carcinoma.  The synthesis of glycoconjugates, i.e. GPI anchor, proteoglycan, glycosphingolipids, and N- and O-linked glycoproteins, is dependent on the supplies of substrate glycoconjugates and nucleotides sugars, and the enzymatic activities of glycosyltransferases and glycosidases.  In addition, the cancerous tissues are the mixtures of cells with the different proliferation- and differentiation-stages, connective tissues, blood vessels and so on.  Same as true for normal cervical tissues.  Based on the present data, further evaluation on the expression of glycoconjugates and the characterization of cervical carcinoma 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: HALLMARKS OF GLYCOGENE EXPRESSION AND GLYCOSYLATION PATHWAYS IN SQUAMOUS AND ADENOCARCINOMA CERVICAL CANCER
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript by Mertinez-Morales et al. investigated the glycogene expression and glycosylation pathways in squamous and adenocarcinoma cervical cancer using microarrays and RNA-Seq data. The purpose of the study is rather interesting, since glycogenes still remain one of the under explored area as compared to other genes. However, there are some drawbacks in the designing, data analyses and interpretation of the results.

The introduction lacks a proper definition of glycogenes. In addition to the two enzymes mentioned by the authors on line 67, other types of genes are also classified as glycogenes (https://doi.org/10.1186/1471-2164-10483). It should be highlighted if authors want to focus on any specific set of glycogenes.

Line 111: Please provide mean age and standard deviation for both samples.

The discussion is extremely long which makes the manuscript a difficult read.
Experimental design: It is not clear what was the aim of carrying out microarray analysis. Bulk of their results and discussion is based on the analysis of publicly available RNA-Seq datasets. Besides, the microarray platform used consists of only 10,000 genes, and therefore may comprise only a limited number of glycogenes. This might explain why only 15 glycogenes were identified as differentially expressed in their microarray dataset. It would be interesting to know how many were actually glycogenes from the list of 10,000 genes.

Additionally, why did authors chose Microarrays over the RNA-Seq?

The authors must mention the version number of databases and tools for reproducibility. For example, using the list of 6 up-regulated glycogenes I could not find any interaction between these 6 genes (interaction score = 0.9). It would have been better if the interaction network was generated using 401 glycogenes and then map the 15 genes identified from microarray onto that network.

What fold change is represented by the 15 altered glycogenes? Moreover, the authors did not validate by experimental methods (e.g. RT-PCR) any of the 15 altered glycogenes.The authors should also improve on the presentation of protein-protein and pathway enrichment analyses.
Validity of the findings: It is also not discussed how many out of their 15 glycogenes were also identified in RNA-Seq analysis. Reasons for any discrepancies should be discussed. Please report adjusted p-values for enrichment analyses.

Line 220: Table Supp 2 is cited before Table Supp 1.

Figure 2 and 3: Since the genes belong to different clusters in these figures, it is suggested authors highlight these genes (probably by different colors) in supp. Tables 1 and 2. It is difficult to follow which genes from supplementary data belong to which clusters.

Line 265: impllicated -> implicated.

Information in Table 5 and supplementary tables is limited. It is recommended to include the gene/protein names and their functional annotations.
Additional comments: The authors should look at some of the representative papers focusing on glycogene (
https://doi.org/10.1371/journal.pone.0013002; https://doi.org/10.1186/1471-2164-10-483) analyses as well as papers with PPI network and enrichment analyses (https://doi.org/10.1038/s41598-020-65909-x). This will help them to improve the manuscript substantially by properly analyzing the gene expression data.


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: HALLMARKS OF GLYCOGENE EXPRESSION AND GLYCOSYLATION PATHWAYS IN SQUAMOUS AND ADENOCARCINOMA CERVICAL CANCER
Review round: 1
Reviewer: 3

Output:
Basic reporting: The article needs revison for English langaguge, not thourgh though.

Introduction and Discussion are very long and need extensive revision.
Experimental design: Needs more studies to include.

Exclusiion and inclusion criteria of subjects are poorly defined.
Validity of the findings: Hypothesis and experimental design is good, however, there is so much repetition of results. Needs extensive revision and cutting down the introduction and discussion.
Additional comments: The article needs revison for English langaguge, not thourgh though.


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: HALLMARKS OF GLYCOGENE EXPRESSION AND GLYCOSYLATION PATHWAYS IN SQUAMOUS AND ADENOCARCINOMA CERVICAL CANCER
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript looks improved now and seems acceptable for publication.
However, please add a legend for the supplementary data.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: No additional comments


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

Input:
Title: GENOMIC COMPARISON OF NON-PHOTOSYNTHETIC PLANTS FROM THE FAMILY BALANOPHORACEAE WITH THEIR PHOTOSYNTHETIC RELATIVES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper presents an interesting and innovative information for Balanophoraceae, showing relevance to be published by the PeerJ. However, even though the study has a lot potential, significative amount of changes and corrections are required in order to improve understanding and flow.

The authors use a somewhat informal terminology throughout the manuscript, sometimes implying personal opinion: for example, in the abstract we can see “a tremendous AT content” and “we supposed that it would be interesting”. I would advise to keep a clear and professional English language.

The background presented in the introduction could be improved. A better explanation of the family and the species investigated in the study could be added. The statement used in Line 95 does not justify the choice of the organisms investigated:
        “We supposed that nuclear genomes of plants with such unusual plastid genomes   also deserve being studied.”

 Moreover, the text is full of strong statements that have no citation as support. I would advise to re-write the introduction using the literature as support, removing any personal opinions, and “tone down” general statements.
Experimental design: The study presents an original primary research. However, the research question could be better described (better written) to point out its relevance and how the research fills a knowledge gap.

I appreciate the effort of the authors, however the M&M section could be re-written in order to facilitate understanding, please follow examples such as Mahmood et al. (2020) (https://doi.org/10.1038/s41598-020-70406-2).
Validity of the findings: The study has potential, however I can not fully state about the methods and the validity of the findings, since there is a great amount of strong statements that are not supported by any literature throughout the manuscript (some speculations, some results from other studies without citation). In addition, the language is confusing which does not help interpretation.
Additional comments: General comments:

The paper presents interesting and innovative information for the Balanophoraceae family, showing relevance to be published by PeerJ. However, significative amount of changes and corrections are required in order to improve understanding and flow.

There is a great amount of strong statements that are not support by any literature in both the introduction and discussion sections. Another concern that I have is about the language used in the manuscript. The authors use a somewhat informal terminology throughout the manuscript, sometimes implying personal opinion: for example, in the abstract we can see “a tremendous AT content” and “we supposed that it would be interesting”. I would advise them to re-write the study and keep a clear and professional English language.

The material and methods section could also be reorganized and re-written. It is also necessary to say that there is a significant amount of “result-like” sentences within the M&M.

I present below a point-by-point comment to help in the correction process:

Abstract:
General comments:
Line 20: the word tremendous imply personal interpretation, please keep formal language. Maybe changing it by “high” or something similar.

Line 21: greater by “one order of magnitude” instead of “an order”

Line 22: Be careful with general statements, no one can possibly exhaust the literature, since there are so many papers published every day. So, it would be more acceptable to say: “To our knowledge, none of these features have yet been investigated” instead of “All these features have no definite explanations.”

Line 24: Please keep the formal language, please re-phrase “we supposed that it would be interesting”, same for Line 31 “is for an unknown reason several times larger than”, in this case, language in unprecise, one could say: “X amount”, or “X percent”, or “an average of X% higher”. The abstract should present more data, not only: “several genes”, “several times larger”

In addition: The conclusion could be improved in the abstract. What are the biological implications? What are the main “take home messages” of the study?

Introduction:
Besides informal language, the authors do not respect the format for citation. Please correct it.
•	Since informal and ambiguous language was present throughout the whole manuscript, I marked the sentences that need to be changed in yellow in the pdf file.

Consider change one of the two: “this is called” on lines 41 and 43

I would advise to “tone down” general statements, and also use citations to support your statements. For example:
•	Was the entire first paragraph based on Leake (1994) and Tesitel (2016)?
•	Second paragraph: “This is the reason why most plant genomes sequenced and assembled to date are plastid ones.” Please be careful.

In addition, the authors say: “they do need light for survival”, however heterotrophic species do not need to be exposed to light for their survival, but they need light, since their host needs light.

Line 63 to 66: paragraph containing only one sentence. Keep formal scientific language. Same for Line 191 to 192, and phrases from lines 245 to 251.

Line 67 to 68: Personal opinion. In addition, I understand the position of the authors, however it will depend on the features chosen for sequencing. One can choose to have a high depth sequencing of a plastid and it will increase the sequencing cost.

There is no connection between the phrase on line 67 to 68 to the following sentence. In addition, transcriptome sequencing can also be expensive, sometimes more expensive than genome sequencing.

Lines 72 to 78: no literature to support strong statements and results. Same for lines 82 to 94.

Lines 95 to 109: Full of personal opinion statements. In addition: there are results and methodology mixed up in the introduction in an unusual manner and the citations are confusing and in a wrong format.

Material and Methods:
I appreciate the effort of the authors, however this section should be re-written in order to facilitate understanding, please follow examples such as Mahmood et al. (2020) (https://doi.org/10.1038/s41598-020-70406-2).

There are results described in the M&M. For example: Lines 123 to 131, and 139 to 148.

The authors did not perform transcriptome sequencing for the species used in the comparison, correct? Maybe adding a table to summarize the information for this data would facilitate understanding.

Line 153 to 155: Could be explained in the results section. Please re-write to a more scientific sound statement.

Line 170: How did the authors selected “minor isoforms” to be discarded? What were the parameters used?

Lines 262 to 274: Discussion like. Please restrict your information to ensure that the methods are explained with clarity.

Results and Discussion:
Strong statements without literature support (i.e., lines 331-336; 391-397).

For example: “The RBH analysis revealed no transcripts of the lost genes, implying that they were not transferred to the nuclear or the mitochondrial genome, but instead disappeared completely.” Such strong statements should be avoided and substituted by a more conservative and “scientific sound” statement. For example: “Our results suggest that … which corroborates the hypothesis that such genes might not be present in the studied species, such as found by X et al. (20XX) or X et al. (20XX)” (using evidences in other species to support your statement).
•	Just to think about: Would it be possible that during your filtering steps some sequences have been removed, or during extraction the transcripts were not sampled given the low quantity of 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: GENOMIC COMPARISON OF NON-PHOTOSYNTHETIC PLANTS FROM THE FAMILY BALANOPHORACEAE WITH THEIR PHOTOSYNTHETIC RELATIVES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper “Genomic comparison of non-photosynthetic plants from the family Balanophoraceae with their photosynthetic relatives” describe a comparison of transcriptomes features among five species: two non- photosynthetic species Rhopalocnemis phalloides and Balanophora fungosa and their close photosynthetic relatives Daenikera sp., Dendropemon caribaeus, Malania oleifera. The motivation for the study was the unusual features found on the plastid genome of Balanophoraceae family, such as high AT content and substitution rate.
As for the writing, in many cases I consider the text being extremely informal. I suggest the authors to carefully check all text to improve the scientific writing. I will provide some examples in the General comments section.
As for the background provided, figures and tables I consider satisfactory.
I could not find any link to the raw data on the manuscript. This is mandatory for publication on Peer J.
Experimental design: On the general basis the research question was well defined and relevant. The authors provided sufficient information to replicate the analysis, except for the raw data not provided as afore mentioned, and some punctual notes that I raised in the General comments section.
Validity of the findings: Although the subject of the paper is very interesting and relevant for the academia, the authors did not provided accession to the raw data. As the data on which the conclusions are based were not provided the paper did not fit for this criterion.
The detailed points regarding the results and conclusions are in the General comments for the author section.
Additional comments: Introduction

Line 98-109	I consider this text fragment confusing. I suggest it could be rewritten.

Materials and Methods

Line 117 and 132	As a matter of comparison, the plant tissues used for the plant transcriptomes was the same as the present study? Tissue-specific differentially expressed genes were already identified in literature. (See Yu et al  2017- 10.3390/genes8120372 - for example)
Line 123	I couldn’t find the SRA accession numbers for the sequenced Rhopalocnemis phalloides
Line 160	“Bases with sequencing quality less than 3 were trimmed from the 3' end.” I do not understand what “quality 3” really means. Is that a phred score? If so, I suggest that it be clearer.
Line 165	I suggest change the word “removed” to “discarded”
Line 156-166	Is that a reason why the author did not use the command LEADING (trim the reads from the 5’end) for Trimmomatic? I noticed only the TRAILING command, which trims the reads from the 3’end.
Line 184	I suggest change the word “met” to “found”
Line 191-192   “CDSs with proteins that had no matches in NCBI NR, and consequently no definite taxonomic  assignment, were kept.” Were the CDSs really kept? Even though they don’t match the NCBI database? I’m not sure if I understood this sentence, could you please check.
Line 215 	“DIAMOND as a tool for similarity calculation.” Is that a separated tool used for similarity calculation? Or it is an algorithm incorporated into OrhtoFinder software? Please let this information clearer and inform the citation and software version.
Line 262-274 The information present in those lines are not proper to the Materials and Methods section. I suggest to incorporated then into the Discussion.

Results and discussion
Line 278	I suggest you specify which figure you are referring in the text (Example “The results for all transcripts indicate the near absence of missing genes (Fig 2-A)”.
Line 294 	“thus the completeness of the assembly may be overestimated” To ensure the completeness analysis of the assembly I suggest you to align the transcriptome obtained to a transcriptome already available in the literature of a close species and calculate the amount of CDS aligned and the mean percentage of the CDS length that were aligned.
Line 311	“We do not have an  obvious explanation for this phenomenon.”  The text fragment gives the impression that the authors did not make an extensive search for explanations on the literature. I strongly recommend changing this kind of argument on the manuscript. It does not sound good. A better statement could be such as: “We recommend futhers analysis/studies to enlighten this phenomenon…”.
Line 313	Interesting how? It would be better if you explain why this genes were selected.
Line 331	“The plastid genomes of Rhopalocnemis phalloides and Balanophora fungosa were previously  shown to be approximately 10 times shorter than plastid genomes of their close photosynthetic  relatives” Is that information on literature? If so, you should quote the paper.
Line 359	“It was logical for us to have a look…” Text fragment consider informal to a scientific writing.
Line 366 	“do not differ much from  that of their photosynthetic relatives.” Is the numbers statistically different? I think is valid for the authors to test this and incorporated the pvalues on the manuscript.
Line 481	“The increased substitution rate of the nuclear genome (Fig. 3), in our opinion, may be explained in several ways” Another example of text fragment consider informal to a scientific writing.


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: GENOMIC COMPARISON OF NON-PHOTOSYNTHETIC PLANTS FROM THE FAMILY BALANOPHORACEAE WITH THEIR PHOTOSYNTHETIC RELATIVES
Review round: 2
Reviewer: 1

Output:
Basic reporting: English: The English could still be improved throughout the manuscript.

Introduction: The structure of the introduction is sound, and so is the amount of information for the most part. The only thing that is missing is some information on the family Balanophoraceae and the Santalales. The reader might want to learn a bit more on how closely the studies species are. The respective papers should be cited.

l. 40 – “…create organic substances…” – I don’t thing “create” is the right word here, “generate” or “synthesize” might fit better

L. 49 – “The majority of heterotrophic species spend most of the year completely underground…” – There are a few exeptions: Rizanthella garnerii and Hydnora triceps, both flower belowground.

L. 50 - “…since they do not need light for survival.“ – not corrected according to the previous reviewer – it should be stated that they don’t directly depend on light, but indirectly they do through the host (as a previous reviewer suggested).

L. 54 – „…plants have three genomes: the plastid, the mitochondrial and the nuclear.“ – Please rephrase. This is not good language. You mean the plastid genome, the mitochondrial genome, and the nuclear genome. This might be confusing to a native speaker.

L. 55 – „The plastid genome is the smallest and has the highest copy number, thus alleviating sequencing and assembly.“ – Add reference

L. 56 – „These as well as other features of plastid genomes are the reason why most plant genomes sequenced and assembled to date are plastid“ – The conserved structure and the gene density are also reasons why they can be easily assembled. Add respective references.

Section Line 54-64 – There are a lot of relevant references missing.

L. 65 – “The mitochondrial genomes of non-photosynthetic plants are poorly studied…“ – This is a harsh statement and does not acknowledge people who have worked on mitochondrial genomes of non-photosynthetic plants. It is certainly a challenge and hard to get published unless there is one circular mt genome.

L. 69 – „Sequencing of whole nuclear genomes is sometimes expensive due to their large sizes. Therefore, to obtain information about the nuclear genes, scientists often sequence the transcriptome instead of the genome itself.“ – While this might still be true to plants with very large genomes, I still would not state it that way. A proper transcriptome study including various stages and tissues including sufficient replication requires a lot of sequencing as well. Please rephrase. This is again disrespectful to other scientists working on high quality transcriptomes!

L. 75 – „…genes participating in chlorophyll synthesis…“ – To participate implies this is an concious process, but it is not. Please rephrase. See also line 432

L. 76  - „It is hypothesized that the chlorophyll may have some other function apart from photosynthesis“ – Add some examples and references.

L. 87 – „These plastid genomes are about 10 times smaller than..“ – They are a tenth of the size of…

Section L. 85-97 – Please add references! You have to acknowledge other people’s work!!! There are none mentioned in this text!

Literature: There is a lot of literature missing in the introduction. Given the length of the ms, I can’t provide a comprehensive list for the authors.

Structure: The lack of hypothesis early on in the present paper, at the end of the introduction makes it hard for me to understand what the authors are after here. The paper is very descriptive and lengthy up until the last paragraph starting on line 428. Especially the first half of the Results and Discussion section is quite dragging. The structure is clear, but the joint Results and Discussion paragraph is not beneficial to the present manuscript.

Figures : The figures support the data, although they are very minimalistic. The font size in Figure 1 and Figure 3 should be bigger.
Experimental design: Research question:
L. 98  - „Given the unusual features of the plastid genomes of Balanophoraceae, we supposed that their nuclear genomes may also provide information valuable for understanding the evolution of non-photosynthetic plants.“ – This is not a clear hypothesis. The last section of the introduction should contain the hypotheses what is going to be studied and how. It should provide information on what exact data is necessary to answer the specific questions in focus. Most of this is missing here, please work on this section again.

Methods: The RNA for the sequenced species were extracted from very different tissues: Rhopalocnemis phalloides – inflorescence, seedlings, fruits, leaves, and some are unknown. This is not ideal for comparison. I get that the authors took what was available and added Rhopalonemis. So seeing how complete the five transcriptomes are in the BUSCO analysis is quite surprising. However, I wonder if the same analysis using the plant set would reveal in fact genes or gene classes that are missing due to losing the ability to photosynthesize. On the other hand one can only comment on what is present and not what is absent because discrimination between absent and not detected is not possible with the present heterogenous dataset.
Validity of the findings: Overall the analyses are valid for getting a first overview of the situation, which I believe is what the authors went for. When calculatign the dN/dS ratios using over 1,000 genes, it would be interesting to see if there are genes that have accelerated rates and some that have the same rates. More  differentiated results would be valuable in the future. Also, I think it would be helpful to include plants outside the Santalaes (like Arabidopsis, where it is possible and makes sense) to be able to pinpoint more genes affected by loss of photosynthesis.

There is a recent study on another parasitic plant Sapria with a similar overall idea to the present ms - Cai, Liming, et al. "Deeply Altered Genome Architecture in the Endoparasitic Flowering Plant Sapria himalayana Griff. (Rafflesiaceae)." Current Biology (2021). The authors take a detailed look at what nuclear genes are present and what they (presumably) do, specific pathways are discussed. This study might be relevant to the present ms.

L. 370 – “The increased AT content of Balanophoraceae is a feature both of  non-coding and of coding regions,” – When comparing the AT-content to nuclear genes the AT-content of plastid genes should be used, not the complete plastome (even if it doesen’t make much of a difference)

L. 381 ff – “…it safe to assume that the AT contents of plastid protein-coding genes of Daenikera sp. and Dendropemon caribaeus are somewhere in the range of 60.9%-63.7% or close to that range, because this range is based on 29 plastid genomes of photosynthetic Santalales known to date. – I would leave this out or rephrase this speculative statement. You just never know until you have the data.

L. 451 – “The disappearance of plastid RRR proteins gives an explanation for why..” – change to “possible explaination”

L. 475 – “Analyses of substitution rates in plastids…” – change “plastid” to “plastome”
Additional comments: In the present manuscript with the title  “Genomic comparison of non-photosynthetic plants
from the family Balanophoraceae with their photosynthetic relatives” transcriptomic data from the non-photosynthetic plant Rhopalocnemis phalloide was generated and compared to another species from the same family, Balanophoraceae, and three other species from the same order, Santalales. Overall, this is a valuable study. Especially the last paragraph in the Results and Discussion section has some interesting and original ideas. The rest of the text leading up to this could be more concise.


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: CIPROFLOXACIN REDUCES TENOCYTE VIABILITY AND PROTEOGLYCAN SYNTHESIS IN SHORT-TERM EXPLANT CULTURES OF EQUINE TENDON
Review round: 1
Reviewer: 1

Output:
Basic reporting: Pls. see document attached
Experimental design: Pls. see document attached
Validity of the findings: Pls. see document attached
Additional comments: Pls. see document attached


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: CIPROFLOXACIN REDUCES TENOCYTE VIABILITY AND PROTEOGLYCAN SYNTHESIS IN SHORT-TERM EXPLANT CULTURES OF EQUINE TENDON
Review round: 1
Reviewer: 2

Output:
Basic reporting: I want to congratulate the authors for the manuscript entitled: "Ciprofloxacin reduces tenocyte viability and proteogycan synthesis in short-term explant cultures of equine tendon" reference 58014.
The manuscrpit is well-structured, good background has been provided.
The hypothesis is very relevant, since the results justify the clinical observations of fluoroquinolones on tendoncyte viability/repture.
Although some references are older than 10 years, the articles are referred to some recognized or classical in vitro and in vivo studies on the effect of FQ (fluoroquinolones) on tenocyte viability.
Experimental design: I want to congratulate the authors for the manuscript entitled: "Ciprofloxacin reduces tenocyte viability and proteogycan synthesis in short-term explant cultures of equine tendon" reference 58014.
The design is very interesting and the results confirm that FQ affect tendon viability even at 8 days after discontinuation of treatment.
Fernandez-Cuadros et al has stated that pathlogy could be present since 2 hours of treatment to as far as 6 moths after stopping treatment (Fernández-Cuadros, M. E., Casique-Bocanegra, L. O., Albaladejo-Florín, M. J., Gómez-Dueñas, S., Ramos-Gonzalez, C., & Pérez-Moro, O. S. (2019). Bilateral Levofloxacin-Induced Achilles Tendon Rupture: An Uncommon Case Report and Review of the Literature. Clinical Medicine Insights: Arthritis and Musculoskeletal Disorders, 12, 1179544119835222). You could add this paragraph on introduction.
Material and methods are clearly explained, that I have nothing more to add.
Validity of the findings: The authors have stated that FQ inhibit tenocyte viability and inhibits synthesis of proteoglycans.
In discussion, Corrao et al 2006 was cited. You should describe that although you have seen that latency of recovery after FQ therapy is not observed at 8 days, other authors have described even FQ effect at 6 moths of FQ treatment (Fernandez-Ciuadros et al).
Additional comments: The manuscript is well written. The results are in accordance to the objectives of the study.
Discussion is linked to the matter of the manuscript.
Conclussion is not concise, some authors have been cited in such a section (Bank et al, Khaliq and Zhanel et al). I strongly suggest that the paragraph where authors are referenced should go in discussion part. I see techically innapropriate to cite/reference authors in conslussion section since this part is reserved to the final results of the manuscript, not for observations from other authors.
Some other paragraphs of conclusion refer to strength and limitations of the study. I would suggest authors to put those paragraphs in Discussion section, and to limit Conslussion to the main results observed in the study (viability of tenocytes at 96h and 8 days and PG synthesis) based on biochemical and gene evaluation.


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: CIPROFLOXACIN REDUCES TENOCYTE VIABILITY AND PROTEOGLYCAN SYNTHESIS IN SHORT-TERM EXPLANT CULTURES OF EQUINE TENDON
Review round: 1
Reviewer: 3

Output:
Basic reporting: 1. Overall:
-This manuscript is fairly clearly written.  A few specific comments:
-line 81: write out “approx.”
-At the start of a sentence, don’t use abbreviations or number (write-them out).
-Line 329: insert “in” between reduced and equine.
-Line 334: Please reword the first sentence of the conclusion for clarity

1. Introduction:
- very thorough.
2. Results:
-Effects on tenocyte viability (line 212): please specify that this is compared to controls.
-Line 231-232: is the change in versican mRNA in the treated or controls?
3. Discussion:
-The discussion is too long and includes information that is not relevant to this publication. For instance, on line 256, the authors make reference to ibuprofen and its affects on FQ accumulation.  This is not relevant to this paper, nor is ibuprofen used in horses with any regularity.
-The mention of renal failure and renal transplant also seems misplaced in this paper.
-Removal of extraneous information will shorten this section and make it easier to read.
-Line 314: Since the study is cited, please provide a suggestion as to why the contradictory results between the current study and the Juras study.
4. Conclusion:
-extremely long and includes a lot of extraneous information. Would suggest making it more succinct rather than a repeat of what is found in the discussion.
5. Figures:
- clear and well described
Experimental design: 1. The study begins to fill the gap in our knowledge regarding the potential toxicity of FQs on tendons in horses.
2. Suggest adding the reason for the selection of ciprofloxacin concentrations along with the aims in the introduction or in the methods section when the specific concentrations are first mentioned.
3. Methods are clear.  One specific area that could be more clear is the “RNA extraction and reverse transcriptase” section.  The authors state “in a separate of experiments.”  Does this mean a completely different culture and were the treatments the same?
4. With respect to doses, are these really clinically relevant with respect to the concentrations that actually reach the tissue.  The authors cite studies done in humans but no PK studies in horses.  Furthermore, the concentrations that the authors suggest the tendon is exposed to in vivo seems very high.  In looking at the references, there are only a couple that describe tendon concentrations and they are for other FQs and concentrations appear lower.  In my mind this raises the question of the clinical applicability of these concentrations.  Justification for doses should be more clear or at the very least acknowledgement that these may or may not be clinically relevant.
Validity of the findings: 1. While this is a good first step in understanding the effects of FQs (specifically Ciprofloxacin) on tendon cells, the findings and the significance are a bit overstated.
-The findings from this study show an effect on tenocyte viability but the authors appear to over speculate as to the mechanism.
-Line 275: the authors suggest that suboptimal concentrations of decorin may be contributing to tenocyte viability.  Unless I missed something, they looked at mRNA expression (not concentrations which suggest protein) and even then, there was not a significant difference in decorin expression.
-Line 282: the authors comment that the study provides insight into the mechanism underlying the latency between cessation of FQ therapy and development of tendon pathology.  Please either remove or provide data to back up this statement from this study.
Additional comments: This paper attempts to begin to address an issue of importance in veterinary medicine but one that we don’t have much information about, especially in horses.


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: ANTI-CLUSTERING IN THE NATIONAL SARS-COV-2 DAILY INFECTION COUNTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: OK
Experimental design: See comments on analysis method below
Validity of the findings: See comment below
Additional comments: In this manuscript, the authors analyze data regarding the national daily COVID-19 infection number from the Wikipedia WikiProject COVID-19 Case Count Task Force data. They look for a criteria to establish if the noise in the data is consistent with reliably reported data. As a bench mark, they postulate that the noise should correspond to Poissonian noise, and test it by a one-parameter Poissonian \phi_i model, which basically assumes that the number of daily infections should  grouped in clusters, each containing\phi_i counts per cluster. \phi_i is determined in different regions by choosing the value which leads to the best fit to Poissonian  distribution. Applying this analysis to different countries reveals that for most countries a super-Poissonian behavior emerges.

Since much of the decisions relating to COVID-19 policies are based on daily infection numbers, it is worthwhile to develop a better understanding of their statistical characteristics, which can be used to draw attention to questionable reporting or unusual circumstances. Therefore, there is no doubt that the manuscript is timely and of interest.

Nevertheless, I feel less confident about the assumptions and method. Based on my experience of analyzing noisy data from complex dynamical systems (albeit very different than the one discussed here), Poisson noise usually arise in non-correlated simple dynamics. For correlated complex systems (which I suspect corresponds also to the infection dynamics) I expect different noise characteristics. Indeed this seems to be the authors conclusion from their analysis. What I am missing is why did they base their analysis to begin with on a deviation from Poisson noise .Moreover, once it is clear that Poisson noise does not capture the behavior, why use a one-parameter Poissonian \phi_i model, where \phi_i is a single cluster size, although one would expect a wide range of clustering (power law?) to describe the data?

I believe that the authors should clarify their rational for choosing their analysis method more clearly, and if possible, supplement them by an analysis more suitable for super Poissonian (clustered) noise. This will greatly enhance the value of their study.


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

Input:
Title: ANTI-CLUSTERING IN THE NATIONAL SARS-COV-2 DAILY INFECTION COUNTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is a very interesting analysis, focusing on the properties of noise in epidemic reporting, which is an often under-appreciated source of information.  While it does provide a basic notion super-Poissonian vs sub-Poissonian noise, it appears to does side-step several issues that may warrant further discussion. I will comment on these below.
Experimental design: 1. The assumption that the expected value of cases on one day is the median of 4 neighboring days can lead to significant overestimate during exponential growth or decay. I suggest fitting an exponential (or a quadratic form for log(n_j)) instead, to test for this effect.
2. Statement in Line 130, suggesting that relative noise in Poisson process is 1/\sqrt{N} is only for large N, and the exact Poisson probability \lambda^N\exp(-\lambda)/N!, can be used for any N, including N=0.
3. I do not understand why a KS test is needed to determine \phi'. A product of (rescaled) Poisson probabilities gives the likelihood for \phi', which can be used to constrain \phi' using Bayes theorem.
4. What does "sufficient fit" in Line 201 mean?
5. Many reported cases have weekly cycles (e.g. a lull in weekends). Is there a way to test for this effect?
6. Does the analysis apply to multiple epidemic waves?
7. While the article focuses on daily reporting, it would be useful to see how the results change if we look at longer periods, such as those consisting of multiple days
8. Translation of acronyms to country names are missing from the manuscript
9. Link in Line 429 is broken
Validity of the findings: The main premise of this paper is that some countries appear as outliers in the distributions of a statistical measure, and they are interpreted as due to (intentional or unintentional) administrative reporting  practices. What is missing from this is any measure of the epidemic itself, which has proved highly unpredictable and chaotic, and can vary from region to region, for a multitude of reasons. This seems premature. For example, the US and Brazil exceeding the extrapolation from small epidemics may well be a feature of a larger more heterogenous epidemic, leading to more daily variations.  The grouping of low variance vs high variance countries appears arbitrary and does not follow from a clear quantitative criterion.

Line 320 says "It is in this sense that the sequence can be considered sub-Poissonian. ", through comparison of Algeria with other countries, even though the data is NOT actually sub-Poissonian. As such, I believe this terminology is misleading: sub-Poissonian suggest doctored data, while Poissonian noise may be natural for the  epidemic in one country, even though it may not be in others. The COVID-19 pandemic has shown a huge diversity of behavior across different countries and continents, and simply being an outlier (e.g. New Zealand)  doesn't suggest doctored data.

Overall, my recommendation is discuss the extent to which the underlying unknown epidemic dynamics may confound the findings here. Furthermore, I recommend a quantitative p-value, with a clear threshold, that could be used to reject Poissonian and super-Poissonian hypotheses.
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: ANTI-CLUSTERING IN THE NATIONAL SARS-COV-2 DAILY INFECTION COUNTS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The paper is well written and the flow of the paper is logical.

The paper lacks comparison with the vast non parametric statistics literature that can be used to assess the proposed anti-clustering behavior.
Experimental design: The research question is well defined. The proposed solution is statistically weak, for at least three reasons:

1. One of the many non parametric tests available should be attempted (see any introductory statistics book that have. chapter on non parametric statistic)

2. Taking the median of the counts over five consecutive days to estimate the Poisson parameter is data-driven and ad hoc assumption which may bias the results of the Kolmogorov test. The Authors should avoid this and use a time dependent model such as an autoregressive one

3. The issue of over/under dispersion wrt to the Poisson can be tackled with a negative binomial distribution, and several authors have used this to model covid-19 count data
Validity of the findings: The findings are well described. However, the methodology on which they are based is weak and there is no comparison with alternative non parametric or semi parametric methods
Additional comments: The problem of anti-clustering of the covid-19 data is a relevant one. The methods proposed to ascertain statistically the presence of an anti-clustering behavior in covid-19 national data of a set of countries is weak and there is no comparison with alternative methods. The background statistical literature is very scarce.  The paper should be rejected in the present 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: ANTI-CLUSTERING IN THE NATIONAL SARS-COV-2 DAILY INFECTION COUNTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The paper concerns a relevant problem, it is original and well written. The authors have taken my previous comments into account in their revised version
Experimental design: The experimental design is clear and improved after the revision
Validity of the findings: After the revision the findings are more robust
Additional comments: The paper could be accepted conditionally on a further enlargement of the bibliographic references, especially on the comments introduced after revision

For example, concerning autoregressive methods, the authors should quote Agosto,Campmas, Giudici, Renda,
Monitoring covid-19 contagion growth, statistics in medicine, 2021:
https://doi.org/10.1002/sim.9020

And the references therein.


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: ANTI-CLUSTERING IN THE NATIONAL SARS-COV-2 DAILY INFECTION COUNTS
Review round: 3
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The Authors have satisfactorily addressed my previous 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: LOW WINTER PRECIPITATION, BUT NOT WARM AUTUMNS AND SPRINGS, THREATENS MOUNTAIN BUTTERFLIES IN MIDDLE-HIGH MOUNTAINS
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: General comments
The manuscript entitled “Low winter precipitation, but not warm autumns and springs, threaten mountain butterﬂies in middle-high mountains” explores a series of climatic factors that could shaped population trends of mountainous butterflies. It also connects changes in species abundance and their phenology through an interesting approach of seasonal variation on regional climatic conditions, assuming an effect on early life stages as well.
Overall, this is a well-written manuscript that I much enjoyed reading. Experimentally the study is well-designed and very well-performed, while the analyses are relevant and sampling size robust. The authors are aware of up-to-date literature and have a deep ecological understanding and scientific knowledge with regards to the paper scope.
This study is an outstanding contribution to the existing literature on phenological patterns and population trajectories because first, it provides data on cold adapted organisms that are less studied compared to their counterparts in lowlands and second, it gives access to long term data that are largely lacking from Central Europe.

I provide below some general thoughts and minor suggestions, hoping to be of use of the improvement of the paper.

Minor comments
84-85: Isn’t it the case for lowland species as well?
113-114: This part is somehow misleading. After reading it, I was expecting to see a combined field and lab work where all developmental stages of the species would have been monitored but it’s clearly not the case here. I’d suggest rewriting this part and try to include a term like “hypothetically all phases of larval development” or something similar.
164: Please correct the number of years. It is not 3 but 5.
168: Does this mean that you were in the field from month June, but you had no observations expect for the warm year 2018? If so, then I think you should need to clarify that as it increases the robustness of your sampling effort.
187: I think it might be easier to write population abundance index here. In general, I would try to minimize the use of acronyms as one can easily get lost especially when reading the methods and results.
188-193: It would be really useful to provide the script for onset and duration as well as the script for abundance index. I know that I would like to try your 20% (onset) and 20-80% (duration) approach in my next attempt to calculate phenological indices.
206: Please add the word “autumn” before total precipitation so that readers can understand what Aut stands for. Otherwise, you might want to replace “Aut” by the first letters of the two months such as SO (September, October). Please consider to add this explanation to the corresponding Table as well.
In addition, please replace “and” by “as well as”
207: Please delete “the”.
231: Please delete Rv.3.4.4 as it is mentioned earlier. I would suggest to report the version of R you used for your analysis either in the end of statistical methods or at the beginning of methods section. For example, “All statistical analysis were conducted in R v….”.
257: Please explain the four data sets, it is not clear here.
281-282: For all cases but E. epiphron in K mountain, right? Please correct.
288-289: Please consider including the whole time series as it might be more informative.
296: Please correct Table, it should be Table S4.
307: Is it again of the next year? Please clarify.
316-317: It might help if Table S3 was added here.
318-320: Maybe I am missing something here, but what I’m getting from Table S3 is certainly an increasing abundance during warm springs but not in summers (strong predictor effect has negative slope). With regards to winter precipitation, patterns seem to be increasing (positive slopes)
332: Please add “shortened” after the last word (and) of the sentence
350-351: It is not clear from Fig.3 that populations trends are stable for E.epiphron J. What about the decreasing trend in panel (f)?


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: LOW WINTER PRECIPITATION, BUT NOT WARM AUTUMNS AND SPRINGS, THREATENS MOUNTAIN BUTTERFLIES IN MIDDLE-HIGH MOUNTAINS
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: It is an important study, with interesting results to understand the effect of climate change on mountain biological systems. There is a considerable amount of data, the result of many years of monitoring. The analyzes are as expected for this type of study. The presentation of the figures and the results is done in a satisfactory way as well.

Some general comments:
I suggest adding a general conclusion, with the main points of the work.

As the authors use a large number of variables, it would be interesting to make the results clearer for readers, perhaps using a simple table (especially when presenting the relation between abundance and climatic variables), so the reader would be able to follow the discussion more easily.

There are some parts of the introduction that would fit in the methods, and of the discussion that is results. To avoid repeating the same information in the text, I would carefully review these two sessions.

Specific comments:
Line 54: “Middle-high”
Line 69: why not use “micro-habitat”?
Line 72: the same, why not use “sub-alpine”?
Line 106 to 110: would be better in the “methods” section.
Line 114: replace the phrase “We ask, which…” with “We tested which climate…”.
Line 133: “Permissions for the field…” would fit better in acknowledgment.
Line 136: Erebia sudetica does not occur then in J? Make this information clearer.
Line 163: “Together with students…” also would fit better in acknowledgment.
Line 168: The area is monitored in other months too then? It’s important to mention this, to show how the authors discovered that adults emerge before...
Line 229: And also separately for each species, correct?
Line 271: The information on the hours/sampling would also be relevant.
Line 320: Perhaps consider putting the tables S3 and S4 in the scope of the article, in some simplified way. It helps follow the text.
Line 345 to 348: This part is a bit of repetition of the results.
Line 358: higher or lower?
Line 366: Is this sentence correct “…were not affected by snow cover duration based on our results”? Because there was a snow cover effect, right?


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: LOW WINTER PRECIPITATION, BUT NOT WARM AUTUMNS AND SPRINGS, THREATENS MOUNTAIN BUTTERFLIES IN MIDDLE-HIGH MOUNTAINS
Review round: 1
Reviewer: 3

Output:
Basic reporting: This is a well written manuscript of high relevance in the fields of ecology and conservation biology. All in all, it is well performed and I have only some relatively minor comments given at the general comments. Figures and tables are OK and the results allow the drawn conclusions. The discussion in general is interesting and relevant. The referring to the existing literature is well done and up-to-date.
Experimental design: Experimental disign is sound and well suitable to address the resarch question. The latter is well defined and relevant. The ms is performed at generally high standard.
Validity of the findings: The findings are solid and novel. They fulfil all necessary standards and thus fully merit publication in PeerJ.
Additional comments: L63: add 2with many of its species" after clade
L71: simplified --> lower
L85: Rephrase "advanced adult period onset". Maybe simply add flight after adult or exchange these two words.
L104: Add population after E. epiphron
L110: Rephrase, sounds odd.
Introduction in general: Maybe work out at the end the research question a bit more clearly.
L136: Not only Monts Du Cantal, but at several places (e.g. Puy Marie etc.)
L137: In Fagaras Mts, I also think in Rodna Mts, not sure abaout Retezat
L144: List incomplete; England must be northern England, missing Cantabrian Mts, Massif Central, Vosges, Central Apennines; maybe also mention the strong differentiation at nuclear (Schmitt, T., Hewitt, G.M. & Müller, P. (2006) Disjunct distributions during glacial and interglacial periods in mountain butterflies: Erebia epiphron as an example. Journal of Evolutionary Biology 19: 108-113.) and mt level (Minter, M., Dasmahapatra, K.K., Thomas, C.D., Morecroft, M.D., Tonhasca, A., Schmitt, T., Siozios, S. & Hill, J.K (2020) Past, current, and potential future distributions of unique genetic diversity in a cold‐adapted mountain butterfly. Ecology and Evolution 10: 11155–11168.).
L150: Normally end of June
L259: delta normally needs no "-" as it refers to a difference which is not negative
L344: over --> more than
L356: insulation --> isolation
L358: "higher lower lethal temperatures" sounds really odd; rephrase
L360: insulated --> covered
L368: periods --> flight
L400: indices --> indications
L435: precipitations --> precipitation
Figure 3, 4 and 5: Please add p and r in all cases in the figures.
Table 2: Why best model with negative delta AIC and not 0 with all others being positive?
In addition, I suggest to give the GLM results in one or two additional tables in the main ms.


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: LOW WINTER PRECIPITATION, BUT NOT WARM AUTUMNS AND SPRINGS, THREATENS MOUNTAIN BUTTERFLIES IN MIDDLE-HIGH MOUNTAINS
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors accepted most of the suggestions, properly justifying those they did not. Thus, I consider the paper ready and suitable for publication.


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

Input:
Title: THE STABILITY OF SOIL ORGANIC CARBON ACROSS TOPOGRAPHIES IN A TROPICAL RAINFOREST
Review round: 1
Reviewer: 1

Output:
Basic reporting: English writing should be improved.
physical protection of SOC such as soil texture should be included.
Experimental design: this manuscript is within the aims and scope of the journal.
the research question is meaningful for understanding SOC stability in tropical rainforest.
the research method is suitable for the question and the description of method is sufficient.
Validity of the findings: Conclusions are well stated.
Additional comments: Comments:
This manuscript explored the underlying mechanism of topography effects on chemical fraction of SOC and SOC stability in Jianfengling, Hainan island. The authors found that the chemical fractions proportion of SOC showed the trend of alkyl carbon > O-alkyl carbon > carboxyl carbon > aromatic carbon. The decomposition index were greater than 1 in different topography types. Moreover, the influencing factors of SOC stability were SOC content, soil moisture, and ammonia nitrogen in relative flat area (including flat and slight steep topography) and soil moisture, and ammonia nitrogen in relative steep area (including steep and top topography). Overall, these findings are helpful to understand the mechanism underlying the effects of topography on SOC chemical fractions and stability in tropical rainforest.

The experimental method is suitable for this study and could provide support for the conclusion. The topic is meaningful and provide potential mechanism involved in the effects of topography on SOC chemical fractions and stability. However, the major concern of this manuscript is that the authors only consider the chemical factors including SOC content, ammonia nitrogen and neglect physical protection of SOC including soil texture (sand, loam, clay proportion). I suggest that the authors should add the soil texture data to re-analyze the influencing factors. In addition, the authors should substantially improve English writing and I therefore recommend it for publication after major revision.

Specific comments:
1.	Abstract section. The authors should add results of differences of four different topographies not only in RF and RS.
2.	Line 17, “the chemical fractions of SOC showed …”the meaning is not clear.
3.	Line 20-23. The influencing factors of SOC ….in the relative steep areas. Please  change this sentence into short sentences.
4.	Line 24, Inertia index also higher in Rs than that in RF. This sentence is wrong in grammar.
5.	Line 41-42. Add reference behind this sentence.
6.	Line 57. “Studies found topography influences nutrients availability…” problems in grammar.
7.	In introduction section, the authors should add the effects of physical protection on SOC stability.
8.	Line 92.  Fe3+ and Mn2+
9.	Line 134. Is this classification into two types eligible? Please verify it from references.
10.	Line 156. Should describe the differences of o-alkyl carbon and aromatic carbon content between topography types.
11.	Line 169. Should add “AN and SM explained **% variability of SOC stability in RF areas”
12.	Line 180. “ are characterized by the trend with alkyl carbon…”
13.	Line 199-200. The SOC only affected SOC stability in RF areas might be due to the SOC content in RF areas was significantly higher than that in the RS areas. Is this description contrary to Fig. 2? Fig.2 showed that steep and top had the highest SOC content.
14.	Line 219. “Stubborn” here is not suitable. Change into another word.
15.	Line 224-226. Studies found increase soil water content benefits dissolved organic carbon dissolution….. in organic carbon decomposition. This sentence has grammar problem.
16.	 Line 241. “We hypothesized nutrients be the key influencing factor in study areas was not fully confirmed.” Please paraphrase this sentence.


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 STABILITY OF SOIL ORGANIC CARBON ACROSS TOPOGRAPHIES IN A TROPICAL RAINFOREST
Review round: 1
Reviewer: 2

Output:
Basic reporting: Stable mechanisms of tropical rainforest soil organic carbon were still unclear. In addition, this study is interesting and significant, I recommend reconsideration of this manuscript following minor revision and modification. Furthermore, this manuscript needs a linguistic revision before it is resubmitted and it is suggested that the authors seek some professional help in doing this.
Experimental design: This study analyzed the chemical fractions of SOC by novel 13C CPMAS/NMR technology. Driving factors of SOC stable process were revealed under different topographies.  Experimental design and statistical analysis was correct.
Validity of the findings: Abstract, some abbreviates were not necessary, and it was redundant. “soil was” changed to soils were
Introduction, the second paragraph lacked specific data regarding soil organic carbon fraction and its stability under different topographies. Moreover, this section should add its potential driving factors.
Discussion section, please adding some relative results by 13C CPMAS/NMR technology.
Additional comments: No additional comments


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

Input:
Title: THE STABILITY OF SOIL ORGANIC CARBON ACROSS TOPOGRAPHIES IN A TROPICAL RAINFOREST
Review round: 2
Reviewer: 1

Output:
Basic reporting: literature references should be made carefully.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Comments:
The English writing and the text structure have been improved in the revised manuscript. I have no further comments on this manuscript, but modifications of references citation formation should be made before finally accepted.
The authors should especially pay attention to the references with 3 authors.
Line 53. ChenXu & Mathers 2004
Line 114. ChaudhariSingh & Kundu 2008
Lin3 199. Chapin IIIMatson & Vitousek 2012


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 STABILITY OF SOIL ORGANIC CARBON ACROSS TOPOGRAPHIES IN A TROPICAL RAINFOREST
Review round: 2
Reviewer: 2

Output:
Basic reporting: Thanks to the careful and powerful revises of  authors. I insist that the manuscript could be accepted at this form.
Experimental design: satisfied.
Validity of the findings: satisfied.
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: SC-JNMF: SINGLE-CELL CLUSTERING INTEGRATING MULTIPLE QUANTIFICATION METHODS BASED ON JOINT NON-NEGATIVE MATRIX FACTORIZATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: This paper develops a new method named SC-JNMF for integration of multiple quantification methods to perform stable clustering of scRNA-seq data.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Please see my comments:
1, Authors point out that there are many gene expression quantification pipelines, like cufflink, RSEM, etc, and they provide inconsistent results. But those quantification methods are designed for bulk RNA-seq data and not for single cell data. On scRNA-seq, most research just use the read count instead of FPKM, RPKM, or TPM. It is not clear what is the benefit of using those quantification methods on single cell data.
2,NMF is widely used in single cell clustering analysis. Important citations are missing in introduction:
A.	Shao, Chunxuan, and Thomas Höfer. "Robust classification of single-cell transcriptome data by nonnegative matrix factorization." Bioinformatics 33.2 (2017): 235-242.
B.	Duren, Zhana, et al. "Integrative analysis of single-cell genomics data by coupled nonnegative matrix factorizations." Proceedings of the National Academy of Sciences 115.30 (2018): 7723-7728.
C.	Jin, Suoqin, Lihua Zhang, and Qing Nie. "scAI: an unsupervised approach for the integrative analysis of parallel single-cell transcriptomic and epigenomic profiles." Genome biology 21.1 (2020): 1-19.
3, The update role in Eq. (7-9) looks incorrect to me. Please check the equations carefully. For example, D1 and H are cannot be directly multiped since the dimensions are not matching.


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: SC-JNMF: SINGLE-CELL CLUSTERING INTEGRATING MULTIPLE QUANTIFICATION METHODS BASED ON JOINT NON-NEGATIVE MATRIX FACTORIZATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: see General comments
Experimental design: see General comments
Validity of the findings: see General comments
Additional comments: The authors proposed a joint-NMF based method to integrate expression matrix with different quantification methods and applied join-NMF for cell clustering.

Although the idea of the shared matrix of “modules x samples” is already applied for multimodal data (https://www.nature.com/articles/s41467-020-20430-7 for example), such idea is not applied for multiple expression data with different quantification methods.

Although the integration of such expression data is interesting topic, I have several concerns for publication.

Major comments
1. In my opinion, the experimental design is not appropriate for clustering validation. I think the cell-type (label) of the Segerstolpe dataset is determined with the original expression data, for example. In such a case, it is obvious and not interesting that the ARI values with original expression data (or data with similar quantification methods) are better than those with Salmon and kallisto. Therefore, the conclusion that “ARI tended to be low only when using Salmon as the quantification method” (line.205) is inadequate. Overall, I think this bias makes us difficult to interpret the effects of different quantification methods and different clustering methods.

2. To overcome the above problem, I recommend the authors use the bulk RNA-seq dataset of sorted cell types such as https://dice-database.org/downloads#info_anchor and https://www.sciencedirect.com/science/article/pii/S2211124719300592 for clustering evaluation.

3. The scRNA-seq dataset in this article is not the latest. I recommend that the author add the evaluation with the latest scRNA-seq data because the quality and properties of the data are improving.

4. I have a concern about the merit of joint-NMF. In the current results, I cannot follow the effect of “joint” because the ARI values result from comprehensive procedures. I want to know the impact of “joint”-NMF by comparing the ARI values for such as
- Combining two expression matrices into one matrix, using general NMF and applying the same clustering method.
- Using only one expression matrix, using general NMF, and applying the same clustering method.
- Averaging the expression matrix, using general NMF, and applying the same clustering method.

Minor comments
Line.57: It is inappropriate to say “without mapping” about Salmon and kallisto because these methods roughly map reads to the transcripts with k-mer. It is appropriate to say “without alignment”.

Line.132: The authors say “log2 transformed the data”, but I wonder it produce negative values ?

Line.137: The dimension of D2 and W1 are wrong?

Line.142: The notations such as W_{1k} are confusing because the authors use H^{kj} in the following description. I want the authors to rewrite the united notation.

Line.143: I think lambda_1 is not related to “column vector sparsity regularization”.

Line.180: The reference to the table does not work well.

Line. 188: I cannot follow the detailed clustering setting. For example, I think the authors used FindNeighbors() and FindClusters() of Seurat Clustering, and I want to know the setting and effects of “dims” parameter. In addition, the other clustering method is described in the result section that makes me difficult to follow the result. Please divide the method and result into different sections.


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: SC-JNMF: SINGLE-CELL CLUSTERING INTEGRATING MULTIPLE QUANTIFICATION METHODS BASED ON JOINT NON-NEGATIVE MATRIX FACTORIZATION
Review round: 1
Reviewer: 3

Output:
Basic reporting: The language needs to be improved. page 5 line 159, what is "the ratio of the total of".

Also some terminologies are not standard. The authors used "biased" in many places, where "variation" is more appropriate.  For example, in page 2 line 70, "we need ... that reflect individual cell biases", but the author meant the gene expression variation among individual cells. To refer to a RNASeq dataset, "RNA-seq library" is more commonly used than "RNA-seq reads". In the factor analysis literature, "latent factor" is more common than "latent feature" used by the authors. For example, on page 3 line 108-110, "features" was used in many places in this sentence but they have different meanings. The authors could use "cell-derived factors" instead of "features". "Rank" is more common than "Rank number". On page 6 line 228, the authors could use "factor loadings" instead of "factor values".
Experimental design: On page 4 line 133, the author normalized gene expression by L1 norm instead of L2 norm. The author could explain the rationale of that.

Equation(3), the author should clarify which matrix norm is used for W and H. Also in their experiments, \lambda_2 and \lambda_3 are always set to be zero. So they don't actually need to include these two terms. If they include, they need to show how to select these two hyperparameters.

It's unclear how to derive equations 7-9 from 4-6.

As quantification by STAR has better performance than others, \lambda_1 should have important role on the performance of joint NMF. The authors fixed \lambda_1 to match the number of genes, but it would be interesting to show how the performance varies with \lambda_1.

The author mentioned the sample size can affect the clustering performance (page 7 line 240), but it's unclear from the manuscript in which case their method is more beneficial. The authors could subsample the data set and compare their joint NMF with single NMF varying data size.

The method of choosing the rank is interesting but lacks justification. why the rank when the sparseness hits the elbow point is a good choice.
Validity of the findings: The improvement of joint NMF on two quantification methods compared with only using STAR is not clearly shown. Fig 3 only has aRIs for combination of two methods, it'd better if including the result only using one of the quantification methods and including all three methods.

The author stated that using similar gene expression profiles is worse than combining different gene expression profiles. However, different gene expression profiles have different performance on their own. The reason combining with STAR has better performance is likely because STAR is a better quantification method. Instead of comparing different combinations directly, it makes more sense to compare the performance of each combination with the performance of each individual method. Also line 248 on page 7 is quite confusing.

In figure 5C, several genes have quite different weights in two quantification methods. It would be interesting to provide several examples with further diagnosis.
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: SC-JNMF: SINGLE-CELL CLUSTERING INTEGRATING MULTIPLE QUANTIFICATION METHODS BASED ON JOINT NON-NEGATIVE MATRIX FACTORIZATION
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Authors have solved all my comments, I have no further 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: SC-JNMF: SINGLE-CELL CLUSTERING INTEGRATING MULTIPLE QUANTIFICATION METHODS BASED ON JOINT NON-NEGATIVE MATRIX FACTORIZATION
Review round: 2
Reviewer: 2

Output:
Basic reporting: see comments
Experimental design: see comments
Validity of the findings: see comments
Additional comments: The authors tried to resolve all of my concerns, but I still have a few concerns.

1.	I cannot follow why the authors used Monaco and CellBech dataset only in Fig.2. In my opinion, these datasets will be useful for other analyses (such as Fig.3).
2.	The ARI values of “salmon and kallisto” are lower than those of “kallisto” or “salmon” for the Pollen dataset. In my opinion, it is important to analyze and discuss this case for clarifying the usefulness of “joint” NMF.
3.	It is difficult to see and compare the values of different methods in Fig.5. I want the authors to revise Fig.5 so that the boxplot are arranged for the same dataset and same subsample rate, likewise Fig.3.


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: SC-JNMF: SINGLE-CELL CLUSTERING INTEGRATING MULTIPLE QUANTIFICATION METHODS BASED ON JOINT NON-NEGATIVE MATRIX FACTORIZATION
Review round: 3
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors tried to resolve all of my concerns, and now, I recommend that it be accepted for publication.

I understand it isn't easy to clarify the reason for comment.2, and I hope the authors will find the reason in future 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: DIFFERENTIAL REGULATION OF ANTIOXIDANT ENZYMES IN FRANKLINIELLA OCCIDENTALIS (THYSANOPTERA: THRIPIDAE) EXPOSED TO THERMAL STRESS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors studied the effect or thermal stress on the activity and expression of three enzyme/superfamily: SOD, POD and GST in the invasive species Frankliniella occidentalis. The experimental work performed to meassure enzymatic activity is technically correct. However, the meassurements of gene exprssion present important issues. Please see my comments below.
The more important observation is that GST is not a single enzyme, but an enzymatic superfamily with tens of representatives in each insect genome (see for example Shi et al. Genomics 100 (2012) 327–335. Furthermore, GST superfamily is classified in different clades (Delta, Epsilon, Omega, Theta, Sigma and microsomal clades). Each one of this clades has been associated with different physiological role, particularly in insects. Hence, when authors describe results on “GST gene”; one particular GST gene can be responsible of a small part of GST activity, depending on the process under study. They should explain wich GST gene they are studing, to wich clade it belongs, and why they decided the study of this particular GST from the complete GST repertoire of the species. Otherwise all the results on GST expression cannot be interpreted and are not robust enough to be published.
Lines 98-99: How the sequences where searched in the transcriptome? Wich sequences were used as queries? Details on this experimental part of the work are relevant in order to understand the complete work. Moreover, positive hits on the database search must be reported as complete sequences. Also, information about the database should also be maked public: completeness of the transcriptome, how it was generated (from which tissues? wich life stage? wich sequencing platform was used? provide the basic parameters of the sequencing strategy (long of reads, million of reads generated, assembly strategy, etc.). If the authors prefer to not publish their transcriptomic data, they shoul work with the published genome of the species, but not refer as “unpublished data”, given that all the data used must be published in order to admit reproducibility of the results.
qPCR: a minimum of two house keeping primers is the standard for the technique. Information on efficiency for every qPCR primer must be provided.
Experimental design: as stated above, the transcriptomic and genetic analysis fail to meet the standards
Validity of the findings: werease the enzymatic activity assays could be vaild, the genetic analysis must be revised, as I stated above.
Additional comments: no comments


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

Input:
Title: DIFFERENTIAL REGULATION OF ANTIOXIDANT ENZYMES IN FRANKLINIELLA OCCIDENTALIS (THYSANOPTERA: THRIPIDAE) EXPOSED TO THERMAL STRESS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment.
Experimental design: Methods should be described with sufficient information to be reproducible by another investigator. This manuscript fails at this point. Procedures are referenced to other papers but should be explained here, at least its basics.

The statisitical analyses is where the main failures are evinced. No information regarding how the 4 replicates were taken into account. Clear and detailed information on the ANOVA should be stated. In fact, it seems that a GLMM would be better suited for this analyses to take into account possible effects of the replicates.

Also, the F statistics of the ANOVA have different number of degrees of freedom between the 3 enzymes and it is unclear why.

Survival or mortality results are not reported but mentioned in the experimental design.
Validity of the findings: no comment.
Additional comments: This paper adds relevant information of effects of thermal stress on an agricultural pest. The rationale behind the experiments are clear and the objective fulfilled. However, some decisions on which treatments were used as controls and details on experimental procedures have not been mentioned.

Additionally, mortality results have not been reported. The main flaw of this paper is the poorly detailed statistical analyses. Authors should invest much more effort in detailing the models used, albeit GLMM seem to be the correct approach to take into account the effect of replicates.

In general, this paper is a good report on the dynamics of expression of the enzymes and genes involved in thermal stress. Perhaps more contextual framework would help understanding the importance of the results.


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

Input:
Title: DIFFERENTIAL REGULATION OF ANTIOXIDANT ENZYMES IN FRANKLINIELLA OCCIDENTALIS (THYSANOPTERA: THRIPIDAE) EXPOSED TO THERMAL STRESS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comments
Experimental design: no comments
Validity of the findings: no comments
Additional comments: Unfortunately, the gene-identification procedure is not clear in the revised manuscript. Given that this is a central part for the interpretation and reproducibility of the results, this should be clarified in the work in order to be suitable for publication. Authors should: a) provide complete information about how the database was generated and sequenced (how the transcriptome was assembled, how many million reads where used, paired or single reads, etc); b) make public the raw reads in a public repository such as SRA in NCBI; c) specify how the transcript in the transcriptome where identified (i.e the method used to assign a name for each transcript in the database); d) provide the complete sequences of the transcripts (“genes”) that they are using for primer design. Furthermore, is not clear why the authors speculate that a particular GST is involved in the response to temperature stress; they cannot affirm that this is a delta GST without a phylogenetic analysis.
Lines 109-110: “Expression levels were normalized using reference genes GAPDH and EF-1 for high and low temperature stress, respectively”. As I stated in my previous revision, both house-keeping genes must be used as reference genes for all the determinations (at least two reference genes must be used in qRT-PCRs studies for comparisons on gene expression), not alternatively for different experiments.
I cannot find supplementary file 3.
In conclusion, unfortunately the authors did not address any of the previous requirements that I posed in order to consider the manuscript suitable for publication.


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

Input:
Title: DIFFERENTIAL REGULATION OF ANTIOXIDANT ENZYMES IN FRANKLINIELLA OCCIDENTALIS (THYSANOPTERA: THRIPIDAE) EXPOSED TO THERMAL STRESS
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Although some more detail on the statistical analysis was included, I would like authors to specify if assumptions of the methods were checked. And, if those were not met, how did they managed to analyse them.

Also, they declare in the rebuttal that: " When analyzing the data, if there is an obvious large deviation between one data and the other three data, we will choose to delete this data to ensure the accuracy of the final result."

What would be obvious large deviation? and, more importantly, why would you even discard data. I am uncomfortable with this procedure and would never recommend 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: DIFFERENTIAL REGULATION OF ANTIOXIDANT ENZYMES IN FRANKLINIELLA OCCIDENTALIS (THYSANOPTERA: THRIPIDAE) EXPOSED TO THERMAL STRESS
Review round: 3
Reviewer: 1

Output:
Basic reporting: I really appreciate the effort made by the authors in the improvement of the manuscript; I think that present version is much robust than previous ones in the presentation of results. However, published scientific literature must contain enough information to be replicated by readers, and this is still lacking in the manuscript. I feel that authors are not familiarized with the bioinformatics field, and for this motive this part of the work is weak and must be corrected for publication:
Lines 103-106: Raw reads must be assembled in a transcriptome for database searches: please provide basic information on how this transcriptome was assembled, at least which program was used. Also, I suppose that you performed Blast searches with all the transcripts as queries, to obtain the information provided in Supplementary file 3. Otherwise you could not make searches using key words. The information on how searches were performed to assign a hit for each contign must also be provided. It is not the appropriate way to make database searches by key words; a scientific work should make blast searches that are the correct approach. In Blast searches you can evaluate false positives and false negatives; not all the sequences of interest will be named according to a key word. However, if you identified the sequences by key words (which is not correct but is already done in this work), you must check that they are not false positives, by performing blast searches in other databases (NCBI for example). I have tried to check it for your sequences but they are not still available in Genebank. In the case of GST gene family, you cannot affirm that is a delta GST without a phylogenetic analysis. At best, you can provide the best hit in the Blast search, and inform the level of sequence identity.
Authors inform that the contig 16150 has a hit with glutathione S-transferase D1 [Apis mellifera]. This is not informative without further information on e-values.
Authors write that they used “GST” as key word for database searches. Using this key word a single hit is obtained (Supp file 3) in contig3573, which seems to be a glutathione-s-transferase theta from Aedes aegypti. However, using the key word “glutathione-s-transferase”, you can have several hits. One of this hits is the mentioned glutathione-s-transferase delta from Apis mellifera, but this hit is not obtained using the key word mentioned by authors. Even though I know that this is not the focus of the work, the information provided is not enough, confuse and/or wrong.
In my previous review I posed the following questions: a) provide complete information about how the database was generated and sequenced (how the transcriptome was assembled, how many million reads where used, paired or single reads, etc); b) make public the raw reads in a public repository such as SRA in NCBI; c) specify how the transcript in the transcriptome where identified (i.e the method used to assign a name for each transcript in the database); d) provide the complete sequences of the transcripts (“genes”) that they are using for primer design.
Point a is partially addressed (information lacks on how samples were prepared and how the transcriptome was assembled). Point c has not been addressed; without this information the description of the methods is incomplete and the manuscript is not suitable for publication.

Throughout the text, please take in account that insect species have tens of GST encoding genes; avoid generalizations about “GST”. You are working in the better case with a particular GST gene.
Experimental design: see my comments
Validity of the findings: see my comments
Additional comments: see my 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: DIFFERENTIAL REGULATION OF ANTIOXIDANT ENZYMES IN FRANKLINIELLA OCCIDENTALIS (THYSANOPTERA: THRIPIDAE) EXPOSED TO THERMAL STRESS
Review round: 3
Reviewer: 2

Output:
Basic reporting: Ok.
Experimental design: Ok.
Validity of the findings: Ok.
Additional comments: Thank you for explaining and taking into consideration the suggestions. I would suggest for the future that the authors avoid transforming data and explore the use of GLM and GLMMs for dealing with troublesome data. Also, to try and use other software like R or python for analyzing data.


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: COMPUTED TOMOGRAPHY ELUCIDATES ONTOGENY WITHIN THE BASAL THERAPSID CLADE BIARMOSUCHIA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Please see attached PDF file.
Experimental design: Please see attached PDF file
Validity of the findings: Please see attached PDF file
Additional comments: No additional comments