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: PATIENT-REPORTED FATIGUE IN PATIENTS WITH RHEUMATOID ARTHRITIS WHO COMMENCE BIOLOGIC THERAPY: A LONGITUDINAL STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is generally well written, but could still be improved, and some sentences need clarification:
In lines 124-125 you write: "In a previous study of both self-reported and physician reported data,…". I assume that it was the outcomes that were studied, not the data, and suggest that you re-write this sentence.
In general, I would prefer the concept patient-reported outcomes (PROMs) rather than self-reported. You will find this in many publications.
Moreover, you use both physician-reported and health personnel-reported data. You refer to DAS28 as “The most common physician-reported measure”. As far as I can see, it was a nurse that collected the clinical data in your study. Maybe you could use one term throughout the manuscript to avoid confusion?
In line 132 you state that “One study found higher prevalence of RA in women than men”. The gender difference in prevalence of RA is well-documented in many studies. I suppose that this is not your point here?
Study design: The setting for the study is not reported. I assume that it was a rheumatology specialist department?
Ethical considerations: How were the patients informed? Oral and/or written information?
Minor comment: The in text citations do not consequently follow the required reference style (3 Authors et al....)
Experimental design: Inclusion criteria: Could the patients consent to participate in the main study and decline participation in your study, or did they consent to participate in both studies at the same time?
Assessments: Does the FSS have a defined cut-off for severe fatigue?
Do you know if the psychometric properties of the instruments have been tested in Norwegian populations?
Lines 196-198: You refer to DAS28 as physician-reported data. To my knowledge DAS28 also includes Patient Global Assessment of Health (PGA), see for example https://www.nras.org.uk/the-das28-score. Did you include that?
Your second aim was to identify possible predictors for change in fatigue. Did you have any hypotheses that guided your selection of possible predictors?
In the abstract (lines 35-36) you state that you performed bivariate and multivariate regression analyses. In line 205 in the Methods section you state that you have used “Linear mixed effect analyses to identify associations between change in fatigue level and clinical variables, such as…” I suggest that you reformulate this sentence to “…analyses were used to identify possible predictors of…” to make clear that these analyses are related to your second aim. Moreover, I suggest that you use predictors when you refer to the results of these analyses in the Discussion.
Validity of the findings: The results are sufficiently reported in tables and text and are consistent with other studies of fatigue in patients with RA.
In line 239, you write that you "did not observe any effect of DAS28 on fatigue". I guess you mean that DAS28 was not a significant predictor of fatigue, i.e. change in fatigue was not explained by disease activity?
You might also have a look at a study by Olsen, C. et al Arthritis Care an Research 2016, which is related study With similar results in Norwegian RA patients.
Discussion, line 268: I do not think that “Patient reported versus reports by health professionals” is a subheading that covers the content written below. Neither is it related to the aims of the study. I suggest deleting this subheading and rather using the two aims as subheadings. Moreover, I miss a description of the strengths of the study.
Line 299: I do not think that the reference to a study of fibromyalgia patients is relevant here as your study focus on patients who commence biologic therapy.
Line  315: The sentence “… as the Norwegian population is rather homogeneous” could be supported by a reference, for example to studies of other RA populations in Norway. I am not sure if the population in general is rather homogeneous.
In the last paragraph, you might as well refer to the study of a group cognitive behavioural therapy program for fatigue by Hewlett et al 2011 and a Norwegian mindfulness-based programme by Zangi et al 2012.
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: PATIENT-REPORTED FATIGUE IN PATIENTS WITH RHEUMATOID ARTHRITIS WHO COMMENCE BIOLOGIC THERAPY: A LONGITUDINAL STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: none
Experimental design: With regards to #3 The authors should have adjusted the analysis using baseline FSS. You answered that "Thank you for pointing out this. We used a linear mixed effects model including all time points, i.e. also FSS at baseline. In time domain we used a simple contrast, which means comparison of each follow-up time point with baseline. We have added a sentence to clarify this. See page 10, last paragraph."
I am still unsure whether the baseline difference was adjusted for, in general the default (in most software) is to look a them independently form each other, this would mean that the adjustment wouldn't be taken into account. My concern is that there is a statistical difference at baseline between gender, which could be due to other factors. Females have worse fatigue at baseline and treatment would lead to greater improvement in more severe conditions with biologics. Given the small number in each group it wouldn't take much to for this to be overestimated hence the importance of getting the analysis right - is it severity or gender? This would not be taken into account in default analysis some other methods would have to be used (e.g autocorrelation) to include baseline ffs. This may have been carried out but it is not clear from the text further clarity is needed, and if not done correctly should be redone.
Validity of the findings: none
Additional comments: none


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: PATIENT-REPORTED FATIGUE IN PATIENTS WITH RHEUMATOID ARTHRITIS WHO COMMENCE BIOLOGIC THERAPY: A LONGITUDINAL STUDY
Review round: 2
Reviewer: 2

Output:
Basic reporting: My major concerns in this section have been sufficiently addressed. I have only one comment to the inclusion of study section: "The study was carried out at a rheumatology specialist department (not by a department). I would also suggest to include in which part of Norway this was, as you refer to this in the Discussion section.
Experimental design: Re. my comment on hypothesis for selection of possible predictors: I think that the sentence you have added on pg. 7does not provide any further information and may be deleted. I realise that this is an explorative study and that the possible predictors are sufficiently accounted for in the Background.
Validity of the findings: I have no further comments to this section.
Additional comments: No additional comments


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

Input:
Title: THE SYNERGISTIC EFFECT OF CONCATENATION IN PHYLOGENOMICS: THE CASE IN PANTOEA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is very well written, and makes use of a well-characterised case study to address a topic of current relevance to microbial phylogenetics. The figures are informative and not excessive (i.e. only used to highlight relevant points in the manuscript) and there is generally ample citation to past research and literature. All results and discussion are clearly framed in the context of the research questions. Given that the manuscript focuses on a single genus, I feel that the title is too broad in scope (this would be more of a case study than a universal finding), but this is my only criticism of the writing.

The authors have also included (in my opinion) one of the best justifications for concatenated trees in the context of incomplete lineage sorting - that a speciation event does not mean that every gene in the organism diversifies, and hence the true evolutionary pattern of an organism may be distributed unevenly across many genes, which may not be captured in aggregate single-gene trees.

As a small note, Figure 3 is referred to before Figure 2 in the text of the manuscript, although it was probably intended that Figure 2 be first cited somewhere in LN215-220.
Experimental design: The experimental design and statistical analysis are rigorous and adequate to tackle the hypotheses proposed. The overall approach is also logically broken down into three main testable hypotheses, each of which is further subdivided as required. My only concern with the approach of the manuscript is that findings are evaluated in comparison to a Pantoea species hypothesis that is grounded in a concatenated tree. While I accept that we can never know the evolutionary history of a lineage it does seem circular to assume the concatenated model is true and to then evaluate the individual genes in the context of this model. The incorporation of independent lines of evidence to place weight on the concatenated model would add to the manuscript even if they are not specifically phylogenetic means. For example, genome-genome ANI or synteny comparisons could be used to predict the branching order of Pantoea species, against which both the concatenated and single-gene approaches could be compared.

This is particularly relevant in light of the work of Thiergart et al. (2014; https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4302582). While it could certainly be argued that the authors are providing an explanation for why the 'disappearing tree' phenomenon occurs, it would be more powerful to control for this criticism of concatenated trees within the methods of the study.
Validity of the findings: The largest criticism of the results is likely that the authors have relied on approximate maximum likelihood trees (i.e. FastTree) as the foundation of their analysis. While they have provided a justification for this choice, and have verified findings using the more rigorous RAxML program where uncertainty arises, there are other methods of tree inference which can match or surpass the quality of RAxML with much lower compute times (for example, ExaML and IQ-Tree). FastTree also supports a very restricted range of evolutionary models (JTT, WAG, and LG) which may lead to reduced accuracy in the findings.
Additional comments: No additional comments


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

Input:
Title: THE SYNERGISTIC EFFECT OF CONCATENATION IN PHYLOGENOMICS: THE CASE IN PANTOEA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Some minor comments

* Line 103: I think that saying that the analysis accounted for all known phylogenetic errors is a bit of an overstatement. This sentence could be probably rewritten in a less

* Line 14-145 How much was "not robustly supported". Was there a bootstrap analysis? How much was the cutoff? --- PS (I see in the figure that this was a SH test, but how much was the value of the non-highly supported nodes?)

* Line 197-198 and the reference to Figure S1. This figure is too important to be left as the first figure in the Supplementary materials. It would stand to reason to make a larger figure 1 that includes in the first section the species tree and in the second section the net analysis so that both objects can be inspected visually right away. I would understand the decision of leaving the Species Tree for the SM only if the inclusion of it in Figure 1 makes the Figure to hard to read. Maybe some color guidelines below the main groups would be helpful

* Lines 211-212 The assertion would be easier to verify by having the two structures next to each other, see previous comment

* Lines 214-215 It is probably worth quoting here “Concatenated alignments and the case of the disappearing tree”. Thiergart et al. use a very similar approach you are using. It would be worth also pointing out the differences (which explain why yours make sense and theirs do not)

* Phylograms for limited sets of genes: I feel it is really a pity that the authors did not present their results in a better way. Please, see my comment in the section: Experimental design

* Please, share the file to open the SplitTree analysis

* Please, discuss (much more), the way that people are dealing today with phylogenetic incongruence (reconciliations). See the other sections
Experimental design: I have two major comments:

* Phylograms from limited set of genes

Although the approach that the authors used is not bad, I think that they could improve it quite a bit and obtain a potentially very robust and interesting result.

With the same experimental setup (20, 50 and 100 genes), obtaining the consensus phylogeny and then measuring the RF distance of the “true” species tree. Then plotting that to see the increasing trend in the similarity with the "true" species tree when more genes are sampled. Using more categories (e.g. 20, 40, 60, 80, 100 and 120) would probably allow the authors to obtain a very neat plot in which they can try to adjust a regression model. If the findings of the current paper are true, we shouldn’t see a great improvement by randomly sampling more than 100 gene trees.

* What about phylogenetic reconciliations?

One of the most popular ways of dealing with phylogenetic incongruence is using phylogenetic reconciliations. I was very disappointed to see that the authors completely left out this whole topic. For example, Szollosi et al 2012 (Phylogenetic modeling of lateral gene transfer reconstructs the pattern and relative timing of speciations) use a probabilistic reconciliation model that accounts for the phylogenetic incongruence to recover the species tree phylogeny. Even if the gene topologies are very often completely different from those of the species tree, the authors managed to recover a robust tree of cyanobacteria. I think a proper discussion on this topic is completely missing, as well as a proper analysis using some of these techniques to really quantify how many transfers can be found in this dataset (especially relevant when the authors set as one of the main goals of the paper, in line 113, to determine whether the observed conflicts could be ascribed to organism-level evolutionary processes). it is possible to answer this question using some of these techniques. See also Williams et al 2017 (Integrative modeling of gene and genome evolution roots the archaeal tree of life) used a phylogenomic analysis to study the evolution of Archaea genomes in which they dealt with the phylogenetic incongruence.

The authors might argue that they deal with HGT and ILS when using the network approach, but I would slightly disagree. They do indeed deal with this issue but in an insufficient manner. For example, the discussion in the lines 325-336 says that if more transfers or ILS were affecting this dataset, we would expect to see more boxes in the network analysis. I find this disappointing. I wonder if there is not a more numerical way of expressing these results instead of the highly subjective “low” or “high” number of boxes. Reconciliations analysis can give very precise estimates on the number of transfers affecting the dataset.
Validity of the findings: My biggest concern with this paper is that I feel that the importance of phylogenetic incongruence has not been dealt with properly. The network analysis does not show much more than saying than there is some incongruence between the gene trees and the species tree and that the major relationships are still preserved. This is not a surprising result.

The paper would benefit much also from a better analysis in the section Phylograms from limited set of genes
Additional comments: On the good side, I would point out that the authors deal with a very interesting topic and they performed a very exhaustive work in some areas. They explored the impact of recombination in their datasets, the origin of the phylogenetic incongruence at various level and the possiblity of obtaining a good species tree even when there is no apparent signal in the individual gene trees.

On the "down" side, I feel that the HGT issue was completely left aside, that they did not discuss a major topic on the uses of phylogenetic incongruence and that the approach of some of the experiments they carried out should have been presented in a more numeric way.


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

Input:
Title: THE SYNERGISTIC EFFECT OF CONCATENATION IN PHYLOGENOMICS: THE CASE IN PANTOEA
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: In general, the work in this manuscript is well described, the aims clear, and the approaches sound. However, I would like to bring an issues to the authors' attention: In my opinion the authors overstate the accuracy of Maximum Likelihood (or AML) methods for phylogenetic reconstruction based on concatenation. It is true that ML/concatenation is often regarded as a nearly infalible gold standard in phylogenomic studies where the final tree is the main objective. However, this manuscript being concerned with the statistical robustness of the methods applied (rather than the final tree), should at least consider the observation that such methods are statistically inconsistent. This is problematic in the current manuscript in at least two points:

(1) The authors appear to take the concatenation-based phylogenetic reconstruction in Palmer et al (2017) as incontrovertible truth. Although I consider that work to present a high-quality phylogeny of the group, it remains a (likely) phylogenetic hypothesis. I'd like to invite the authors to consider the possibility that their previously published reconstruction be flawed.

(2) The authors do not consider other methods that could account for ILS, such as those based on multi-species coalescent model. It would be fantastic to see these methods used side to side in this study. However, if that's simply beyond the scope of the current study, the scope of the study as stated must be revised and this limitation should be made explicit. For example, note that the authors generate reconstructions with different numbers of loci and compare those to the reconstruction generated with all core genes of the larger group in Palmer et al (2017; 1,039 genes). This comparison is valuable, as it tells at which point the concatenation-based reconstruction saturates (i.e., it doesn't change anymore with additional loci), but it cannot be taken as a metric of loci necessary to reconstruct the true species tree (i.e., it's error-free). There is good evidence in the literature that ML/concatenation doesn't necessarily tend towards the true species tree regardless of the number of loci; i.e., it's statistically inconsistent. This is not true for MSCM, which is guaranteed to tend towards the species tree when the number of loci increases given true gene trees (although the absolute error may at some points be higher than that of ML/concatenation).
Additional comments: Minor issues detected:

L115: contains -> contain
L136: extra comma
L144: "When the relationships obtained from AML analyses were not robustly supported, ...": please make the criterion used explicit. Was this based on the bootstrap supports? Did the authors use a minimum, maximum, or average node support as the threshold?
L165: was -> were
L199 and elsewhere: Alignment lengths should be given in "columns" or "amino acid columns", unless the authors mean to indicate all characters in the alignment matrix excluding gaps and missing data
L329-334: This section appears very subjective. Perhaps the authors could provide more quantitative statements to strengthen this section
L384-386: "... [gene and species] phylogenies are clearly not comparable and no conclusions regarding species evolution should be drawn from such comparison": I suspect the authors mean that direct comparison (e.g., raw tree topology distances) shouldn't be considered as robust tests for the quality of species tree reconstructions. However, I must disagree with the statement as is. Gene and species phylogenies are comparable: indeed, that's the fundamental idea behind MSCM-based reconstruction, to compare gene trees to (potential) species trees in order to reduce deep coalescence.

Signed: Luis M. Rodriguez-R


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

Input:
Title: THE SYNERGISTIC EFFECT OF CONCATENATION IN PHYLOGENOMICS: THE CASE IN PANTOEA
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors have addressed my previous concerns. I have no further comments or criticisms of the manuscript.


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

Input:
Title: THE SYNERGISTIC EFFECT OF CONCATENATION IN PHYLOGENOMICS: THE CASE IN PANTOEA
Review round: 2
Reviewer: 2

Output:
Basic reporting: I feel that the paper has improved much after the reviews. I especially enjoyed the discussion of Thiergart et al. 2014. I want to congratulate the authors for the good job
Experimental design: No comment
Validity of the findings: No comment
Additional comments: No comment


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

Input:
Title: THE SYNERGISTIC EFFECT OF CONCATENATION IN PHYLOGENOMICS: THE CASE IN PANTOEA
Review round: 2
Reviewer: 3

Output:
Basic reporting: I would like to echo Dr. Waite's observation on the title. Although this study may serve as a specific example that can be used more broadly, all of it is circumscribed to the genus Pantoea. Not only are all the results derived from a single genus, but nothing in the discussion attempts to address potential differences in other taxa, making this manuscript very specific to Pantoea. I respectfully invite the authors to reconsider the title. I have no other comments on the basic reporting.
Experimental design: No comment.
Validity of the findings: No comments.
Additional comments: The authors repeatedly indicate that the branch lengths in the MSC tree indicate the discordance between gene trees. I don't believe this is accurate, and is not supported by the cited reference. The coalescent units are simply mutation units scaled by population sizes. Since population sizes are not necessarily constant, the relationship is not necessarily linear, but they're not a measurement of discordance between gene trees.

On a related note, the new Figure 1 presents the MSC tree. In the tree the authors decided to present terminal branches with a length of 1 coalescent unit. This decision should be indicated in the legend. Note that ASTRAL does not (cannot) compute lengths of terminal branches.

Signed: Luis M. Rodriguez-R


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: SIMULATING THE EFFECTS OF CLIMATE CHANGE ACROSS THE GEOGRAPHICAL DISTRIBUTION OF TWO MEDICINAL PLANTS IN THE GENUS NARDOSTACHYS
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1. The English language should be improved to ensure that readers can clearly understand your text. Clear and professional English is not used. For examples, "RPC" should be "RCP".
2. The introduction and background descriptions are too simple to introduce the biological characteristics of species and the current research status of SDMs, nor to provide relevant references. Such as, "Species distribution models can help solve this problem. The main ecological niche models for species distribution predictions include Bioclim, Climex, Domain, Garp, and MaxEnt maximum entropy model."
3. Figure 1 has no latitude and longitude. The label of Figure 2 has some problems. The pictures and texts do not correspond.
4. The frame structure of the article is unreasonable. For example, the relevant description of RCPs should be put in the introduction.
Experimental design: 1. " After duplicate and invalid records were removed, a total of 183 records were collected. " How many sampling points are there for the two species respectively? How to identify them as wild species?
2. " Nineteen bioclimatic variables with 2.5-minute resolution of the current conditions and future conditions... " Why not use data with a resolution of 30 seconds? The environmental data is not filtered to reduce the correlation between the data. Why not consider topography, soil, light and other factors?
3. “ the results were divided into four levels using ArcGis 10.2: 0-0.05, 0.05-0.25, 0.25-0.5, and 0.5-1. ” What is the basis for the results of suitability classification?  Why are the first three categories defined as unsuitable areas?
4. " Representative Concentration Pathways (RPCs) 2.6 and 8.5, were selected..." Please explian why RCP 2.6 and RCP 8.5 were chosen.
5. From the aspects of research content, model construction and research results, there is no innovation in this paper.
Validity of the findings: 1. Comparing the impacts of climate change on the area of suitable habitats , the specific scope and reasons of the area change are not explained.
2. "Therefore, developing artificial cultivation techniques for N. cdinensis is more beneficial from a long-term prospective."  The reason for choosing N. cdinensis for artificial cultivation is illogical.
3. " precipitation is an important factor in the impact of climate change on N. jatamansi and N. cdinensis." The results show that the effect of precipitation on species distribution is greater than that of temperature, but the basis is insufficient, which needs further explanation.
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: SIMULATING THE EFFECTS OF CLIMATE CHANGE ACROSS THE GEOGRAPHICAL DISTRIBUTION OF TWO MEDICINAL PLANTS IN THE GENUS NARDOSTACHYS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors have presented a good background on this topic. The language use is professional, and the paper is structured conform journal guideline. Please increase the size of the text in Figure 1, 2, 4 and 5.
Experimental design: The manuscript is within the journal’s scope. It has well-defined research questions considering the current knowledge gap in the literature, followed by a rigorous investigation. Methods are well described however I would recommend explaining the method through the flowchart but in a more symbolic way, not just text.
Validity of the findings: Data and statistical analysis are robust and controlled.
Additional comments: Authors of this manuscript have presented a study concerning the impact of the effects of climate change across the geographical distribution of two medicinal plants in the genus Nardostachys. The author has applied entropy models MaxEnt to predict the potential geographical distributions of the genus Nardostachys under current and future climatic conditions based on two representative concentration pathways (RPC2.6 and RPC8.5) for the 2050s and 2070s. The authors have found that the potential distribution of the two species will increase, thus more suitable habitats will be present in China. The topic is interesting, an important issue and generally well written, well structured and contributes to the existing knowledge. However, there are still some occasional grammar errors through the manuscript especially the article ‘’the’’, ‘’a’’ and ‘’an’’ is missing in many places, please make a spellchecking.

The results and discussion section needs further improvement, compare your findings with the other author's conclusions.
•	In general, the manuscript needs to shorten, there irrelevant and redundant information in the text.
•	Please provide more deep discussion about your results, compare your findings with the other author findings.
•	Please clearly state the novelty of this work.
•	Please check the reference style, some of the references are not according to the journal style, especially the journals abbreviations.
•	At present, neither the original contributions of the paper nor its practical significance is clear for me.
•	As said, it could be a failure to comprehend the main argument; and lightening the discussion would enhance comprehensibility and make the authors’ point clearer and stronger.
•	Therefore, the reviewer recommends to further improve the manuscript before accepting it for publication. Some of the specific comments are listed below.
•	Please consider citing following literature; reviewer believes that it may help the authors to have a more holistic overview about climate impact on plants:

Rivaes, R., Rodríguez‐González, P. M., Albuquerque, A., Pinheiro, A. N., Egger, G., & Ferreira, M. T. (2013). Riparian vegetation responses to altered flow regimes driven by climate change in Mediterranean rivers. Ecohydrology, 6(3), 413-424.
Lv, X., & Zhou, G. (2018). Climatic Suitability of the Geographic Distribution of Stipa breviflora in Chinese Temperate Grassland under Climate Change. Sustainability, 10(10), 3767.
Rivaes, R. P., Rodríguez-González, P. M., Ferreira, M. T., Pinheiro, A. N., Politti, E., Egger, G.,& Francés, F. (2014). Modeling the evolution of riparian woodlands facing climate change in three European rivers with contrasting flow regimes. PloS one, 9(10), e110200.


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 AND EXPRESSION ANALYSIS OF THE GDSL ESTERASE/LIPASE FAMILY GENES, AND THE CHARACTERIZATION OF SAGLIP8 IN SEDUM ALFREDII HANCE UNDER CADMIUM STRESS
Review round: 1
Reviewer: 1

Output:
Basic reporting: see general comments for the author
Experimental design: see general comments for the author
Validity of the findings: see general comments for the author
Additional comments: The manuscript is well developed and clearly structured, and the conclusions are supported by the results. I only have a few comments.
1. For the title, I suggest to  change " Identification and expression analysis of the GDSL
esterase/lipase family" to "Identification and expression analysis of the GDSL
esterase/lipase family genes", otherwise, it sounds like the the manuscript is about identification and purification of GDSL esterase/lipase proteins, not genes.
2. What does GDSL stand for?
3. The English language needs to be improved. For example, Line 29,  I suggest change "experimental materials" to roots of S.alfredii seedlings. Line 31 and 32, "were" should be was, "investigate the resistance and accumulation to Cd" change to "investigate its role in resistance and accumulation to Cd". Line 59 "are adapted to" should be  "adapt to". Line 243 "For the..." change to "due to the..".
4.  The genes studied are described as both GDSL gene/family/enzyme and GELP gene/family/enzyme, are GDSL and GELP equivalent?
5  Line 292, "As shown in Figure 6, SaGLIP8 is also an inhibitory protein". Figure 6 only describes the gene expression level, hence, it is not clear to me why SaGLIP8 is an inhibitory protein, does SaGLIP8 inhibit the function of any other protein? Please rephrase this sentence and add more explanation.
6. Line 293 and 294, "Some reported GLIP genes can be regulated in a variety of ways to enhance their defense functions" needs a reference.


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 AND EXPRESSION ANALYSIS OF THE GDSL ESTERASE/LIPASE FAMILY GENES, AND THE CHARACTERIZATION OF SAGLIP8 IN SEDUM ALFREDII HANCE UNDER CADMIUM STRESS
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this manuscript, the authors have performed extensive genomic analysis and identified 80 genes that belong to GDSL esterase/lipase protein family in S. alfredii. Furthermore, authors have identified the gene, SaGLIP8, involved in the cadmium detoxification. The manuscript is well-written and provides sufficient background for readers.
Experimental design: The methods are properly described. This study will be of interest to researchers working in this specific field. Furthermore, the authors provide all the raw data files with their analysis and mentioned all the tools and databases used for the study.
Validity of the findings: No comment
Additional comments: 1. line 74: the number of GELP in Arabidopsis is written 108 whereas in line 180: 105 known sequences in Arabidopsis is mentioned.
2.  line 195: Table S7 is mentioned. Is it correct?
3. line 254-255: the sentence seems to be incomplete. What are the "corresponding proteins"? Mention the name of the proteins.
4. In the result section: Gene structure and conserved motif analysis": The authors should show the conserved motifs representation in the three-dimensional structure.
5. In Fig. 7, the authors show the bar graphs based on heat map as shown in Fig. 6. The authors identified some specific genes highly expressed in roots, stems and leaves. It is better to show the RT-PCR gel image of these specific genes also.


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 AND EXPRESSION ANALYSIS OF THE GDSL ESTERASE/LIPASE FAMILY GENES, AND THE CHARACTERIZATION OF SAGLIP8 IN SEDUM ALFREDII HANCE UNDER CADMIUM STRESS
Review round: 1
Reviewer: 3

Output:
Basic reporting: This is a very well-written manuscript showcasing preliminary data of the function of SaGLIP8 gene under cadmium stress.
Some comments are as follows:
Abstract:
1. abbreviations are used without defining them first.
2. under "methods" in abstract, line 29 - "...experimental materials...." - please mention what these are.

Introduction:
1. Lines 52 - 59 - Authors introduce Cd as a pollutant that has serious impacts on growth and development. Then talk about sources of Cd as pollutant and methods of accumulation in organism followed by what the harmful effects are. Re-arrange these sentences such that impact on growth and development is immediately followed by examples of those impacts.
2. Lines 59-60 - what are some examples of how plants tolerate high metals?
3. Lines 60-61- examples of hyperaccumulators
3. Lines 67, 69 - some genes are mentioned that improve tolerance for Cd. However, what are their basic functions? Is it known? Please define what these genes are.
4. Line 71 - what are lipid proteins? It seems to be a vague term. Does it imply proteins involved in lipid metabolism or proteins that contain fatty acid modifications? How does the lipid part of the organism tie into studying heavy metal stress?
5. Line 73 - Please define GDSL before using the abbreviation.Also please introduce what the GDSL family is before discussing GELP family proteins.
6. Line 79-81 - When discussing the structure of the protein, please have a figure, either molecular or schematic to refer to. How does the protein structure tie in with the motifs that have been discussed later in the discussion section of the manuscript? This is not discussed or mentioned anywhere. A discussion would make the study more complete.
7. Line 87 - Authors say "....has not yet been systematically identified under Cd stress..." Doe this mean that the gene has been studied when there is no heavy metal stress?
8. Line 90 -  Please define SaGELPs before using the abbreviation.
9. Line 90 - Authors say ..."obtained..." Should it instead read "discovered" instead? As the authors are the one who are analysing the genome?
Experimental design: Good experimental design.

Line 133 - spelling mistake. "promoters" instead of "promotres"
Line 149 - in the description of this method, relate back to "Plant materials and stress treatments" as that is where the roots, stems and leaves are from.
Validity of the findings: The results and discussion section is well written. Some comments are as follows:

Results section:

Line 235 - What does "untreated" mean? Please state clearly.
Line 252 - Is there a numerical value to the "significant difference"?

Discussion section:

Line 266 - Please examine the sentence and make sure it is complete.
Line 276 - Please define what "samples" mean.
Line 276 -277 - please discuss shortly how the heat map was obtained.
Line 291-292 - How does figure 6 show that SaGLIP8 is a inhibitory protein?
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: RISK OF ACUTE EXACERBATION BETWEEN ACETAMINOPHEN AND IBUPROFEN IN CHILDREN WITH ASTHMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The data reported in the manuscript are improper formatting (i.e. puntuaction).
Introduction is clear and well written.
Literature is well referenced.
Methods are adeguately described.
In the table 2 the legend (a, b, c) is not clear.
Experimental design: The experimental design is clear
Validity of the findings: No comment
Additional comments: The manuscript entitled  ” Risk of acute exacerbation between acetaminophen and ibuprofen in children with asthma” by Yung-Chieh Huang et al., is a retrospective analysis regarding the acetaminophen or ibuprofen use on risk of acute exacerbation in children with asthma.
The study is very useful to answer the important practical question from clinicians regarding which medication to use, acetaminophen or ibuprofen, in children with asthma that necessitate a treatment with an antipyretic, analgesic medication.

However, I have some comments.
1)	The study to be appropriate and specifically designed should include the dosage of acetominophen and ibuprofen.

2)	The authors should explain the adherence to asthma-controller medications among the patients.

3)	Is there a history of aspirin sensitivity among the patients? Since there is cross sensitivity to all classes of NSAIDs (Kowalski ML et al., Allergy. 2011 J;66:818-29), this aspect could be considered .

4)	The authors do not perform a randomized study.  Therefore they cannot rule out the possibility that the asthma exacerbations are caused by the respiratory infections themselves.

5)	The study intends to retroprospectively evaluate the effect of the use of acetaminophen versus ibuprofen in young children with persistent asthma. The authors should clarify that their study may not generalizable to other age groups or children with intermittent to mild asthma.


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: RISK OF ACUTE EXACERBATION BETWEEN ACETAMINOPHEN AND IBUPROFEN IN CHILDREN WITH ASTHMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this study the authors compare the risk of asthma exacerbation between1-year use of acetaminophen and 1-year use of ibuprofen in children aged 1–5 years with persistent asthma, focusing on the risk factors, including severe asthma exacerbation in the past.
The paper is widely interesting since raise imporatnt questions regarding pediatricians prescriptions of the use of antipyretics among children with asthma withouth a full evaluation of the risk.
Experimental design: This study is a retrospective database analysis where the authors correlate asthma exacerbation and the therapeutic use of  acetaminophen or ibuprofen. Asthma exacerbation is assessed as an acute or sub-acute episode of a progressive increase in asthma symptoms,associated with airflow obstruction. Althought factors contributing to asthma exacerbation include poor asthma control, severe exacerbation in the past year, viral infection, allergen exposure, virus–allergen interaction, smoking, and air pollution the authors include as clinical parameters only emergency room visits and hospitalization
Validity of the findings: They conclude thet Ibuprofen carries a higher risk of asthma exacerbation  than acetaminophen in  children with asthma, especially for those who have severe AE with an ER visit or hospitalization, or systemic corticosteroid prescription in the previous year.

The data are very interesting but the results difficult to follow how they have been reported.
It is know and also the authors confirm thet asthma incidence is sex related ( e.g. in the study the numbers of male patients is doubled respect to female) and also the therapy is widely different. In order to further demonstrate their hypothesis it might be better express the final results separately in male e female patients.
Additional comments: The data and the study are interesting and of considerable therapeutic importance given the wide use of antipyretics in children, in any case considering the different incidence of the disease between the two genera, it would be interesting to present a separate data in order to highlight useful differences in therapeutic approach.


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: RISK OF ACUTE EXACERBATION BETWEEN ACETAMINOPHEN AND IBUPROFEN IN CHILDREN WITH ASTHMA
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors made all the changes required by the reviewers.


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: DISTRIBUTION OF CRABS ALONG A HABITAT GRADIENT ON THE YELLOW SEA COAST AFTER SPARTINA ALTERNIFLORA INVASION
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article would benefit from revision by an English speaker as there are a number of instances of strange word use and incorrect subject-verb agreements.
Literature references are sufficient.
Figures 3 and 4 are extremely challenging to interpret in their current form. In particular, navigating between the figure legend and the figures themselves to figure out what the letters mean in the figures requires too much effort. I encourage the authors to move away from these analytical frameworks and instead simply present the data in figure 3 as multiple panels of bar charts that show the average densities of each species in each of the habitat types (i.e. one small figure for each species, with column color referring to habitat type). I would also group the crabs into functional groups (e.g. predators versus herbivores/scavengers) so that at least your reader can get an idea if there are certain functional groups that seem to thrive in the invaded habitat
Experimental design: The research question is well defined and relevant. The implications of this study are significantly limited by the fact that the data are only derived from a single site, however. It would help if the authors at least better justified why this field site is perhaps representative of other marsh systems in the area.

While I appreciate the temporal sequence of the monitoring - showing crab densities on a monthly basis - the authors do not make it clear why they do not sample during the winter. I imagine this is because the crabs are not active due to lower temperature. However it would be helpful if this were stated. In addition, can the authors propose hypotheses related to the temporal dynamics of the crab communities - for instance do they expect the community composition to be more stable in one habitat type relative to another based on the stability of environmental conditions or food availability across habitats? The authors could calculate crab community stability metrics to evaluate such a hypothesis. In essence, I would like to see them better leverage the temporal data that they do have.

In addition, it needs to be clear in the methods and figure legends if the data being presented has been summed across all pitfall traps deployed over the entire monitoring period and thus what their units of measurement are.
Validity of the findings: As mentioned above, I found the third and fourth figures impossible to interpret and strongly encourage the authors to change their approach to presenting these data.

The discussion and conclusions are fine in my opinion.
Additional comments: I encourage the authors to reach out for help with editing to take care of some of the issues with English language and calculate community stability metrics to evaluate the stability of individual crab species and the entire community over time to better leverage their data set. And, justify if/how their study site is representative of other invaded wetlands in the region.


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: DISTRIBUTION OF CRABS ALONG A HABITAT GRADIENT ON THE YELLOW SEA COAST AFTER SPARTINA ALTERNIFLORA INVASION
Review round: 1
Reviewer: 2

Output:
Basic reporting: It's clear that the authors are not native English speakers. The paper has a number of grammatical errors and typos. These could easily be fixed, but I am not going to try and do it on a pdf file. A native speaker could fix many of these problems fairly quickly. The article as a whole is interesting and provides background on the effects of Spartina alterniflora on crab communities.
Experimental design: The design is straightforward, sampling crabs in different habitats in pitfall traps.
Validity of the findings: The data is ok. I am not familiar with the analyses cluster analyses used and it is not clear to me how figures 3 and 4 are interpreted. I would encourage the authors to explore a multivariate approach such as ANOSIM and SIMPER to more rigorously test their patterns.
Additional comments: This paper would be suitable for publication after additional data analyses and heavy editing on grammar.


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: DISTRIBUTION OF CRABS ALONG A HABITAT GRADIENT ON THE YELLOW SEA COAST AFTER SPARTINA ALTERNIFLORA INVASION
Review round: 2
Reviewer: 1

Output:
Basic reporting: grammatical errors fixed, article much improved
Experimental design: appropriate
Validity of the findings: the multivariate stats are a strength, the results section is nicely written and presented
Additional comments: The authors have done a good job addressing my concerns, the multivariate stats in particular, and the paper reads much better now. I do not think addition revisions are needed.


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: NO ASSOCIATION OF COMT WITH INSIGHT PROBLEM SOLVING IN CHINESE COLLEGE STUDENTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article is well written, although literature references should be implemented to provide adequate field background/context.
Experimental design: no comment
Validity of the findings: A more careful discussion of data is suggested (see "General comments for the author").
Additional comments: In this study Yang and coll. attempt to replicate findings of a recent study (Jiang et al., 2015), reporting an association between the COMT polymorphism and insight problem solving performance. After a concise, although thorough description of their results, which show no association, the authors outline some discrepancies between their study and Jiang’s one.

General comment
Although the functional influence the COMT polymorphism on dopamine clearance at the prefrontal cortex level is likely well known by scientists working in the field of behavioral neuroscience, an overview of this topic is totally missing in the present version of the study.
A brief overview of this topic on the introduction section would clarify the biological rationale underpinning the interest of this study (see Chen JS et al., Am. J. Hum. Genet. 2004; Bellgrove MA Exp Brain Res 2005 studies, among others).
In addition, although the study investigates a specific behavior, i.e. the insight problem solving, a brief overview of previous studies reporting association/no association between the COMT polymorphism and cognitive abilities, with particular reference to those that also investigated sex-dependent influences, should be briefly summarized in the introduction section (see Mione V. et al., Frontiers in Behav Neurosci 2015; Zabelina D.L. et al., PlosOne 2016; Jaspar M. et al., Cerebral Cortex 2015; White TP et al. Neuropsychopharmacology 2014; Elton A et al., Front. Hum. Neurosci 2017).
Of note, the authors of this study do not quote a recent study, which being related to their field of interest they might know, i.e. Han W et al., PeerJ 2018.

This implementation would provide the present study a wider audience.

Specific issues

- It is well known that the last part brain to mature is the prefrontal cortex. The authors should consider the age of participants as a possible cause of the discrepancy between their findings as compared to those of Jiang et al., 2015 study.
Indeed, the Mage is significantly different in the two studies: here18.9, SD=0.84 (Jiang et al: Mage=16.54 years, SD = 0.70).
Considering that the maturation of the prefrontal cortex networks is a late achievement, being completed at 20-22 years of age, the age factor should be carefully discussed, also within the perspective of future studies.

- The authors should comment on the possible influence of the robust imbalance between female (76.7%) and male fractions in the cohort of participants to their study.

- The authors should verify that genotype frequencies within male and female groups were consistent with the Hardy-Weinberg equilibrium using a specific test. The sentence ‘No significant deviation from Hardy-Weinberg equilibrium was observed (data not shown)’ is not sufficient.


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: NO ASSOCIATION OF COMT WITH INSIGHT PROBLEM SOLVING IN CHINESE COLLEGE STUDENTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. The introduction is meagre to a fault. Only a bare skimming of the theoretical background in the topic.

2. Methodological standards are questionable.
(a) Important details about the task are missing. The reader is given no indication as to what the task is and how the fit the criterion of being an insight task.

3. Results: Key results are missing (no information provided on how many problems out of the 10 were classified as familiar problems and how many were classified unfamiliar problems.

5. Several grammatical and semantic errors throughout the manuscript.

6. Several instances of #NULL fields in the data where the behaviour results should be.
Experimental design: 2. Methodological standards are questionable. For instance, at the level of the task alone there are there severe problems:
(a) Important details about the task are missing. The reader is given no indication as to what the task is and how the fit the criterion of being an insight task.
(b) No non-insight tasks were employed as control tasks.
(c) Only 10 problems (which translates to 10 trials) were used as a whole which is very low.

3. Results: Key results are missing (no information provided on how many problems out of the 10 were classified as familiar problems and how many were classified unfamiliar problems. So it is impossible to tell what exactly the accuracy rate reflects (percentage correct on unfamiliar problems). In addition, the accuracy rate was extremely low (e.g., total insight problem solving for all participants around 27.5%). So the data is very sparse and potentially based on only 1-2 correct responses of the participants.
Validity of the findings: 4. The findings are not congruous with the study their are trying to replicate (Jiang et al.). However, it is questionable whether this can be considered a replication study when not even clear that the same materials were used in this study as in the Jiang study. Certainly this study has less insight problems than the Jiang et al. study.
Additional comments: 1. The introduction is meagre to a fault. Only a bare skimming of the theoretical background in the topic.

2. Methodological standards are questionable. For instance, at the level of the task alone there are there severe problems:
(a) Important details about the task are missing. The reader is given no indication as to what the task is and how the fit the criterion of being an insight task.
(b) No non-insight tasks were employed as control tasks.
(c) Only 10 problems (which translates to 10 trials) were used as a whole which is very low.

3. Results: Key results are missing (no information provided on how many problems out of the 10 were classified as familiar problems and how many were classified unfamiliar problems. So it is impossible to tell what exactly the accuracy rate reflects (percentage correct on unfamiliar problems). In addition, the accuracy rate was extremely low (e.g., total insight problem solving for all participants around 27.5%). So the data is very sparse and potentially based on only 1-2 correct responses of the participants.

4. The findings are not congruous with the study their are trying to replicate (Jiang et al.). However, it is questionable whether this can be considered a replication study when not even clear that the same materials were used in this study as in the Jiang study. Certainly this study has less insight problems than the Jiang et al. study.

5. Several grammatical and semantic errors throughout the manuscript.

6. Several instances of #NULL fields in the data where the behavioral results should be.


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: HEAD LOUSE EGG AND NIT REMOVER—A MODERN “QUEST FOR THE HOLY GRAIL”
Review round: 1
Reviewer: 1

Output:
Basic reporting: Head lice are specific and permanent parasite insects that live in the human head. Their eggs are firmly attached to the hair by means of a resistant glue-like substance, that holds the empty eggshell fixed to the hair long after the nymph has emerged.

In this article, authors evaluated in a laboratory test, the effectiveness of a formulation based on isononyl isononanoate to facilitate egg and nit removal from the hair, in a comparative study with various commercial products.

Moreover they evaluated in an in vivo test, the number of eggs and nits removed by combing for the head of 15 children in which one half of the head was treated with the formulation and the other half was combed only on damp hair.
Experimental design: The manuscript is relevant due to the cosmopolite medical importance of pediculosis and falls within the scope of the journal.

It is clear and well written, the introduction is adequate to the objectives of the article, and the bibliography is updated.

The methodologies are based on previous reports and are carefully described with sufficient information in the article.

The results are summarized in two figures and two tables, all of them are relevant to show the obtained results and the statistical analysis performed.

The conclusions are consistent with the results and respond to the objectives of the study.
Validity of the findings: However, I consider that some information is missing in the table 2 where the demographic characteristics of the participants are described.

Although some of the characteristics of the children are simple to determine (sex, age), and others are well described (length, colour and curl of the hair), there are others that are not well defined.
For example, it is not clear how authors established the thickness and the dryness of the hair.

Y strongly recommend to the authors to add some indication or criterion to define both variables in Table 2. This information will be useful in the application of this procedure in new studies.
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: HEAD LOUSE EGG AND NIT REMOVER—A MODERN “QUEST FOR THE HOLY GRAIL”
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is written in correct English. However, the manuscript is not clear in many paragraphs, mainly in the Materials and Methods section (see General Comments for the author).

The manuscript include sufficient introduction and background and is structured in a standard way. Relevant prior literature is appropriately referenced.

The figures and tables presented are consistent with the correspondent studies, however, there are studies/evaluations carried out that are not presented in tables, figures or text; that is, all the results that correspond to all the bioassays described in Mat and Met are not reported in the manuscript
(see General Comments for the author)
Experimental design: The identification and description of the knowledge gap is well presented, and this gap is relevant and meaningful; however, the objective of the study is not clearly established in the introduction. I suggest that the authors present, at the end of the introduction, clearly and concisely the objective of the study and, if possible, the hypothesis to be corroborated.

There is no clarity in the description of the experimental design and bioassays of the laboratory studies. I suggest that the authors: a) improve the order in which the experimental design and bioassays are described, b) reduce/unify paragraphs where the experimental design and the laboratory bioassay are described (a large number of paragraphs, where some descriptions are repeated, conspire against the expository clarity).
(see General Comments for the author)
Validity of the findings: The data and results of a large number of bioassays described in Materials and Methods (e.g. the primary screening and the evaluation of different versions of the formulation chosen to obtain an optimal final formulation) are not presented in the manuscript in any formal way (i.e. text, figures or tables). These results are only referred to in sentences without any quantitative and statistical information (see General Comments for the author).

On the other hand, there is no information on the statistical tests used, except in one case (lines 264-272) (see General Comments for the author)

The authors make an excellent discussion of the problem of the persistence of lice eggs in the hairs, even if the embryo has already died. However, they do not discuss their own experimental results, neither in terms of the meaning of themselves nor in relation to results of other studies (except lines 277-280). Nor do they discuss whether their results corroborate or refute the hypotheses proposed. Finally, the authors also do not attempt to discuss how the original hypothesis, or some alternative hypothesis that has arisen from unexpected results, allow explaining and/or can be explained by other results and hypotheses in their study area.
Additional comments: COMMENTS OF THE MANUSCRIPT: HEAD LOUSE EGG AND NIT REMOVER – A MODERN “QUEST FOR THE HOLY GRAIL” by Elizabeth R Brunton, Ian P Whelan, Rebecca French, Mark N Burgess, Ian F Burgess

This article presents laboratory studies of the effectiveness in the removal of eggs and nits from the head louse of different compounds and formulations. In a second part and after a selection based on the results of the initial screening, the article reports on the effectiveness, based on laboratory bioassay and in a kind of clinical study, of the selected compound and its final formulation.

The manuscript has serious problems in the presentation and dicussion of the study that prevent me from recommending it for publication. These problems can be summarized as follows (details are below in this review):

1) The objective of the study is not clearly established in the introduction.
2) There is no clarity in the exposition of the bioassays and experimental design of the laboratory studies.
3) There is no description of the statistical tests used.
4) No data, quantitative results and statistical tests of the primary screening, formulations and of the evaluation of the variations on the relative proportion of the components of the formulation are presented. In general, these results are only referred in sentences without any quantitative and statistical information. This is, perhaps, the most problematic of the manuscript. The only reported results in a formal way of the laboratory bioassays are the evaluations of the final formulation compared to some marketed products from Europe (i.e. Fig. 2).
5) Finally, the manuscript seems to be the presentation of a study that starts from the evaluation of a great variety of compounds (whose results are not shown in full), after which one candidate is selected and evaluated; but, after reading table 2 it is observed that the final candidate is a commercial product in the market; so the objective of the study is not clear.

Specific comments
Abstract
Line 26-30. This description is more appropriate for the results section
Introducion
Lines 57-59. Please, give some bibliographical reference.
Lines 67-68. I am not against the use of comparisons or other literary resources (i.e. rhetorical figures, tropos, etc.) but, since it is not recommended or optimal for a scientific manuscript, then it has to be used exceptionally; that is, in cases where the comparison or analogy used is adjusted in a very precise and unambiguous way to the study developed allowing a better understanding of the phenomenon studied, the question asked or the proposed objective (precisely, this requirement explains its infrequent use in manuscripts scientists and their unadvised use). In this case: a) the analogy between "nit remover" and "holy grail" is not entirely clear; b) the phrase "much myth and mystery as the original" is not very precise; e.g. How was the amount of myth and mystery quantified? In this way, I think that this comparison/analogy between the "nit problem" and the "mystery of the holy grail" does not make much sense since it does not help to better understand the proposal and the development of the research.
Materials and Methods
Lines 85. -18 ° C, does not affect the structure of the glue-like substance?
Lines 102-108. a) Writing should be improved to clarify the experimental design. For example, first a primary screening was carried out for evaluate the x1, x2, x3, and x4 compounds. Then, the selected compound was formulated with the y1, y2, y3 or y4 excipients and each formulation was evaluated. Different combinations and concentrations of excipients were also evaluated. Finally, the final formulation obtained was compared with commercial products z1, z2, z3, z4 and z5. The bioassay 1, which consisted of…., was used for the primary screening. The bioassay 2, which consisted of…., was used for the evaluation of final formulation.
b) What was the criterion to select the optimal compounds after primary screening?
Lines 118-120. Please, give some bibliographical reference.
Line 127. a) Why were only intact eggs used in this part of the study?.
b) Why do the authors give a bibliographic reference for the final formulation? Is this final formulation the result of the bioassays of the present research or is it the result of a study already published? Actually, this doubt is key in the understanding of the study.
Line 137-138. The phrase is not understood.
Lines 177-184 and 186-195. I suggest fusing both paragraphs.
Results
Lines 201-211. The authors do not present data, results and statistical information of each measured variable in each bioassay carried out; they only describe, without some numerical data, the obtained in a general way.
Lines 213-214. It is not an appropriate phrase for the results section, but rather it is a phrase for the introduction or for the discussion.
Lines 213-220. It is the first time that the authors refer to: 1) the vehicles that they used for the different formulations, 2) to the presence of a gel and 3) to that this gel was diluted with water. The authors should detail in the Mat and Met section the vehicles used to obtain each formulation, the different combinations they made, and the manner in which the selected formulation was diluted with water.
On the other hand, again there are no data, quantitative results and statistical significance of all the bioassays carried out.
Line 222-227. a) If Figure 2 is an example of the comparisons made, what were the other comparisons? What results were obtained?.
b) What does "p = 0.000764 to 0.006132" mean after saying that "Compound X was significantly more effective"?
c) In figure 2 it is not clear which is the compound that contains "isononyl isononanoate"
d) When reading table 2 it is clear that the product evaluated is "Stubborn Eggs remove"
1) Is this the "final formulation" referred to in the Mat and Met section?
2) Is this the formulation that contains isononyl isononanoate? If so, it should be specified in the Mat and met section.
3) Is this "final formulation" a commercial product? If so, it obviously does not seem to be an experimental formulation as described in the Mat and Met section.
4) And if the previous point is correct, then the study should be presented in another way
Lines 263-271. The statistical development is not clear (I insist that this must be described in the Mat and Met section). The design requires an analysis of paired comparisons. This analysis will be parametric or nonparametric according to whether the errors fulfill the assumptions of normality and homoscedasticity. The errors meet or not such assumptions, and there are objective evaluations based on specific tests. In this way, the decision to use parametric or non-parametric tests is not subjective but follows criteria established and shared by the scientific community. For all this, it is not correct to express "if we consider parametric, we have this result; if we consider it non-parametric, we have this other result. "
On the other hand, the Mann-W is not a nonparametric test for paired comparisons; one of the most used is Wilcoxon Test for paired comparisons


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: HEAD LOUSE EGG AND NIT REMOVER—A MODERN “QUEST FOR THE HOLY GRAIL”
Review round: 2
Reviewer: 1

Output:
Basic reporting: Regarding my previous revision:
The manuscript has improved notably, mainly the Materials and Methods and Results sections.
Minor revision are needed (see General Comments for the author)
Experimental design: Regarding my previous revision:
The objective is clearly established in the introduction.
The description of the experimental design and bioassays of the laboratory studies has improved markedly.
Validity of the findings: Regarding my previous revision:
The authors incorporated new information into the results that have improved the presentation and understanding of the study. Minor revisions are needed (see General Comments for the author)
The Discussion section was also improved
Additional comments: COMMENTS OF THE MANUSCRIPT: HEAD LOUSE EGG AND NIT REMOVER – A MODERN “QUEST FOR THE HOLY GRAIL” (#32808)  by Elizabeth R Brunton, Ian P Whelan, Rebecca French, Mark N Burgess, Ian F Burgess
The revised manuscript has been significantly improved and is now suitable for publication in PeerJ. I thank the authors for their good work and for attending to all the comments and questions I have made..
Minor revisions are needed (but important when the development and effectiveness of a product is scientifically presented)
1)	Please, the authors should clarify how many replicate (i.e. groups of 10 eggs) were made for each compounds/treatment in the bioassays of the figures 3, 4, 5 and 6.
2)	 Please, the authors should present the results of the tests with 5, 10 and 30 minutes of application described in lines 151-153. If these results are not presented, the authors should eliminate this description.
3)	Lines 255-298. Please, the authors should refer to significant or non-significant differences between treatments by post-hoc comparisons of the bioassays corresponding to figures 3, 4 and 5 (the only statistical reference for these bioassays is on line 296). That is, the authors must specify the statistical significance (in words and/or with the P-values) of the results/comparisons described in these paragraphs.
4)	Figs 3, 4, 5 and 6. The authors should show the significant differences between treatments in figures 3, 4, 5 and 6 (e.g. by means different letters).


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: MARINE BIODIVERSITY RESEARCH IN THE RYUKYU ISLANDS, JAPAN: CURRENT STATUS AND TRENDS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article describes the relative paucity of research in the RYS, highlighting under-studied sub-regions, taxa and ecological topics. I enjoyed reading it, thank you!

The article is generally clear and well written.  There are a few grammatical errors/typos and the authors should check for these more carefully.  Ones I spotted are:
Line 20: change to ‘spanning protists to vertebrates’
Line 58: across
Line 176: change ‘less’ to ‘fewer’

Figure 1.  I wonder if this figure might be clearer if the authors split it into two, i.e. Figure 1a would be the map and Figure 1b the pie charts.  It’s not a huge issue but it feels a bit cluttered to me.  Pie charts are also notoriously difficult to assess by eye apparently although I appreciate your choice since it allows the reader to simultaneously visulalise the number of publications. Another option could be a barplot similar to ggplot’s “geom_bar”.

Unfortunately I was not able to open the Supplementary eps file.  I apologise - I should have checked earlier and have now run out of time to get the file in a different format.
Experimental design: The authors provide an overview of the state of marine biodiversity research in the Ryukyu Archipelago (RYS), investigating sub-region, taxa and ecological topic.  Additionally they look at the spatial bias of records with respect to marine research stations and institutes.
The research is well defined and methods well described.

The authors give considerable discussion to the problem of a sizeable number of research articles published solely in Japanese.  These articles are not considered in their study.  It is therefore hard to know whether the RYS is genuinely as under-studied as their results suggest.  I see two options here.  The first, which would be preferable, would be for the authors to extend their study to the Japanese language publications.  I appreciate that if these journals are not databased this could be somewhat laborious – and would also of course require fluency in Japanese and access to the journals.  However, I have manually searched journals not included on WoS myself for articles relating to a particular region (in this case Sundaland) and it actually only took a few days.  Adding in these Japanese language articles would add real value to the study. It would be interesting – and important - to know whether these articles contain the same biases as English-language articles and also what proportion of research on the RYS is unavailable to a global audience.

The second, easier option, would be to be much more upfront that the articles are English-language. So, beginning in the abstract and continuing throughout, ‘this study provides an overview of the state of English-language marine biodiversity research in the RYS’ or similar.

It may be that a compromise between the two options is possible, e.g. count the number of Japanese publications that relate to the RYU without going into detail on ecological topic.  However, I would still prefer the first option.
Validity of the findings: See my comments on the English/Japanese language publications above. That aside, the data appear robust and conclusions well stated.
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: MARINE BIODIVERSITY RESEARCH IN THE RYUKYU ISLANDS, JAPAN: CURRENT STATUS AND TRENDS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper is very well written - there are however a small number of places where sentence structure could be adjusted to achieve greater clarity.
Examples from the abstract:
First line ... Could be reworded as "Coral reef of the subtropical Ryukyu Archipelago (RYS; also known as the Nansei Islands) in Japan have been shown to harbour very high levels of marine biodiversity".

Last line... Could be reworded into two senteces i.e. "Coral reefs in the RYS feature high levels of both endemism and anthropogenic threats, subsequently they contain a concentration of some of the worlds most critically endangered marine species. To protect these demonstratably threatened species, more data is needed to fill the research gaps identified in this study.

When you reference to 4.0 times (rate of increase) could this be expressed as 4-fold?

Please consider adding an additional sentence at the front end of the abstract to justify the significance of biodiversity in the RYS and to set up why its important to explore the extent of reserach and to identify gaps. In order to fit that sentence it I would encourage revisting the rest of the abstract with the view of writing it a bit more succintly.

Intro
line 58 - spelling error "across"
line 62 - please split into two sentences.
line 64 - could be rewritten for clarity. e.g. "In 2002, a global analysis of regional conservation priority ranked southern Japan and Taiwan first due to the high levels of both endemism and threats, highlighting that rapid conservation action is needed".
line 73 - can you state the numbers of visitors in each region for context?
line 76 - any refs to substantiate the diversity in RYS
Experimental design: The paper appears to be a "gap analysis" - have the authors considered using this phrase as it may help further conceptualize the entire study.

For the benefit of audiences outside of Japan, is there any way you can include a satellite image of the RYS - potentially showing well known places such as Tokoyo and Okinawa and places of interest that are mentioned in text such as Sekisei Lagoon or some of the other marine field stations mentioned in Table 1.

I note data us used from WoS and OBIS - I am aware in my region the OBIS records are very scant and there is much data in museum databases that has not been included in OBIS to date - how utilized is the OBIS dbase for this region and have you considered exploring releveant museum databases or additional species records?
Validity of the findings: All the findings are sound but I would encourage the authors to include some additional discussion points. For example - does the higher number of fish, cnidaria and crustacean research papers reflect there is a bias towards these areas that stems from these being the areas where there is local and national academic expertise? Hence this has led to teaching and learning biases within Japanese universities?  If so, does that mean more capacity building and investment is needed in other subject areas?

How many of the papers include international collaborations? I dont expect the authors to go back and recalculate this across the 960 papers but it could be worth mentioning that the current global landscape of research direction is constantly moving towards collaborative, multi-institutional projects - hence in order to address the knowledge gaps that have been identified here, it could be worthwhile making a statement such as ... Marine biodiversity reserch in the RYS will benefit from continuing to build international collaborative research projects to broaden the breadth of taxonomic studies and scope of ecosystem based and conservation planning studies....

I am curious to know how well utilized OBIS is - there is definatlely the opportunity here to further encourage the scientific community to utilize this dbase.

line 257 - please quantify the term "higher numbers" when referring to population density between GBR, RES and RYS.

It might be worth mentioning some of the other "non-charasmatic or cryptic" taxa that are not specifically examined in this paper to highlight the extent of marine biodiversity and how existing research is just stratching the surface. Also the Cnidaria is a huge phylum is it worth mentioning any biases within - such as bias towards Anthozoa or hard corals over other classes/subclasses/families?

Is it worth mentioning promising new technologies such as eDNA for auditing a wider variety of marine biodiversity?
Additional comments: It was interesting to read where the historical research has been focussed and where the research gaps are - this type of study could be very important for shaping the future of marine research in the region to ensure important knowledge gaps begin to be plugged.

I have one suggestion that I would highly encourage the authors to consider with a view to maximising  the overall uptake of the finding of this study. While the reserach gaps are referred to in the discussion I would encourage the authors to go one step further and include an additional summary section/table along the lines of "Key Research Priorities to support marine biodiversity conservation in the RYS".

While this will no-doubt not be an exhaustive list - I think it would be really useful and make the document even more accessible if such a 'future research roadmap' could be created. The authors may want to consider including notes about the importance/significance/value of each research priority (or research question) and potentially rank each according to their perceived 'achievablity'. Within this table priority regions could be included (aka more reserach on all taxa is needed in the Tokara Group), or for example you could highlight more research on Echinodermata is needed in the Yakutane and Miyako Groups.


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: MARINE BIODIVERSITY RESEARCH IN THE RYUKYU ISLANDS, JAPAN: CURRENT STATUS AND TRENDS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Overall, I struggle to find the significance and relevance of the work as currently written in the paper.  The authors need to do a better job of describing the significance of findings and placing results in the context of specific past work done. Replace general statements with some specific details, while also making the writing more concise.

Hypotheses are not clearly described and validated.

What are you trying to show with Table 1?- it is very difficult to read. Wikipedia is not an acceptable source of information-- do more research to locate acceptable references. Some of the information is relevant and should be referenced more in the paper, such as the relative sizes of the regions and population, but it currently seems buried in this difficult to read table.  If you do not reference certain information in the paper, decide if it provides relevant information that would help explain research patterns, and use it in the discussion instead of burying it in a table. Much of the information would be much better presented as a figure. For example, you could show the marine research stations, labeled islands, land area and/or population size in a map relative to the number of papers or OBIS records found in these regions.

Your figures/table should clearly demonstrate the taxonomic and regional bias that you found, such that the reader does not have to search and struggle to find for the evidence of bias.

Overuse of terms like “urgent”, “stark”, “critical”, “clear” especially given the lack of clear patterns as currently presented in the text/figures.
Experimental design: Literature search methods are unclear, and methods section needs to be  revised.  Some more specific comments are in the attachment.

We need more information on relative rates of research… I am not convinced about the lack of research in the region because you need to back it up with relative figures (relative to land area, distance to research stations/university, population size, overall known biodiversity) and relevant background work. Figures that clearly present some of this information would help.

The analysis of OBIS records should not be described as an indicator of research rates, but instead as online accessible occurrence records. This is valuable in a different way, as these records can be easily downloaded and used for species distribution models, etc. In contrast, data from published research is often relatively inaccessible.  As I mention in specific comments, OBIS records come from a variety of sources, not all a result of scientific research. I suggest including a figure with the number of OBIS records for one or more taxa that are particularly interesting in the paper, instead of supplement. Label the regions more clearly in the figure, and include other information that might help explain bias (i.e. location of research stations, populous cities, etc).
Validity of the findings: The discussion needs to be completely rewritten with more background information and context to concisely describe the significance of work in an organized and logical way.

Clearly lay out all of the potential sources of bias and how they relate to your results.  One key driver of bias is likely the activity of one or more specialists in a region. You should look into whether certain specialists are driving any patterns found.  For example, by looking at the collector/observers from OBIS records.

Why are the numbers of papers increasing?

After reading this paper, I also have very little idea of the content of work that has been published.
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: MARINE BIODIVERSITY RESEARCH IN THE RYUKYU ISLANDS, JAPAN: CURRENT STATUS AND TRENDS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The suggested improvements have been met - thank you.
Experimental design: The suggested improvements have been met - thank you.
Validity of the findings: The suggested improvements have been met - thank you.
Additional comments: Thank you for taking on board my comments and updating 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: MARINE BIODIVERSITY RESEARCH IN THE RYUKYU ISLANDS, JAPAN: CURRENT STATUS AND TRENDS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The current version of this article is much improved, and well written.  I find that the authors sufficiently address comments from previous version.
Experimental design: The methods are more clearly explained and questions defined.
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: DO THE REGULAR ANNUAL EXTREME WATER LEVEL CHANGES AFFECT THE SEASONAL APPEARANCE OF ANABAENA IN POYANG LAKE?
Review round: 1
Reviewer: 1

Output:
Basic reporting: Manuscripts can be described in a more professional English language. The figures and tables can clearly present the research results.
Experimental design: The experimental design of this research is reasonable and can clearly verify the scientific hypothesis.
Validity of the findings: The scientific problems are clear and it has practical guiding significance for the water resources management of Poyang Lake.
Additional comments: The manuscript can be published after revision.
The author needs to answer a few questions.
(1) Why did you only choose northern part between Duchang and Xingzi counties of Poyang Lake for research? Is there any Anabaena occurrence in other water areas of Poyang Lake?
(2) According to the water level data, the water level in 2013 was significantly lower than that in 2014. Why did the authors not compare the difference between two years of Anabaena? Why is there a difference in the peak of Anabaena occurrence in two years?
(3) We recommend increasing the canonical correlation analysis to determine which important environmental factors have significant impact on the biomass and heterocyst frequency change of Anabaena.
(4) We recommend increasing the results of correlation analysis between water level and other water environmental factors.


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: DO THE REGULAR ANNUAL EXTREME WATER LEVEL CHANGES AFFECT THE SEASONAL APPEARANCE OF ANABAENA IN POYANG LAKE?
Review round: 1
Reviewer: 2

Output:
Basic reporting: 'no comment'
Experimental design: 'no comment'
Validity of the findings: 1)	What are the main environmental factors affecting the dynamic changes of phytoplankton community structure? Need to be supplemented in the paper.
2)	 How does the change of water level affect the community structure of phytoplankton? The changes of biomass, community structure and population dynamics need to be further elaborated.
3)	There are several kinds of algae in the composition of cyanobacteria. Is this the change of influence on Anabaena? Is it the same for other cyanobacteria or only for Anabaena?
4)	 Does the change of water level imply the change of other environmental factors? Additional explanations are needed in this regard.
5)	There are many papers on the effect of water level on phytoplankton,
 which need to be further supplemented.
Additional comments: The change of water level has an impact on the structure of phytoplankton community. On the one hand, it affects the composition of phytoplankton community, on the other hand, it affects the size of individual phytoplankton. The author studied the relationship between the water level change of Poyang Lake and Anabaena, which has a good role in guiding the management of water resources, but it needs further modification and can be considered for publication.


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

Input:
Title: IDENTIFICATION AND DIFFERENTIAL EXPRESSION OF PIRNAS IN THE GONADS OF AMUR STURGEON (ACIPENSER SCHRENCKII)
Review round: 1
Reviewer: 1

Output:
Basic reporting: 'no comment'
Experimental design: 'no comment'
Validity of the findings: 'no comment'
Additional comments: Overall, the manuscript is well written and easy to follow. The datasets and analyses are described sufficiently and the presented results are convincing. There are few places, as outlined below, that I recommend the authors to review.

* In the abstract,:"sturgon" should read "sturgeon" (Line 33, 36 and 39).

* In the introduction, the authors should mention what is known about piRNA generation in sturgeons. Molecular mechanisms, like ping-pong model (as mentioned in Line 318) should be mentioned here as well.

* The section 'Small RNA annotation and piRNA identification' is not clear. It says, previously published transcriptome was used to map small RNAs. Does "clean unique reads" refer to small RNAs sequenced for this study? In that case, why were the redundant reads removed from small RNA libraries? I can see why one would remove redundant reads from mRNA-seq, but given this is small RNA-seq wouldn't one expect to see many small RNA reads of exact same length and nucleotide composition to be sequenced? Please make it more clear in the paragraph every time you refer to small RNA seq datasets generated for this study.

* In that same paragraph, the steps taken to remove certain small RNA reads should be stated instead of just saying 'as previously described' (Line 157).

* The link to the piRNA database provided in line 165 is not working.

* In line 211, the authors should write clearly what transcriptome reference sequences were used.

* Re-write the Lines 210-211: "After these results were mapped to the A. schrenckii transcriptome reference sequences, 3.1 - 3.4 × 10^6 perfectly matched small RNA reads remained", with e.g.,
'About 3.1 - 3.4 × 10^6 small RNA reads mapped to the A. schrenckii transcriptome reference sequences with a perfect match.'

* Re-write the Line 220: "A total of 5.7 - 6.2 × 10^5 piRNAs reads, corresponding to 16.6 - 19.8% perfectly matched small RNA reads, were obtained." e.g.,
'In total, we identified 5.7 - 6.2 × 10^5 piRNAs reads in our sequencing results, that account for 16.6 - 19.8% of perfectly matched small RNA reads.'
To further improve the clarity of that sentence, it may be worth repeating, what was the criteria to claim that a given read is indeed a piRNA. Give a short explanation and some references, too.

* Line 221: The authors identify 875,679 putative novel piRNAs from the testis and ovary libraries. Give a short explanation why these reads are considered 'putative novel piRNAs'. What fraction of the reads these novel piRNAs account for? Also, refer the reader back to the methods, by writing, e.g., 'novel piRNAs were predicted by following steps described in the methods section'.

* In Figure 1A.: You should state clearly, whether the figure considers only known piRNAs, only putative novel piRNAs or all together.

* Line 225: It is unclear how the "rate of sex-specific expressed piRNAs was up to 87%" was calculated. There should also be some description of what it implies.

* Figure 2A: Is this figure showing just '875,679 putative novel piRNAs'? Line 236 would suggest so, but the figure legend says it is all small RNAs.

* Line 236: "26-30 nt in length according to the potential piRNA reads" >>> it may be better to write it as follows:
>>> "Both male and female sturgeon gonads displayed a peak in reads of 26-30 nt in length. This is an expected length distribution for potential novel piRNA reads."

* Line 239-240: "showed a peak in reads of 20-24 nt in length corresponding to miRNA reads" >>> ...showed a peak in reads of 20-24 nt in length which is an expected length distribution for miRNA reads.

* Line 241-242 "Moreover, both ovary and testis samples had a strong 5’ U bias in reads of 20-24 nt (miRNAs) and 26-30 nt (piRNAs) in length, whereas only one peak of 5’ U was found in reads of 20-24 nt among somatic samples (Figure 2B-2D)." >>> I think the authors meant "... whereas only reads of 20-24 nt among somatic samples had a 5’ U bias."
Following up, does this figure only include known/infered miRNAs and piRNAs or is it all reads of that size and simply called miRNAs and piRNAs? Please make it clear in the text.
The legend to the Figure 2 should also be more precise and clear about what is being plotted.

* Line 248: "After mapping onto the A. schrenckii transcriptome, " >>> What exactly was mapped? Just the 875,679 putative novel piRNAs? This should be clearly stated.

* Line 248: What criteria/threshold was applied to call a gene a piRNA-generating gene? Also, give a reference to the list of genes used in the mapping.

* Line 250: "up to 4,181 unique piRNAs reads per gene (mean of 18 piRNAs per gene)." The values provided should be scaled at least to the library size, rather than just giving raw number of reads.

* Line 254: What tool was used to perform GO analysis?

* Line 260: Do the authors mean "piRNAs" or sex-specific piRNA-generating genes? Double check.

* Line 263: Name the statistical test used in the calculation of the p-value. Given that the p-value is just 0.01 it is probably better to just say that a statistical difference was detected, although not to a very high degree.

* Line 266: How the 1871 piRNAs were chosen? Repeat briefly and point back to the methods.

* Line 273: "ASY-piR-122 with median of normalized intensity 13.33 in ovaries vs. 4.91 in ovaries" -- I believe the second value refers to testes.

* Line 275: "Further analysis indicated no significant difference in piRNA size distribution among sex-biased piRNAs " --> A paragraph before the authors write there is a difference is sex-specific piRNAs which creates a confusion at first. It would make it easier if the authors could stress more the difference between sex-specific and sex-biased piRNAs.

* Line 278-280: "The correlation analysis indicated that the expression of piRNAs detected by Illumina sequencing was correlated with those identified by microarray (for 1,871 piRNAs: r = 0.202, P = 0.000; 311 sex-biased piRNAs: r = 0.489, P = 0.000)."
>>> Maybe it is better to highlight the fact that sex-biased piRNAs were more correlated. Are these also more highly expressed in the Illumina dataset?

* Line 318: It is unclear how Figure 1A points to a Ping-Pong model.

* Line 319: "we found the presence of piRNAs in both male and female sturgeon gonads, but not in somatic tissues (Figure 2A)". Figure 2A only shows the size distribution of the sequenced reads. This sentence should probably read:
>>> "Small RNA reads with characteristics typical to piRNAs were found in both male and female sturgeon gonads, but not in somatic tissues."

* Again, the statement in Line 323 is based on p-value 0.01: "significant differences in the sizes of sturgeon sex-specific piRNAs (read counts ≥ 10) were observed."

* The statement in Line 345: again, I think it would benefit the readers to write clearly what is the difference between sex-biased and sex-specific piRNAs.

* Figure 4: The authors should include some information regarding the clustering method used and what metric was used for calculating the distance. This can either be in the legend, or somewhere in the main text.

Other comments:

* Increase the label size in the supplemental Figure S1.
* Table S1: enlarge the column 'clean reads' so that the second line can be read.
* Increase the resolution of Figure 1.
* Font size in Figure 2 could be bigger.
* Figure 3 could be split in two or three parts and plotted along side. This way one could make the text bigger and thus more readable.
* Figure 4: The heatmap, in its current state, does not seem to give any more information than said. If there are clusters that are particularly interesting, it may be worth highlighting those and give some description. One could also try including expression of these piRNAs as determined by Illumina sequencing for comparison.