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: PHYTOBIOMES ARE COMPOSITIONALLY NESTED FROM THE GROUND UP
Review round: 1
Reviewer: 1

Output:
Basic reporting: I commend the authors for their clear and professional English used throughout this article. There are relevant literature cited throughout, although several sections, particularly in the introduction, could include additional citations to support background information given. Specifically, Line 65 (and throughout paragraph) states that other work does has yet to determine processes responsible for microbial assembly of plants, yet there are many publications that the authors cite in the beginning of the introduction that are doing just this; please provide relevant citations. See also Line 72, Line 85-91 (no citations are given).

Raw sequence data is shared through NCBI SRA database. This article is self-contained with relevant community ecological results regarding S. taccada microbiomes. However, their methodology of sampling provides somewhat limited inference on the microbiome, as only swabs were taken, even for soil samples, which may purely be from microbial propagule deposition non reflective of the actual endophytic microbiome (this latter approach would require tissue collection and laboratory sterilization). See additional details regarding these issues, among others, in the next section.
Experimental design: The research is within the Aims and Scope of PeerJ. Research questions were well-defined in the Introduction and certainly meaningful in terms of better understanding how microbial communities assemble in a tropical, native plant species. However, there are significant flaws not only in experimental design and sampling, but also in describing molecular methodologies used to prepare and analyze sequence libraries as described below:

1. Plant microbiomes, particularly with high-throughput applications readily available now (at least more so than in the recent past), in light of understanding below- and aboveground communities should be investigated by surface-sterilizing plant compartments (roots - stems - leaves, etc.; in other words, microbes that lie within tissue) and targeting gDNA within tissue. This study only includes swabbing the outer tissue which is going to be potentially primarily reflective of microbial deposition and to a lesser degree, actual differences in the microbiome among compartments. This seems to be demonstrated in the MDS with somewhat large overlap between some compartments that typically are much more differentiated (leaf versus rhizosphere/soil). This may reflective of (i) actual microbiome differences, (ii) an artifact of sampling by swabbing or (iii) low sequence yields (1000 seqs/sample) not providing enough data to differentiate communities.

2. There is relatively no information regarding 16S rRNA gene library preparation and no citations to accompany this section. More information regarding PCR reaction conditions are needed (or cite relevant papers which use such conditions) as well as MiSeq sequencing procedures (PhiX spike?). Since the authors used 2x300 cycles, they should have recovered better yields per sample if prepared adequately.

3. It is not clear how certain bioinformatic steps were completed. Line 162-163 states average quality scores declined after 150 bp and then "not further considered." Does this refer to trimming sequences, and if so, how did the authors do this (e.g., via QIIME, cut adapt, some other program)? Using a V3 kit, sequence quality shouldn't drop off at 150 bp one would think. Furthermore, did the authors investigate other contaminants besides chloroplasts (i.e., mitochondria, unclassified at Domain level) and did they include Archaea? What reference databases were used for alignment and taxonomy? If qiime default parameters were used, it is assumed greengenes, but this needs to be stated clearly.

4. Lastly, 1,000 sequence depth is very low for below ground samples. Assuming there is better sequence yield for more diverse and high biomass samples (rhizosphere, soils), perhaps the authors can, in addition to comparing all samples,  separate out compartments (below ground vs aboveground) to achieve better yields and have a more accurate picture of soil communities. 1,000 sequences is not enough to describe vast diversity found in soils and may not be enough for aboveground communities as well. A rarefaction curve of all plant parts can answer this latter question.
Validity of the findings: Due to issues with methodologies as provided currently, I am not providing any comments regarding findings as I am uncertain whether they may be an accurate reflection of S. taccada microbial communities.
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: PHYTOBIOMES ARE COMPOSITIONALLY NESTED FROM THE GROUND UP
Review round: 1
Reviewer: 2

Output:
Basic reporting: OVERVIEW

This manuscript examines how microbes are distributed within and between plants. Focusing on the beach shrub Scaevola taccada on the island of Oʻahu, it finds that geographic distance accounts for little variation in the composition of plant-associated microbial communities, but that part of the plant does (e.g., rhizosphere versus flowers). More specifically, it finds that microbial communities in plants are nested from the ground up, with microbial communities in flowers being subsets of microbial communities in the rhizosphere. A model of sequential colonization is proposed to account for this nested pattern.

COMMENTS

I enjoyed reading this paper and look forward to seeing it published. It is clearly written, and addresses a pertinent question using an interesting data set. It has only one substantial shortcoming, which should be relatively easy to address, that the citation of existing literature on nestedness is inadequate. In a general ecological context, there is an extensive literature on how to measure nestedness and on its interpretation. Much more of this literature should be cited both in the methods (e.g., the recent papers on partitioning beta-diversity into nestedness and turnover components), and in the discussion in interpreting the causes of the patterns that are reported (e.g., ordered extinctions as an alternative to ordered colonizations). Furthermore, in a microbial context, Thompson et al (Nature, 2017, doi:10.1038/nature24621) recently reported that on a global scale, plant microbiomes tend to be nested -- for instance, as shown here, it reported that plant-associated communities tend to be nested within rhizosphere communities. That said, although some results are similar, this work clearly adds to the results reported in Thompson et al (2017) and hence, the present manuscript just needs to discuss its findings in the context of the previous findings. Additional literature on the nestedness of microbiomes also exists (e.g., Smith et al, 2018, https://doi.org/10.1038/s41396-018-0086-0) and should also be discussed.
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: PHYTOBIOMES ARE COMPOSITIONALLY NESTED FROM THE GROUND UP
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
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: PHYTOBIOMES ARE COMPOSITIONALLY NESTED FROM THE GROUND UP
Review round: 2
Reviewer: 2

Output:
Basic reporting: Pass
Experimental design: Pass
Validity of the findings: Pass
Additional comments: My concerns regarding the initial version of this manuscript have been adequately addressed and I will be happy to see it published.


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

Input:
Title: IDENTIFICATION OF FOUR HUB GENES ASSOCIATED WITH ADRENOCORTICAL CARCINOMA PROGRESSION BY WGCNA
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: In order to replicability of the related results of the study, R scripts should be uploaded as a supplement file.
Validity of the findings: The conclusion is welcome, but should be verified by molecular biology experiment such as quantitative real-time PCR, western blot. Otherwise, the result is not persuasive.
Additional comments: Authors used public databases (TCGA and GTEx) to identify four hub genes (TOP2A, TTK, CHEK1, and CENPA) associated with adrenocortical carcinoma progression by weighted gene co-expression network analysis. However, some questions needs to be pointed out.
1. The manuscript needs polishing, which contributes to improve readability of the manuscript.
2. Results of the present study were only based on mRNA level, lacked confirmatory experiments including quantitative real-time PCR, western blot. Thus, the current result was not persuasive.
3. In order to replicability of the related results of the study,  R scripts should be uploaded as a supplement file. Furthermore, version numbers of bioinformatics tools should be annotated.


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

Input:
Title: IDENTIFICATION OF FOUR HUB GENES ASSOCIATED WITH ADRENOCORTICAL CARCINOMA PROGRESSION BY WGCNA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The overall structure of the manuscript is well-organized, but there are several issues authors should address.
1.	Figure 1A legend: “fold-changes” should be replaced with “log2 fold-changes”. “lines” should be replaced with “dashed lines”.
2.	Figure 1C: the heat map should be better labeled.
3.	Figure 2: the sequence of each sub-panel is inconsistent with the other figures.
4.	Supplemental Table 1: Sheet 1 and 2 should be better labeled.
5.	Some references in the introduction are not appropriate. Line 45 – 46: Authors should use primary sources. Line 47: Authors’ claim is not substantiated in the referenced article. Line 56: Referenced article is on small non-coding RNAs and it should not be seen as the traditional method of identifying functional genes.
6.  Language should be improved  for easier comprehension. Examples include Lines 41-42, 46-47, 86-88, 198-200, where the current phrasing is difficult to understand.
Experimental design: In general, the rationale and methodology seem appropriate, but there are still some issues.
1.	Authors did not clarify what specific types of data they obtained from TCGA and GTEx (Level 1 to Level 3).
2.	If Level 3 data was obtained, the gene expression level from TCGA and GTEx cohorts would not be directly comparable, due to the use of different analysis pipelines. It is necessary to have a unifying pipeline in analyzing the raw sequencing data from the two cohorts (Qingguo et al. https://www.nature.com/articles/sdata201861).
3.	The method authors used to identify differentially expressed genes is not very robust. I recommend a validation of DEGs using EdgeR (http://bioconductor.org/packages/edgeR/) or DESeq2 (http://bioconductor.org/packages/DESeq2/). Detailed information can be seen in the relevant papers.
Validity of the findings: The conclusion authors drew is based on the data they presented. However, the validation of the data is necessary.
1.	I recommend qPCR validation of the 4 hub genes identified in ACC cell lines. Of course, analyzing human primary samples would be more satisfactory.
2.	It will also be helpful if authors could analyze data from other public datasets to confirm the differential expression of the 4 hub genes.
Additional comments: The manuscript is a computational analysis of RNA-seq data in ACC, using tumor samples from TCGA and normal samples from GTEx. Authors described (i) a list of differentially expressed genes in ACC vs normal samples, (ii) 9 gene modules identified by WGCNA, (iii) 4 hub genes associated with ACC progression. Overall, the manuscript is well-organized in a professional manner and provides novel aspect in the progression of ACC. However, a number of issues about the writing and methods (as mentioned above) must be addressed before acceptance.


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

Input:
Title: IDENTIFICATION OF FOUR HUB GENES ASSOCIATED WITH ADRENOCORTICAL CARCINOMA PROGRESSION BY WGCNA
Review round: 1
Reviewer: 3

Output:
Basic reporting: Adrenocortical carcinoma is a vicious and aggressive cancer, so every new study is quite important.
The work done by the researchers here is particularly interesting and well written. The reader easily follows the progress of the study and I find that the whole is very educational. The data is made available, so it's a very successful search.
The introduction is well balanced highlighting the crucial data of the field, without being too boring to read.
The materials and methods are correct, but it would be good to highlight the choice of different parameters.
Results section is well designed and I like to follow each step of the study.
Unfortunately, I find the figures difficult to read.
Figures 1C, 1G and 1H for example are unusable. Similarly, the figures of Fig 2B, the labels of 2E and 3B are too small. It would be good to improve them.
Experimental design: The methodology used is correct and interesting.
An important point is to discuss the various parameters used.
Thus, I see why 2.5 as TPM, but it would be more detailed this choice for the reader, also estimate (give) the implication that would have given a choice with a stronger value (or weaker).
Similarly, the choice of aov must be discussed.
An underlying question is the sensitivity of modules to the choice of DEGs.
Validity of the findings: As said, "most are expressed at extremely low levels", but is it also experimentation (laboratory) dependent? Variations in the provenance of these data must be discussed. It is unclear whether standardization for example is general or by initial experience.
"Average linkage hierarchical" is a classic approach, but would not Ward have been more relevant?

“KEGG pathway analysis, the genes were mainly enriched in cell cycle and DNA replication (Fig. 2F, Table S5) These processes and pathways all play critical roles in cancer progression (Tachibana et al., 2015), implying that genes in this module may be involved in ACC progression. " Can you be more specific and tell us how to achieve this result quantitatively. What statistical test and under which hypothesis can you conclude?

"The top 5% of genes (16 genes) with confidence" were chosen as "hubs for PPI analysis" (Fig. 3A, Table S6). But how is this choice more relevant than 10% or 1%? Similarly, if you remove one of these hubs, is the set still stable or is it terribly sensitive to given hubs?
Additional comments: As a reviewer, I like reading articles that allow me to follow the path of the authors, which is perfectly the case. There is a real choice to search for genes in a supervised way, and I trust the results found. However, it lacks a little criticism on the choices made and especially the influence of the parameters. These corrected points will make this article an excellent article.


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

Input:
Title: IDENTIFICATION OF FOUR HUB GENES ASSOCIATED WITH ADRENOCORTICAL CARCINOMA PROGRESSION BY WGCNA
Review round: 2
Reviewer: 1

Output:
Basic reporting: There are literature references with sufficient field background/context provided; the article is professionally done with appropriate figs, tables and sufficient data and script shared. It is self-contained with relevant results to hypotheses.
Experimental design: it is rigorous and well done, it can be reproduced if needed.
Validity of the findings: in regards to state of art, it is robust and statistically sound and controlled.
Additional comments: The author answered all my questions and we incorporated these modifications to the manuscript, which can be published from my point of view as it stands.


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

Input:
Title: IDENTIFICATION OF FOUR HUB GENES ASSOCIATED WITH ADRENOCORTICAL CARCINOMA PROGRESSION BY WGCNA
Review round: 2
Reviewer: 2

Output:
Basic reporting: The work is very much interesting one. I have found that majority of queries in my mind already raised by three reviewers and authors already provided their responses which are quite satisfactory. So, present form can be accepted now.
Experimental design: Satisfactory
Validity of the findings: Acceptable
Additional comments: The work is very much interesting one. I have found that majority of queries in my mind already raised by three reviewers and authors already provided their responses which are quite satisfactory. So, present form can be accepted now.


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

Input:
Title: IDENTIFICATION OF FOUR HUB GENES ASSOCIATED WITH ADRENOCORTICAL CARCINOMA PROGRESSION BY WGCNA
Review round: 2
Reviewer: 3

Output:
Basic reporting: Clear and unambiguous, professional English used throughout.
Experimental design: The study is well designed, carried out properly, and technically and statistically sound.
Validity of the findings: The results are comprehensible , conclusive and reliable.
Additional comments: By weighted gene correlation network analysis (WGCNA), Wang-Xiao Xia. et al attempted to identified four hub genes associated with adrenocortical carcinoma progression. They found that the hub genes (TOP2A, TTK, CHEK1, and CENPA)  were highly expressed in ACC and negatively correlated with OS. Thus, these identified genes may play important roles in the progression of ACC and serve as potential biomarkers for future diagnosis. The study is well designed, carried out properly, and technically and statistically sound. The results are comprehensible , conclusive and reliable.


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

Input:
Title: IDENTIFICATION OF FOUR HUB GENES ASSOCIATED WITH ADRENOCORTICAL CARCINOMA PROGRESSION BY WGCNA
Review round: 2
Reviewer: 4

Output:
Basic reporting: The authors use an approache based purely on mRNA levels to identify genes related with ACC. They perform differential expression assuming that the Xena database (the repository from which the case/control datasets were obtained) correctly armonizes mRNA from two different datasets. Can we assume that? This is crucial for the paper to be credible.

They follow up with co-expression networks (WGCNA methodology) and PPI to identify 4 genes involved in ACC and negatively correlated with survival.
Experimental design: Line 58 systematics biology -> systems biology

What are those GTEx control samples, where do they come from? I mean, version of GTEx gene expression, tissue, age and gender

In line 77 they say ACC and GTEx data sets were integrated into a single pipeline. But there are no details about that one. Since the paper is about detecting differentially expressed/connected genes, this part of the pipeline is precisely the crucial one and should be clearly detailed, at least in a supplementary. The fact that they mention the Xena database is not enough. They should describe how this is done and make the point clear that both datasets can be compare because of that because this approach may be full of different biases: e.g. project, sequencing technology, softwares for producing the TPM quantification… Genes being quantified in the same units (TPM) does not necessarily imply they can be put together. If no enough details are given on how they integrated both data sources, I wouldn’t believe what is found there at least in terms of differential expression. In summary, the paper has to give more details about how the DDBBs are integrated and processed together. It will make the paper much more credible.

The authors claim that WGCNA is a package from Bioconductor but is not, it is from CRAN. Maybe the authors are referring to WGCNA tailored to TGCCA.

Line 147: the significance of the blue module being higher (I suppose the authors are actually saying that the p-value is lowest). However, lower p-values, although they are correlated with the magnitude of the correlation, it does not necessarily mean that correlation with blue module will be the highest. Anyway, the authors should report the correlation in that line. The authors do not report how they calculate gene significance and other parameters. The title of figure 2.A is incomplete, did not mention ACC.

The number of modules is suspiciously low. There are many grey genes and this was not properly addressed in the paper. Genes in the grey module do not have high correlation between them but are there because they shoudn’t be in any of other modules. This should be discussed.

Line 164, what is confidence?

It is not clear the role of PPI in the whole analysis further than the generation of nice plots. What is the conclusion drawn from this particular analysis and how it combines with co-expression networks? This should be either clarified or removed in the case it says nothing about involvement of the genes in ACC progression.
Validity of the findings: Is the paper showing something new? They themselves cite other papers that lead to the same conclusions, i.e. the four genes selected from the blue module are also seen as differentially expressed in other papers. Lines from 207 to 223 reinforce what I am saying as well.

The authors keep saying that the four genes contribute to the progression of ACC but this is not necessarily true. Because one can not affirm whether these genes are upstream or downstream. There is no causality anchor and the genes may be over expressed as a consequence of ACC. So instead of genes contributing to ACC progression, ACC progression may contribute to over expression of the genes. The genes are involved in ACC progression, this is certain but further conclusions are not realistic. The authors should put some effort in finding the causal anchor. And this is only possible when the genetics & environment information come into play.
Additional comments: The paper may be useful for the community but I doubt it is saying something not know already, and there are some pitfalls in the design that should be properly addressed.


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

Input:
Title: IDENTIFICATION OF FOUR HUB GENES ASSOCIATED WITH ADRENOCORTICAL CARCINOMA PROGRESSION BY WGCNA
Review round: 2
Reviewer: 5

Output:
Basic reporting: The manuscript is well written. Authors handled the gene dataset in an interesting way which would have a clinical impact and more in-depth analysis on the mechanisms underlying different phenotypes.
The abstract is well structured and concise.
Experimental design: Introduction:
First mentioned as abbreviation MEblue module in line 67? (module eigengene E(blue) of the blue module)

Differentiation between the two peaks (childhood and middle age) of the disease in the analysis would be preferential to pick the biological and mechanistic difference between them.
Validity of the findings: "The correlation between gene expression and stage was determined using GEPIA": screen shot please the option of clinical data availability in that link provided.

First 5 lines of result section is repetitive and should be restricted in the methodology.

Fig. 1C is so faint and thus unreadable.
Fig 2C is referred to the info "MEblue and MEyellow modules were highly correlated with clinical stage" and that is not clear in the corresponding figure.

PPI section in the results: line 162-169 is unclear what exactly was done.
Additional comments: No commrnts


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

Input:
Title: IDENTIFICATION OF FOUR HUB GENES ASSOCIATED WITH ADRENOCORTICAL CARCINOMA PROGRESSION BY WGCNA
Review round: 3
Reviewer: 1

Output:
Basic reporting: Reviewer comments were addressed.
Experimental design: Reviewer comments were addressed.
Validity of the findings: Reviewer comments were addressed.
Additional comments: Reviewer comments were addressed.


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

Input:
Title: ACUTE TOXICITY AND RESPONSES OF ANTIOXIDANT SYSTEMS TO DIBUTYL PHTHALATE IN NEONATE AND ADULT DAPHNIA MAGNA
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: Experiment is designed properly.
Validity of the findings: No comment
Additional comments: I think the manuscript is edited properly and it should be considered for publication. However, author should clarify one point regarding  Daphnia culture.
It is mentioned that a single Daphnia was used to get progeny. How is it possible to get enough synchronized progeny from a single Daphnia in a single day?
It should be clarified how the experiment was setup.


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: ACUTE TOXICITY AND RESPONSES OF ANTIOXIDANT SYSTEMS TO DIBUTYL PHTHALATE IN NEONATE AND ADULT DAPHNIA MAGNA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript was written well, in clear English. Reference is updated and clearly reported the current background on teh investigated topic. Manuscript structure is ok, the data have been shared and are usable to repeat the analyses.
Experimental design: The research fits the aim of the Journal. The experiment is interesting, the tested molecule is of concern and limited information on its toxicity is curretly available. The experiment was performed well. As suggested by the reviewers of the first submission, the authors replicated the experiments to include a t=0 value that in the former manuscript missed. Adding the t=0 baseline data strenghten the results of the paper.

I suggest to add some brief results on the stability of DBP in water, although these data are reported in many previous papers.

I have a little concern about the paragraph oxidative stress status. Oxidative stress raises when oxidative status is unbalanced. So oxidative stress status in my opinion has no sense. I suggest to change the title of the paragraph in materials and metods section as: Oxidative stress assays (biomarkers). Moreover, in the next paragraph I suggest to include T-AOC because this assay measures the non-enzymatic antioxidant capacity. I have also a concern regarding the inclusion of H2O2 and T-AOC results in oxidative damage paragraph. The alteration on these parameters (i.e. the unbalance of the equilibrium between ROS and antioxidant in favor to the former) induces the oxidative stress, leading to oxidative damage. Thus, ROS and non-enzymatic antioxidant measurements cannot be considered as damage.

The applied statistical approach is correct (two-way ANOVA). However, I do not understand why you used Excel. What kind of analyses did you performe with Excel?
Validity of the findings: The results are interesting, data are robust (the number of experimental replicates is appropriate). Interpretation of results is correct and conclusions are generally based on results.

I have only one concern: both in the introduction and in discussion you stated that your results can help to find accurate biomarker to investigate DBP response in environment. In my opinion this is not possible because the suite of biomarker you applied is made by generalistic assays that respond to a plethora of contaminants. Thus, in environment, although you find an alteration in oxidative stress assays you cannot state that this effect was due to DBP rather than other pollutants. I should consider with care this issue within the manuscript.
Additional comments: Please check the common name of the species you referred within the text. Some common name is missing.

Please specify in the figure captions the meaning of capital (or lowercase) letters above the histograms.

Did you check (and report) for the time x treatment interaction effect?

Please include in the result section a Table reporting all the results of the statistical analysis, showing statistical indicator (F), degrees of freedom and P value. The P value is not enough. Alternatively, you can also report the information of statistics within the text (within brackets).


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: ACUTE TOXICITY AND RESPONSES OF ANTIOXIDANT SYSTEMS TO DIBUTYL PHTHALATE IN NEONATE AND ADULT DAPHNIA MAGNA
Review round: 1
Reviewer: 3

Output:
Basic reporting: The article is clear and mostly unambiguous, however there are certain grammatical typos and language issues particularly in the results section (see specific comments below).   Intro and background seem to be sufficient with the exception that the authors do not cite enough of the previous work done with phthalates and Daphnia.  The novelty of this work is not the acute toxicity but is the linking of the acute toxicity data with the anti oxidant systems.   The authors should stress this rather than the toxicity.  The figures are relevant, good quality and sufficiently labelled and described however, some improvement is necessary for clarity.
Experimental design: The primary research is within the scope of the journal, the research question is well defined.   The  relevant and meaningfulness of this data increases our knowledge of effects phthalates, however the authors need to state a little more on how this will be used.   The explanation of the research gap needs some additions.   The research seems be done with reasonable technical and ethical standards.

The methods are mostly described in sufficient details and information to replicate,  See specific comments below.
Validity of the findings: The findings appear meaningful, and the data appear to be robust statistically sound and robust.
The  Conclusions are well stated, linked to original research question & limited to
supporting results.   However the authors do not comment significantly on the Safe Concentration calculation in conclusion.
Additional comments: General comments
Introduction looks good.
Material and Methods:
The material and methods are good, but I would like a little more detail on each of the anti-oxidant methods as that is a very important part of this work.
Results:
Almost all first sentences of results paragraphs need to be rewritten or deleted. A few specific comments are below and indicated on PDF comments.
Line 255 – “To assed”  should be corrected
Line 264 -265 – sentence sounds like part of the intro.    Rather just report what was done.
Line 281  Line should be removed. (any sentences that just says  “data was shown in figure ()” as its main info should be deleted.)  See PDF

Line 392-395  See comment on PDF.

Table 1.  Add column of how many trials and how many replicates per trial.

Figures: Please explain what comparisons are being made.  It can be interpreted that the 24 h were compared with the 48 hours as stated now, but that does not look like it is the case.
Figure 1
A and B should have same y AXIS
Figure 2 – Explanation of the severe difference between adults and Neonates needs to be explained.
Figure 3
A and B should have same y AXIS
Figure 4
A and B should have same y AXIS
Figure 5
A and B should have same y AXIS
Figure 6
A and B should have same y AXIS


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: CIRCADIAN DISRUPTION AND DIVERGENT MICROBIOTA ACQUISITION UNDER EXTENDED PHOTOPERIOD REGIMENS IN CHICKEN
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is well written, but one sentence "This study is also the first to report differential microbiota acquisition under different photoperiod regimes" in the abstract should be modified  since this report is not the first paper to disclose this phenomenon.
Experimental design: Overall is good. But the last part for the comparison between cecal content and feces is somewhat unrelavant with other parts. The authors should address this point.
Validity of the findings: Data is clear, but I strongly recommend the authors to add new parameters comparison for PCoA to further reveal the difference between N and E (e.g. the PC1 distance for each sample in these 2 groups). The current data is not clear enough to show the overall difference between N and E.
Additional comments: I think this paper is potential interesting but the authors should address all the above points before publishing.


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: CIRCADIAN DISRUPTION AND DIVERGENT MICROBIOTA ACQUISITION UNDER EXTENDED PHOTOPERIOD REGIMENS IN CHICKEN
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English is clear. The article is overall well written.
The Background is sufficient and maybe slightly long regarding the introduction. The references are appropriates.
The structure of the article is standard and professional.
Most results presented are of significance to the hypothesis.
Experimental design: The paper reports original primary research within aims and scope of the journal. The research questions is well defined and meaningful with investigation performed to a high technical and ethical standard. Methods were described with sufficient detail and information to replicate. However, the number of samples taken at each time point (n =2) is too low to ensure the validity of the results.
Validity of the findings: The validity of the results is difficult to assess due to the low number of replicates although rigorous methods are applied.
Additional comments: The paper describe the effect of light time exposure on chicken circadian rhythm and its effect on the cecal microbiome. This is a very interesting study design and the questions raised by the paper are of real interest to the scientific community. However, the authors are encouraged to at least double the number of samples taken at each time point to be able to draw any conclusion from the results and assess the significance of their findings.


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: CIRCADIAN DISRUPTION AND DIVERGENT MICROBIOTA ACQUISITION UNDER EXTENDED PHOTOPERIOD REGIMENS IN CHICKEN
Review round: 1
Reviewer: 3

Output:
Basic reporting: The structure is as required for submission to PeerJ. The subheadings for the discussion are not needed. The introduction is clear and thorough although there are some vague statements that need clarification (line 136 and 149). The addition of reference to previous studies on healthy chicken gut microbiota would strengthen the introduction.
There are a number of sentences in the results section that should be moved (if not already stated) from the results to the methods or discussion section (see annotated PDF), as they give details of the methodology or discuss what the results may mean (respectively).
A number of figures need formatting so that the results are clearer to the reader. Figure 1 has axis labels and splitting the figure into and A, B, C and D (per row) would clarify what photoperiod or sample is being shown. There is no clarification of acronyms used for samples in Figure 2. The lefse output in figure 5 is only shown at family level and a number of families are repeated more than once. Would it not be more informative to show the lefse output with all bacterial groups shown at all phylogenetic levels of classification? There is no rationale in the methods why only family is shown in this figure.
Experimental design: This study is within and the design aims to answer a novel question that has not been explored before. However, aim D states “to determine if fecal microbiota is representative of cecal microbiota” (in the abstract). This is not novel as this previous publications have commented on the similarity of fecal and cecal microbiota representing the cecal microbiota eg. Stanley et al. 2015 BMC Microbiology (DOI 10.1186/s12866-015-0388-6). There is a reference to this work already being published on line 549, if this question does need re-addressing then justification for this should be added to the manuscript. When this aim is stated later in the manuscript, in the introduction, “early life cecal microbiota” is mentioned. If this aim D is to assess whether fecal microbiota is representative of cecal microbiota, solely in young birds, this needs to be clarified and justification to why this may be different to adult birds added.
The paragraph in the methods describing the QPCR undertaken (line 225) does not mention any use of housekeeping genes as controls or state the sequence of the primers used.
Validity of the findings: In the results section it is not clear what defines if oscillation is present or absent in the clock genes studied using QPCR. Statistical testing should be added to show that the lack of oscillation in Per2 is statistically significant.
There is scope for more comparison between the results here and previous chicken microbiota papers in the discussion section. Comparison between the finding here in chickens subjected to different photoperiods and normal or diseased chickens (for example Shaufi et al. 2015 Gut Pathogens and Wei et al. 2013 Poultry Science) would strengthen the discussion section and give better insight into the potential implications of these findings to the poultry industry. In the paragraph beginning line 481, but as this is more a reflection of the normal light periods chickens would be exposed to with no artificial light would it not be more useful to compare the results of the normal photoperiod to the cecal and fecal microbiota previously reported in disease free chickens? The importance of the poultry industry and the reasoning behind different photoperiods in the industry were included in the introduction section. However, this had not been commented on in the discussion. Suggestions on how the results reported here may affect the growth and/or egg laying of chickens and any potential changes that would be needed to be made to current photoperiod regimes would strengthen the discussion.
Additional comments: There is the need for clarification of which photoperiod and/or sample type is being described in a number of sentences in the results section (lines 302 and 340).
The inclusion of figure 2 but at phyla, class, order and family level to supplementary material would give a more complete overview of the fecal and cecal profiles of the samples analysed.


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: CIRCADIAN DISRUPTION AND DIVERGENT MICROBIOTA ACQUISITION UNDER EXTENDED PHOTOPERIOD REGIMENS IN CHICKEN
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: Information on the QPCR methods have been added as requested. Could the primers used be added in a table to supplementary items? Clarity on the novelty of the aim assessing the differences between fecal and cecal microbiota has been added. The final conclusions paragraph (line 580) be used to emphasise how this part of your study is different to the references mentioned.
Validity of the findings: Paragraphs beginning lines 483 and 500 discuss the differences in microbiota seen between the two different phot regimes. These paragraphs include lots of references that have found similar findings to the results of the bacterial community profiling of the two groups of chickens. Could you end each of these paragraphs with a sentence linking this directly back to your study.
Additional comments: The authors have taken on my suggestions and there has been great improvement in the manuscript since the first draft submitted for review. The figures are much clearer and the results section is easier to follow and flows better. I have a few minor suggestions that can be found below a few comments. I believe that if these are addressed the manuscript will be ready for publication.
Line 220: Could you add the primer sequences in a table to supplementary items?
Line 356: Could a figure be added to the supplementary items to complement this description of dominant phyla in the two photoperiods.
Lines 377 and 401: are these references to Figure 5, specific to part A or B?
Line 430: Could a figure be added to supplementary items to illustrate the comparison of fecal and cecal microbiota mentioned here?
Paragraph beginning line 472: This paragraph discusses bacterial diversity and community profiles, could this paragraph solely concentrate on bacterial diversity and include differences in alpha diversity seen.
Line 540: state what these changes you found were to remind the reader of what is being discussed.


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

Input:
Title: CIRCADIAN DISRUPTION AND DIVERGENT MICROBIOTA ACQUISITION UNDER EXTENDED PHOTOPERIOD REGIMENS IN CHICKEN
Review round: 2
Reviewer: 2

Output:
Basic reporting: Generally clear and professionally written manuscript.

As the study focuses on the early life of birds, it is important that the age of the birds is mentioned in the abstract.

From reading the introduction, I made the assumption that the study was performed on broiler chickens; It is not until the start of the materials and methods that I realised this was performed on layer hens. Can it be clearer in the abstract (and title) that this was done in newly hatched laying hens to better inform the reader from the start.

Some inconsistencies in materials and methods on reporting manufacturer of reagents used, for example line 208: "RNAlater" (no further details), vs line 236-237: "Q5 High-Fidelity DNA polymerase (NEBNext High-Fidelity 2X PCR Master Mix, New England BioLabs, Ipswich, MA"

Fig. 1 and Fig. 2 legends: "Error bars are standard errors" - but this is a box-plot.

Good quality of tables and figures presented, although Table 1 would be better placed as supplementary materials.

Line 443: the term "flora" should be avoided.

Some inconsistencies in abbreviation used NP/EP
Experimental design: Original piece of research addressing gap in knowledge.

The study aims to answer a number of relevant scientific questions to the industry.

The main concern lies over the number of replicates with only 2 cages used per treatment (20 birds per cage). It seems that birds were considered as the replicate instead of the cage. Co-housed birds would not be statistically independent, and it could be argued that the effect observed is due to cage or room effect rather than the photoperiod effect.

Good level of details provided in materials and methods.

Line 184: As a strong influencing factor on the microbiome, and to enable comparison with future studies (or replication): basic details of the feed used should be stated.

Line 186; "following the producer manual", this is too unclear.

Line 235: as the gold standard practice, was a bead-beating step used prior to DNA extraction?

For sequencing: did you use a mock community standard and a negative blank (from the DNA extraction)?
Validity of the findings: Interesting findings, well reported and discussed in the context of existing literature.

Level of Firmicutes reported is very high (90-94%), is this comparable to similar studies for birds this age?

Line 461: the term "jetlag" could be misinterpreted by reader and should be defined more clearly

Line 532-534:  "final weights of birds raised in either photoperiod were not significantly different, which shows that the differences observed in microbiota composition was not due to differences in feeding behaviors."  this cannot be concluded from bird weight alone as feed intake was not recorded or observed.

A major limitation of the comparison between faecal vs caecal microbiota is related to the sample collection method used where it is not possible to identify which faeces corresponds to each individual bird from a tray collection. This should be also addressed in the discussion section.
Additional comments: It is a real shame that no data regrading the bird's performance in terms of bird weight, feed intake and feed conversion ratio were reported in this study (weight was briefly mentioned towards the end of the discussion). In order for this study to have a significant impact in the industry, this would have been a critical factor to investigate.
I understand this was beyond the scope of the current studies, but a suggestion for future studies on a larger scale.


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: RECOGNIZING TWO NEW HIPPOLYTE SPECIES (DECAPODA, CARIDEA, HIPPOLYTIDAE) FROM THE SOUTH CHINA SEA BASED ON INTEGRATIVE TAXONOMY
Review round: 1
Reviewer: 1

Output:
Basic reporting: (1) Clear, unambiguous, professional English
language used throughout.

NO. With the exception of the description, the English is quite poor. This is the major problem with this paper. See General comments for the authors.

(2) Intro & background to show context.
Literature well referenced & relevant.

YES

(3) Structure conforms to PeerJ standards,
discipline norm, or improved for clarity.

YES

(4) Figures are relevant, high quality, well
labelled & described.

YES

(5) Raw data supplied (as required by PeerJ policy).

YES
Experimental design: (1) Original primary research within Scope of
the journal.

I think so.

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

YES

(3) Rigorous investigation performed to a
high technical & ethical standard.

YES

(4) Methods described with sufficient detail &
information to replicate.

YES
Validity of the findings: No problems found. This is a good classical and modern taxonomic paper, where everything has been done properly.
Additional comments: (1) general scientific review

This paper is a valuable and significant contribution to the taxonomic knowledge of Indo-Pacific Hippolyte species. The authors use a modern integrative approach and the scientific quality of the paper is good. The interpretations and analyses of the paper are usually correct. The structure of the paper is adequate and the style is usually clear. In other words, it is a good modern classical taxonomical paper (not highly original but scientifically well made).
It certainly deserves publication (after language improvement). Whether PeerJ is the appropriate journal or not should be double-checked by the editor. However I think it is.

I would add a key or a character table for Indo-pacific Hippolyte of the "group ventricosa" (to which your two new species do belong). That key or table do not have to be too elaborate (as more species will probably be discovered). For example, problematic taxa like the cryptic species currently confused under the name Hippolyte ventricosa could be pooled together as "Hippolyte ventricosa sensu lato". At this stage, even a very basic table/key would be of considerable help for other carcinologists working on that difficult shrimp  group.

The line drawings are correctly executed.

On the plates, the photographs are sometimes weakly contrasted (a problem often found with raw photographs of crustacean appendages). Usual imaging softwares have functions that can be used for increasing the contrasts of such structures. With Adobe Photoshop, the most useful function is Shadow/Highlight. I imagine that GIMP has an equivalent function but I am not familiar with that software. I would recommend to the authors to test such functions and, if possible, to submit photographs with increased contrasts in the second version of the manuscript. This is not absolutely necessary but this would be highly preferable.

(If the authors intend to publish photographs in further papers, I would recommend them to test stacking photography, which considerably improves pictures when the depth field is small. Even manual stacking photography would improve photographs of the kind you intend to publish. This does not require a high budget)

There are a few typographical errors in the text and on the cladogram (Italics, space,...). The authors should check that.

(2) Language issue. I have the regret to say that the overall quality of the English is quite poor (except for the descriptions where it is more or less OK). This is the weakest point of this paper (and actually its only weak point). I provide language suggestions for the descriptions only in the line by line comments — but not for the rest of the text, as this is not my job of referee, and as I am not myself an English native speaker. I understand that English is not easy for Chinese authors (I guess that the first author is a young scientist with still little experience; the second author is an experienced scientist), but the next version of the manuscript MUST be checked by an English native speaker with a good knowledge in zoology and marine biology (whether it is re-submitted to PeerJ or to another journal).

(3) Comments line by line.

Line 37–38 "Hippolyte Leach, 1814 are commonly perching in the seaweed of tropical and subtropical oceans"
Not all Hippolyte species are associated with seaweeds. Some are obligatory or facultative symbionts of other organisms. The genus Hippolyte is not only found in tropical/subtropical seas but also in temperate seas. Hippolyte varians has been recorded just above the Arctic circle in Norway (d'udekem d'Acoz 2007).

Line 133 Large-sized shrimp of Hippolyte
I would not say "large-sized". There are much larger species in the genus. For example Hippolyte inermis can reach at least 6 mm CL.

Line 185 "wide, third segment 2.06–2.12 times". use "subsegment" as elsewhere.

Line 2010 "pindling". I am not an English native speaker and that's the first time I see that term. Check with the person who will revise your English that this term is appropriate.

Line 229. "Middle-sized shrimp of Hippolyte". Again, the concepts of large, medium-sized or small are very subjective (I would have said small). Maybe it is better not to refer to size at the beginning of the descriptions.

Line 229. "Outline soft". "Soft" is not the appropriate term. Do you mean robust?

Line 233. "distal teeth in female specimens". "Distal teeth" should become "subdistal tooth".

Line 237: "knoblike" should become "knob-like".

Line 249. I think that "middle-length" should become "mid length".

Line 251. "samll". Do you mean "samll" ?

Line 260. "pudgy palp". I don't think that "pudgy" is the appropriate term.

Line 261 "in low". Do you mean "in low extremity" ?

Line 265. "dactylar segment arch". Do you mean "dactylar segment arched" ?

Line 267. "mid-lenght" should become "mid-length"

Line 271. "mid-lenght" should become "mid-length"

Line 271 "slightly reaching to". Do you mean "just reaching", or "slightly overreaching"?

Line 280. "segment" should become "subsegment".

Lines 280-281 "with tiny setula and long simple setae". I see long distal setae on your drawing, but no "tiny setula". What do you mean by "tiny setula"?

Lines 336-339. " Our specimens of Hippolyte chacei sp. nov. were captured among Sargassum sp. together with Hippolyte cf ventricosa. This may indicate that the two species occupy similar ecological niche, but the ratio of prisal nearly reached to 1:8. We speculate that H. chacei sp. nov. probably remain a predicament in the interspecific competition."

It is quite difficult to explain the difference of frequencies between Hippolyte species in Sargassum beds. Several hypotheses are possible. Here are a few examples.  (1) The one proposed by the authors is correct: it is simply rarer. (2) The main habitat of H. chacei is not Sargassum (Sargassum would only be an accessory substrate). (3) H. chacei would have preference for deeper waters and only low densities of specimens would occur at the depths sampled by the authors. (4) H. chacei would prefer environment with a stronger or less strong wave exposure than on the sampling site. (5) Its very high number of teeth on the incisor process suggests a trophic niche distinct from that of other species. this might also play a role in the frequency and micro-distribution of the species.

Line 377. " During the biodiversity surveys". Please be a bit more explicit about these surveys.

Line 377. Hippolyte cf ventricosa. Have you looked at the following characters: number of teeth on mandibular palp; ratio length/width of scaphocerite; number of teeth on fingers of pereiopods 1–2; proportions of the subsegments of pereiopod 2 ? Perhaps will you find differences with the syntypes on these characters. This is just a suggestion.

Lines 403-405 " And in the future, a new taxon established based on integrative datum,eg morphological data, genetic data, and ecological data and so on, will be more valuable and credible."
Personally I would suppress this sentence.

The figures 2 and 11a (colour photographs) are the same. I suggest to suppress the figure 2.

Figure 6. The scale bar is oblique. It should be parallel with the upper and lower margin of the plate.


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: RECOGNIZING TWO NEW HIPPOLYTE SPECIES (DECAPODA, CARIDEA, HIPPOLYTIDAE) FROM THE SOUTH CHINA SEA BASED ON INTEGRATIVE TAXONOMY
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This study comes from an interesting integrative multispecies analysis that combined ecological and morphological traits with genetic data to describe new species from South China sea. The information is original and as far as I know have not been published before. The title is appropriate and concise and the abstract is representative of the content. The text is well organized and clear, most of the interpretations are justified and the discussion and conclusions are supported by the results and the conclusions are correct. However, I found some points that should be checked by the authors before publication.

I´m happy to see new species described based on integrative analysis (morphology and molecules), since this is a new and good science. However, be careful about inferring phylogenetic relationships from a single gene. The 16Smt is very informative, but in some cases its limitations for resolving relationships should be cautiously told, especially for a group that show biological diversity as the Hippolyte members. This is not a really problem, but be cautious. Another aspect that deserves revision are some photos and drawings, whose quality is bad and needs an improvement.

I have made additional suggestions/comments directly in the text designed to improve the manuscript and that should be checked by the authors.

In conclusion, I consider that the present work is suitable for publication after these revisions, which in my opinion do not question the scientific quality of this work, but just the manner to present some 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: HOXC6 PROMOTES MIGRATION, INVASION AND PROLIFERATION OF ESOPHAGEAL SQUAMOUS CELL CARCINOMA CELLS VIA MODULATING EXPRESSION OF GENES INVOLVED IN MALIGNANT PHENOTYPES
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: To investigating the role of HOXC6 in esophageal squamous cell carcinoma (ESCC), authors examined the expression of HOXC6 in ESCC tissues by immunohistochemistry, quantitative real-time PCR and immunoblotting assays, and found that  HOXC6 was highly expressed in ESCC tissues. Furthermore, HOXC6 overexpression promoted the migration, invasion, and proliferation of both Eca109 and TE10 cells. Further bioinformatics analysis showed that HOXC6 activated several crucial genes implicated in the malignant phenotype of cancer cells. These results were meaningful and would contribute to understanding of ESCC pathogenesis. However, there is a question as follows.
1. In order to  enhance reproducibility and allow for new discoveries, raw RNA-seq data should be uploaded to public database, such as Sequence Read Archive (SRA). Accession number should be supplied in 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: HOXC6 PROMOTES MIGRATION, INVASION AND PROLIFERATION OF ESOPHAGEAL SQUAMOUS CELL CARCINOMA CELLS VIA MODULATING EXPRESSION OF GENES INVOLVED IN MALIGNANT PHENOTYPES
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this manuscript, Tang Li et al., aim to demonstrate a function for the HOXC6 protein in esophageal squamous cell carcinoma (ESCC). Overall, the manuscript is well structured, and provides sufficient evidence to suggest a potential proto-oncogenic for HOXC6 in ESCC. A few minor revisions could help strengthen the quality of the manuscript:

-In figure 1, the authors should provide more detail on the precise tumor data set used. Specifically, is the “expression” of HOXC6 only considering mRNA levels, or also taking into account copy number alterations? Additionally, none of the y axis on the blots are labelled, making it harder to interpret the data

-In figure 2C, do each of the columns in the bar graph represent a single patient derived tumor sample? If so, this should be specified in the legend.
Experimental design: - One of the more intriguing experimental issues with this study is the choice of studying the effect of HOXC6 overexpression in ESCC cell lines. Given that ESCC is associated with higher expression of HOXC6 than normal to begin with (as demonstrated in figure 2), presumably studying the effect of knocking down HOXC6 would be more meaningful. While the overexpression data seems convincing, it would be helpful to know if the authors performed knockdown experiments in the same cell lines, even if the results were not conclusive

-In the wound healing assays (figure 4), it is unclear what metric is exactly being measured in the graphs in panels C and D. Specifically, is this the width of the wound gap, or the recovery width? These values are inversely correlated. The images would indicate it is the latter, but this needs to be clearly specified by the authors
Validity of the findings: Overall, I believe the data presented in this manuscript support the conclusions made by the authors. However, the authors should avoid using broad language that may over interpret the data. For example, in line 194, the authors state that “HOXC6 expression is positively correlated with the progression of ESCC”. Drawing such a conclusion would require a time-course study of the expression of HOXC6 during progression of ESCC. A single snapshot of expression data is not sufficient to make conclusions regarding progression of the tumor.
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: HOXC6 PROMOTES MIGRATION, INVASION AND PROLIFERATION OF ESOPHAGEAL SQUAMOUS CELL CARCINOMA CELLS VIA MODULATING EXPRESSION OF GENES INVOLVED IN MALIGNANT PHENOTYPES
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript #32037 by Tang et. al. use established cell lines and tissue samples and experimental methods including RNA-seq to show that HOXC6 promotes migration, invasion and proliferation of esophageal squamous cell carcinoma (ESCC). The study is not novel, however, the authors repeat the results in this carcinoma cells and establish it.

Figure legend should be made more descriptive. Number of experiment (N) should be mentioned for each graph in the figure legend.

Human participant consent form is in a language other than English, probably in Chinese.
Experimental design: The authors use ESCC lines - Eca-109 and TE-10 throughout the study however comparison to normal (non-carcinoma cells) is missing. I suggest that the authors compare their findings in Eca-109 and TE-10 with control cell line -Het-1A (ATCC).

Authors validate the role of HOXC6 by overexpressing it in Eca-109 and TE-10. However, since these are carcinoma cell line and have already alleviated levels of HOXC6. Hence a comparison with Het-1A (ATCC) is a must and also knock down of HOXC6 will further support the results.

The authors mention about 20 similarly regulated genes (17- upregulated and 3 down regulated) upon HOXC6 overexpression in the two cell line.  These should be provided as a table. Table 3 mentions some of these but the full data should be provided.

Authors should check the cell to cell HOXC6 overexpression variability in the two cell lines. The difference could arise due to different expression level of HOXC6. HOXC6 should be immunostained and intensity per cell from multiple cells should be compared between ECa-109 and TE-10 overexpression cell lines.
Validity of the findings: In fig 1, the source and paper for each data should be explicitly mentioned either in methods section or figure legend. Details of the bioinformatics analysis should also be provided.

In fig2C, how was the histogram obtained. The authors should mention it in the figure legend.

In fig2D, the band size should be mentioned next to bands and quantification of western should be carried out and plotted in a figure.

In fig3. qRT-PCR and western should be carried out for control proteins like vinculin or any housekeeping gene.

In, Fig. 4C, 4D, 5C, 5D, 6A (both graphs), significance should be tested for all time points not just the last time point. Wherever the two curves become significantly different, the time point should be mentioned in the graph or in the figure legend.

In fig 6B (right graph), it appears that OD450 value for TE10-NEO (red curve) was carried out just once. There is no error bar.
Additional comments: Is there any repository for RNA -seq data? Link should be mentioned in the paper.


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

Input:
Title: HOXC6 PROMOTES MIGRATION, INVASION AND PROLIFERATION OF ESOPHAGEAL SQUAMOUS CELL CARCINOMA CELLS VIA MODULATING EXPRESSION OF GENES INVOLVED IN MALIGNANT PHENOTYPES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The author's have addressed my comments, and I believe the article is suitable for publication. I have no further comments
Experimental design: The author's have addressed my comments, and I believe the article is suitable for publication. I have no further comments
Validity of the findings: The author's have addressed my comments, and I believe the article is suitable for publication. I have no further comments
Additional comments: No additional comments


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

Input:
Title: HOXC6 PROMOTES MIGRATION, INVASION AND PROLIFERATION OF ESOPHAGEAL SQUAMOUS CELL CARCINOMA CELLS VIA MODULATING EXPRESSION OF GENES INVOLVED IN MALIGNANT PHENOTYPES
Review round: 2
Reviewer: 2

Output:
Basic reporting: No
Experimental design: No
Validity of the findings: No
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: EFFECTS OF HABITAT FRAGMENTATION AND HUMAN DISTURBANCE ON THE POPULATION DYNAMICS OF THE YUNNAN SNUB-NOSED MONKEY FROM 1994 TO 2016
Review round: 1
Reviewer: 1

Output:
Basic reporting: See in general comments
Experimental design: See in general comments
Validity of the findings: See in general comments
Additional comments: This study reports the population size and distribution of the endangered Yunnan snub-nosed monkey, Rhinopithecus bieti, for a period from 1994 to 2016. In this period the population size increased from less than 2000 to about 3000 individuals. According to the authors, this was mainly due to establishing protected habitats. The authors conducted a study that combined census data from different subpopulations in addition to a genetic analysis to determine the abundances and genetic diversity of this metapopulation. Overall, this paper is interesting, well written and relevant, and should be of interest to the readers of PeerJ and other experts in the field.
The one major criticism I have is that the whole aspect of gathering and analyzing genetic information on the 11 subpopulations is grossly neglected. It is unclear whether this was previously published (referred to Liu et al. 2007, 2009) or whether this was new data that was analyzed according to Liu et al. If new information is reported, then this section needs a much more thorough description, including the methods of gathering and analyzing genetic data. If no new data is reported, then this has to be clearly indicated as well as the reasoning why this information was included in this paper.
After reading this paper, the first thought that struck me was that this was a clear example of a successful conservation effort. Clearly there are still concerns about the genetic diversity and increasing human population encroaching on monkey habitats, but it appears that the conservation measures put in place were successful and the population increased by about 50% in 20 years. Surprisingly, there was no real acknowledgement of this success. I don’t think this is any major concern, but I found it a bit curious.
In line with previous comment, I found the first section of the Introduction somewhat dramatic, I don’t think that your general audience of ecologists and conservation biologists need a lot of convincing that biodiversity loss is an issue. I do agree that losing many species is a concern to all of us, but many recent analyses provide a more measured overview of biodiversity loss. Just for scholarly duty, I would recommend for authors to have a look at some of the work by Maria Dornelas or Brian McGill.
Some minor comments:
Ln 210 – negatively instead of negativity
Ln 227-229 – I found it very interesting to learn that lichen is such an important diet item of R. bieti!
Ln 230-235 – As you have not dealt with disease consequences in your study, I think this section is a bit irrelevant. This could be summarized in one sentence, it does not need a whole section.
Figure 2 – in Ln 38 you state that the fragmentation index is between 0 and 1, yet in Figure 2 it goes all the way to 5. Why this discrepancy? Also, based on the shape of your data, I am not convinced that a linear model is the best choice, have you tried fitting different models? Something more similar to an exponentially declining model could fit better and is consistent with other ecological phenomena. Suggest to either transform the data or use another model for a better representation of the decline in growth rates with the fragmentation index.


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

Input:
Title: EFFECTS OF HABITAT FRAGMENTATION AND HUMAN DISTURBANCE ON THE POPULATION DYNAMICS OF THE YUNNAN SNUB-NOSED MONKEY FROM 1994 TO 2016
Review round: 1
Reviewer: 2

Output:
Basic reporting: The reporting of the manuscript is good with some interesting results. However, much more to be done to help improving the clarity of the manuscript. Figures are relevant.
Experimental design: Research questions/objectives should be revised and more clarified with respective methods. The genetics part of the data seems not original and if so, it should be included in the discussion section.
Validity of the findings: The findings are partly good and useful to help conserving Yunnan snub-nosed monkey.
Additional comments: This manuscript aims to evaluate the effects of habitat fragmentation and human disturbance on the population dynamics of the Yunnan snub-nosed monkey is quite good with some interesting results. However, I have the following suggestions for improving the clarity and validity of the manuscript.

Abstract:
1) I recommend authors to restructure the abstract in to four sections as background, methods, results and discussion. The abstract in its form lacks basic information to representing the manuscript.
2) The method section is missing in the abstract section. Delete GLMs from the keywords list.

Introduction:
1) I recommend eliminating the genetics objective, as the authors have no genetic data in the manuscript. However, the authors can use previous research results in the discussion section elsewhere.
2) Line 77: Please replace “Threatened” with “Vulnerable” as “Threatened” stands for Vulnerable, Endangered and Critically Endangered status.
3) The aim of the study in the abstract section (Lines 28-29) and introduction section (Lines 96-98) are conflicting each other. The objective of the study should be clear.
4) The authors should focus on the data they have at hand and previous studies should be included in the discussion section unless the aim of the manuscript is a review paper.

Methods:
1) Line 109: It would be clearer if the authors start a new paragraph here.
2) The authors should clarify the method used for population count. How many time each subpopulation/group counted/censused? Why? Results should be presented in the result section.
3) Line 110 and 111. Please clarify or correct the word “censured”
4) Line 123-125: It seems that the authors have no original genetic data. If so, you could not include in the method section as well as in the results section. However, the authors could use previous studies in the discussion section.  I recommend deleting the genetics part from method and result sections.
5) Line 126-151: Landscape and spatial data analysis. I suggest the authors to include “landscape and spatial data analysis” in their objectives to make it clearer.
6) The authors should elaborate how fragment index could be calculated. The definition for “fragment index” should be revised with supportive scientific references.
7) Line 165-167: I suggest deleting the sentence about genetic diversity.
8) Line 176-177: Delete the sentence and discuss this result in the discussion section when necessary.
9) Line 180-181: Delete the sentence .. “Overall, our results …” please also discuss this result in the discussion section.

Discussion:
1) The discussion need restructuring, avoid repetitions and need revision corresponding to your results. It would be great to focus on the interpretation, and implications of your findings. Discuss the effects of habitat fragmentation on the objectives of your study and implications. In the discussion, the authors noted that the effect of habitat fragmentation and degradation causes population decline in the introduction/abstract. However, the population of Yunnan snub-nosed monkey increased in number in some sites and extirpated in other sites. Discuss what it implies clearly.
2) Avoid unnecessary information that belongs to the description of the state-of-the-art of the research topic as they belong to the Introduction. For instance, paragraph 1 of the discussion section.
3) Line 236-246: Please revise this paragraph according to your result.
4) I suggest to include one concluding paragraph at the end of the discussion section.


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

Input:
Title: EFFECTS OF HABITAT FRAGMENTATION AND HUMAN DISTURBANCE ON THE POPULATION DYNAMICS OF THE YUNNAN SNUB-NOSED MONKEY FROM 1994 TO 2016
Review round: 1
Reviewer: 3

Output:
Basic reporting: I have put all comments in the "General comments for the author" section
Experimental design: I have put all comments in the "General comments for the author" section
Validity of the findings: I have put all comments in the "General comments for the author" section
Additional comments: The paper presents a rare case of reassessment of a critically endangered species over a period of 22 years. It should be a good contribution to the study of primate conservation. While the data are highly valuable, the presentation needs some modest level of revision, especially in clarifying some key issues and measurements before it can be published. Additionally, a potential problem as I feel (I may be wrong) that the Discussion section reads rather detached from their results. A tighter connection with the Results section will make the paper stronger and more focused.

The following are the details that I find may need some work. Please pay attention to the issues that I have made substantial comments. Hope they are of some help to their revision.

Lines 35, 135-139: quantifying fragmentation has been attempted many times. (E.g., Kevin R. Crooks 2017. Quantification of habitat fragmentation reveals extinction risk in terrestrial mammals. PNAS.) The authors present a new way of indexing fragmentation. However, they do not provide reasons for doing this. Nor do they provide details for how to do so. I find that in the way as they described (Line 137), I can’t get to the value range of 0-1. A formula may clarify this issue instantaneously. Also, the name “fragment index” is awkward. A better one may be “fragmentation index,” as they presented in Figure 2.

Lines 47-48: Some of the keywords appear to be too general (e.g., “population dynamics, subpopulation growth rates, GLMs; landscape”). Consider deleting them or replacing them with more relevant alternatives. This will increase the probability of hit when readers search for relevant articles.

Line 80: “slow” may be replaced with “long.”

Line 90-91: add a coma after “activities” and replace “has” with “have.”

Line 101-103. The sentence may read better after breaking it into two: change “…Autonomous Region, which is …” to “…Autonomous Region. It is…”

Line 110: change “censured” to “censused” or “surveyed.”

Line 121: Consider simplifying “In order to monitor” to “To monitor.”

Lines 124-125: Please provide information for what exactly was measured and how. This relates to Table 1, in which the authors provided two measurements of genetic diversity. Apparently, they measured the level of heterozygosity but they didn’t provide details about how this was done and why the information was important.

Line 135: strike out the species name “(Abies georgei)” which is not “dark-coniferous forest.”

Line 141: strike out “defined as” – redundant typo. Also, provide the minimal width of pasture land included in the analysis. (2000 meters is only the length.)

Line 153: It may be better to define the meaning of “population” in this study in the Methods section and mark out the names of the populations on the map. This will help readers understand where the populations are.

Line 161: “R. bieti” (missing a dot after “R”)

Line 162: add “and” before “Moziping.”

Lines 179-180: Hard to understand the statistical results as they are presented. Please also provide the p-values. Additionally, it may be better to develop more the meaning of the modeling results in the Discussion section. As it is written, these results are barely mentioned in the Discussion section.

Line 187: “severely” may be replaced with “effectively.”

Line 198: strike out “of” in “collecting of forest products.” Change “domesticated animal grazing within…” to “grazing by domesticated animals within…” Also “domesticated animals” may be replaced by “livestock.”

Line 203: add “and” before “Moziping.” Consider changing “In part this has been…” to “This has been in part …”

Line 237-239: The statement that “it still faces a decrease in genetic diversity …” can be potentially confusing. In principle, when a population is extremely small, the bottleneck effect for the loss of genetic diversity is inevitable with or without habitat fragmentation, the latter of which can only exacerbate (or speed up) the loss of genetic diversity.


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: ADAPTATION OF METAL AND ANTIBIOTIC RESISTANT TRAITS IN NOVEL Β-PROTEOBACTERIUM ACHROMOBACTER XYLOSOXIDANS BHW-15
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is well prepared, language is fine, methods are well presented and results are discussed and from my point of view conclusions are justified.
Experimental design: Well conducted and reported.
Validity of the findings: Results are presented and discussed properly. It seems to me robust.
Additional comments: I only have minor comments which should be addressed and hopefully improve the manuscript.
Overall, this work will gain attention by specialist but also by general readers from various sectors as microbiology and omics next to environmental sciences.

Minor comments:
- correct in abstract: “genome. Synteny”
- style of references need to be streamlined in the text
- Line 146 to 150; the sentence could be rephrased to be better readable.
- Line 213: “A.” italics style
Line 222: “P450” correct
Line 302: “ phage regions … “ correct
 Fig 5. Needs some improvement as the network is hard to figure out; maybe the color code and designations can be optimized.
- Suppl. Material files (both) are needed for the manuscript. They are fine.


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: ADAPTATION OF METAL AND ANTIBIOTIC RESISTANT TRAITS IN NOVEL Β-PROTEOBACTERIUM ACHROMOBACTER XYLOSOXIDANS BHW-15
Review round: 1
Reviewer: 2

Output:
Basic reporting: - The english is poor. There are many serious errors in grammar and spelling. Many sentences are difficult to understand in the manuscript. Some corrections are given below, but the text should be checked again.

- References and background are sufficient.

- The article structure is professional and raw data is shared.

- Results are relevant.
Experimental design: - The research is within the scope of the journal.

- The research question is well defined and a knowledge gap is filled (a new strain is sequenced).

- The research was well conducted, but some important details are missing :

1) Line 121-122. What is the name of the reference sequence? Check the accession number given in the text (no results were found on NCBI).

2) Figure 1 : it is said in the text, line 159, that 2049 contigs were obtained. But Fig 1 presents one circular chromosome. Was the genome of strain BHW-15 closed or not? Is it the reference with the 2049 contigs mapped on it? If yes, were all the gaps filled? This is not clear and this point must be clarified. Moreover, in Figure 1, the colors for the prophage types (red, gray and green) are not visible on the circular chromosome.
Validity of the findings: - The discussion concludes that strain is metal-resistant because 35 metal resistance genes were detected in the genome. However, genes are not always expressed or functional and no data is provided concerning the Minimal Inhibitory Concentration for metals in the strain (Cu, Zn, As, Cr, etc.). Before concluding that strain BHW-15 is metal resistant the strain should be cultivated on media with different concentrations of metals, and compared to control strains cultivated on the same media. Alternatively, resistance proteins may be detected by proteomics.
Additional comments: The manuscript is interesting and should be published as it describes the genome of a new bacterial strain. However, in its present state it is not clear in the manuscript if the genome was completely closed or not. In addition, the metal resistance phenotype of the strain (MIC) should be determined before publication (antibiotic resistance was determined, but not metal resistance). Finally, the english should be revised and some corrections are given below.

Minor points :
- Replace "Achromobactor" by "Achromobacter" in the title of the manuscript.
- Introduction, line 55. Replace "it" by "them".
- Line 57 : bad english, rephrase.
- Line 59. "Heavy metals" is a meaningless term. The term "metals" should be used in the text (Duffus et al. 2002. Pure Appl. Chem., Vol. 74, No. 5, pp. 793–807).
- Line 60 : replace "maintain" by "maintains".
- Line 65, bad english. Remove the word "rather" and rephrase.
- Line 70 : replace "are directly polluting the water" by "may contaminate water".
- Lines 79-82. These two sentences are hard to understand. What are the "non-antibiotic agents"?? Please rephrase and explain.
- Line 98. "l" should be replaced by "L" everywhere in the text.
- Line 103 : replace "of brown" by "of a brown".
- Line 103 : replace "harborage of" by "the presence of an".
- Line 107 : to confirm the presence of what?
- Line 144 : What medium was used? What was the size of the discs?
- Line 149 : replace "inferring" by "referring".
- Line 158 : replace " xylosoxidnas" by "xylosoxidans".
- Line 161 : What is "In RAST by RASTtk"? Have you used RAST or RASTtk? Please give a reference for RASTtk.
- Line 176 : replace "arsenic oxidizing" by "arsenite oxidizing".
- Line 177 : replace "arsenic reducing" by "arsenate reducing".
- Line 182 : replace "transporter phosphate" by "phosphate transporter".
- Line 194 : "distiction". Do you mean "distinction"? Also remove the word "high".
- Line 201 : replace "In vitro..." by "Using in vitro..."; Replace "test" by "tests".
- Line 205 : replace "contains" by "contained".
- Line 208 : replace "this" by "these".
- Line 220 : replace "Terpinoids" by "terpenoids".
- Line 251-252 : what are "other high arsenic converting bacteria"? Do you mean other bacteria able to metabolize arsenite and arsenate?
- Line 255 : replace "genomic possession" by "genes".
- Line 267 : Tn21 : please give more details on that transposon (a drawing?)
- Line 305 : replace "facilitated" by "facilitate".
- Line 306 : replace "bacteria" by "bacterium".
- Line 307 : what do you mean by "this two resistance"?
- Line 325 : "It can inferred"... ? Rephrase. Do you mean "It can be inferred"?
- Line 326 : "to thrive in ease"?

- Table 2 : replace "aiS" by "aioS".
- Figure 4 (A) : explain in the legend why two red arrows are represented for aioA in strain BHW-15 (two aioA genes in different overlapping reading frames?).


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: ADAPTATION OF METAL AND ANTIBIOTIC RESISTANT TRAITS IN NOVEL Β-PROTEOBACTERIUM ACHROMOBACTER XYLOSOXIDANS BHW-15
Review round: 1
Reviewer: 3

Output:
Basic reporting: 1.1. The English phrasing, grammar and language should be improved to make sure that the audience understand the message. I suggest rephrasing lines 56-57, 201-202, 217-224, 283-285, 322-325. I suggest paying attention to grammar mistakes such as verb and subject agreement (for example, lines 115, 126,215, 252), missing “s” (ex. lines 181, 236, 248, 252, 258, 299) and italic words (for example, in vitro).  I suggest reconsidering the use of the word “heavy” for heavy metals as this notion is controversial.
1.2. I would recommend the authors to improve the field background by (i) explaining why they investigated groundwaters and bringing relevant information concerning metal and antibiotic contamination of this kind of environment. (ii) reference this background with more recent and appropriated literature.
1.3. Figures should be improved to display sufficient resolution to permit the audience to read text on the figure. It includes Figure 1, Figure 3. Besides, Supplemental file 1 contains a table with to column that are difficult to understand and appreciate due to its untidy structure. Supplemental File 2 is titled “Supplemental file 4”.
1.4. Parts of the discussion lack references such as in lines 297-299, 299-305 or 333-336.
Experimental design: 2.1. For a better answer to the research question, I would recommend authors to provide information from the literature or determine in the present study the characteristics of waters from which the strain was isolated, including metal and antibiotic contents.
2.2. I would suggest providing more information about the growth medium, KMnO3 and AgNo3 screening principles to permit the audience to perform that kind of analysis (line 96)
2.3. No information is provided concerning the network analysis network (Figure 5) nor adequate discussion concerning these results.
2.4.  The research question should be restructured to make sure that the audience understand the aim of the study. In other words, I would insist to say that this study aims to genomic basis to understand antibiotic and metal cross-resistance in Achromobactor xylosoxidans BHW-15 in further experiments.
Validity of the findings: 3.1. Discussion should be enriched and explore links between the other strains of Achromobactor xylosoxidans and its metal and antibiotic resistance potential. I would recommend, for instance,
Jakobsen, T.H., Hansen, M.A., Jensen, P.Ø., Hansen, L., Riber, L., Cockburn, A., et al. (2013) Complete Genome Sequence of the Cystic Fibrosis Pathogen Achromobacter xylosoxidans NH44784-1996 Complies with Important Pathogenic Phenotypes. PLoS One 8: 8–11.
Amoureux, L., Bador, J., Fardeheb, S., Mabille, C., Couchot, C., Massip, C., et al. (2013) Detection of Achromobacter xylosoxidans in hospital, domestic, and outdoor environmental samples and comparison with human clinical isolates. Appl. Environ. Microbiol. 79: 7142–7149.
3.2. Linked with the research question, authors should bring clues to complete these results and decipher cross-resistance mechanisms
Additional comments: The study provides relevant information to decipher the link between metal and antibiotic resistance. It provides robust sequencing raw data and relevant analysis of the genome of Achromobactor xylosoxidans BHW-15. If there are weaknesses, these are the technically incorrect English as mentioned above, the insufficient resolution of figures, the lack of references in the discussion and information concerning antibiotic and metal contents in groundwater ecosystems
 I would also reorganize the discussion to start with general findings and end with specific findings.


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: ADAPTATION OF METAL AND ANTIBIOTIC RESISTANT TRAITS IN NOVEL Β-PROTEOBACTERIUM ACHROMOBACTER XYLOSOXIDANS BHW-15
Review round: 2
Reviewer: 1

Output:
Basic reporting: The revised manuscript improved significantly with respect to language and presentation. A proper number of references have been used and as far as I can judge these cover the field of research in a good way. The article is well prepared and structured thus specialist and general reader can easily follow and take the messages in a good order.

Thus I have no reservation about the style and content.
Experimental design: The design of experiments is appropriate for this study aiming to investigate the drawn hypothesis of As contaminated sites and microbial responses on differnet organisation levels.

Methods are detailed described and I have the feeling they can be reproduced.
Validity of the findings: As mentioned above the work is solid!

The results are clear and easy to follow and it adds novelty to the field.

The experiments were proper to gain these results and this allowed to draw a number of conclusions - to my optinion the correct one have been suggested and stated. Here additional references were discussed.

It seems appropriate in terms of length and content.
Additional comments: The authors improved the manuscript quite a lot and the presentation is now more clear. It will be interesting to follow this topic as I think there is room to identify more As resistance correlations - it would be interesting to even study more in detail the antibiotica resistance and occurence of (heavy) metal realted issues.