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: COMPARING TRANSCRIPTOME PROFILES OF HUMAN EMBRYO CULTURED IN CLOSED AND STANDARD INCUBATORS
Review round: 1
Reviewer: 2

Output:
Basic reporting: There are multiple grammatical issues throughout the manuscript that need addressing, particularly with respect to tense, when to use singular versus plural, and overall sentence structure. A native English speaker should review the manuscript beforehand.
Experimental design: Please see below regarding transfer degree, embryo pooling, morphokinetic measurements, and reporting of raw data.
Validity of the findings: The authors state that the validation of the RNA-seq data by quantitative RT-PCR (RT-qPCR) was not performed due to human embryos being precious and that there is no solid evidence that this “provides extra significance to the results.” It’s fairly well-accepted that RT-qPCR should be conducted regardless due to errors that RNA-seq platforms can produce and that the sensitivity of the RNA-seq data is based on the read depth. Therefore, RT-qPCR of leftover RNA or RNA isolated from new embryos should be completed to confirm the findings.
Additional comments: The main objective of this study by Li and colleagues was to examine the transcriptomic profiles of human embryos cultured in a time-lapse imaging (TLI) incubator and a standard incubator (SI) to determine whether a closed culture system has a positive impact on embryo development. Using RNA-sequencing (RNA-seq) of pools of zygotes and TLI versus SI 8-cell embryos of similar morphological grade, the authors determined that the whole transcriptomes of TLI and SI embryos were similar, but distinct from that of zygotes. More importantly, they show that there was differential expression of 539 genes in TLI and SI 8-cell embryos. Gene Ontology (GO) analysis identified higher expression of genes involved in cell differentiation and DNA methylation in TLI embryos, whereas genes associated with RNA transcription, splicing, and DNA repair were enriched in SI embryos. Overall, the manuscript is not well written and there are multiple issues that need to be addressed in order to support the authors’ conclusions of the study as follows:

Major:

(1)	The authors state that “All human embryos used in this study exhibited normal morphology, however didn’t reach the transfer degree.” This is contradictory and additional morphological details with regards to what constitutes the transfer degree and whether there were differences in the number of embryos with different transfer degrees between embryo groups needs to be clarified. Under the limitations in the Discussion section, it also needs to be stated that all the human embryos used in this study were not of the highest quality and therefore, their findings require further investigation.

(2)	It is stated that 5 embryos per group were pooled for RNA-seq analysis. Why was this done when there are several low-input sequencing protocols available, including those for even single cells? How can the authors discount possible embryo heterogeneity within the same or different pools? Also, how many patients are represented in each embryo group to ensure the results are not patient specific?

(3)	Besides providing a closed culture system, TLI incubators enable the measurement of morphokinetic parameters and yet, the authors did not do this. Since the embryos were pooled for RNA-seq analysis, were there any differences in mitotic timing or cytokinesis (bipolar versus multipolar divisions) between embryos within the same pool or different pools? The findings of the study would be more impactful if these analyses were included.

(4)	The authors report the percentage of raw sequencing data that remained after trimming and quality control assessment. It’s the average genome coverage that should be calculated instead and based on this coverage, will determine whether the authors actually performed “deep sequencing.”

(5)	The name of the differentially expressed genes are identified for the first time in the Discussion. This data to should be moved to the Results section in a logical manner to support the GO findings.

(6)	From the methods section, it sounds as if the zona pellucida was removed and the embryos transferred to lysis buffer after freeze-thaw and pooling. Is this accurate? There are no methodological details provided for ICSI.

Minor:

(1)	The use of the term “sequencing fragments” could be confused with cellular fragments. I would suggest that the authors be more specific in their language.
(2)	On lines 90 and 95, the authors refer to “injected oocytes,” but it’s not until line 119 that the term ICSI is first introduced.
(3)	On line 117, the authors state that the embryos were collected from “volunteers.” I think the more appropriate term is “patients.” Also, does this age range refer to maternal age only? Typically, the average age is given. The authors do not state the total number of patients that donated embryos.


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: COMPARING TRANSCRIPTOME PROFILES OF HUMAN EMBRYO CULTURED IN CLOSED AND STANDARD INCUBATORS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The re-submitted version of the article submitted by Li et al has improved dramatically . The major issues with the previous version have been addressed by the authors.
Major Comment: It would be helpful to have some more specific functions of upregulated genes in the discussion section.
Minor Comments:
Line 65: delete 'of'
Line 169: check grammar
Line 176: check grammar
Line 180: check grammar
Line 311: change exposure to exposed
Line 322: change cultured to culture
Line 355: Change leaded to lead
Line 362: Missing citation/ reference
Line 384: Check grammar
Line 385: Check grammar
Figure 4: Label Y axis
Experimental design: In the previous version 2 of the major concerns were 1) English, and 2) lack of qPCR validation of the upregulated genes. Both have been addressed.
Validity of the findings: This study is impactful since it gives a snapshot of differentially expressed genes in the embryos cultured in the SI and TLI system.
Additional comments: All the previous concerns have been addressed in the rebuttal.


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: COMPARING TRANSCRIPTOME PROFILES OF HUMAN EMBRYO CULTURED IN CLOSED AND STANDARD INCUBATORS
Review round: 2
Reviewer: 2

Output:
Basic reporting: (1)	There are still numerous grammatical issues throughout the manuscript that need addressing. For example, see:
(a)	Line 125: “more information of on the process”
(b)	Line 183: “which in turn lead”
(c)	Line 276: “ICSI was performed with 5 h of oocytes retrieval”
(d)	Lines 331-332: “without family heredity case history and smoke history.”
(e)	Lines 333-453: “Every RNA-Seq pools have 5 zygotes or embryos from 5 different patients, which can discount possible embryo heterogeneity from patient factor.”
(f)	Line 454: “were vitrification frozen”
(g)	Line 458: “immediately every five embryos each tubes.”
(h)	Lines 459-460: “present study exhibited normal morphology, however didn’t reach the transfer degree in this cycle.”
(i)	Line 652: “human embryos transcriptomes”
(j)	Line 828: “especially the biological processes terms”
(k)	Line 918: “changed when exposure”
(l)	Line 1021: “possessed several different transcriptome.”
(m)	Line 1170: “which contain”
(n)	Line 1176: “leaded to the blockage”
(o)	Lines 1183-1184: “were several evidence showed”
(p)	Line 1226: “needs to further research”
Experimental design: (2)	The authors do not specify if a DNase step was included or exon-spanning primers used to remove contaminating genomic DNA (gDNA) and avoid amplification of gDNA, respectively, for quantitative RT-PCR (RT-qPCR).

(3)	A minimum of 2 genes, preferably more, should be used for the normalization of RT-qPCR data in order to avoid individual gene variation between embryo groups. This is particularly important since the authors use GAPDH to normalize all the RT-qPCR data, but GAPDH has been shown to be more highly expressed in aneuploid human embryos (Vera-Rodriguez et al. Nat Comm 2015) and aneuploidy is common in cleavage-stage human embryos.

(4)	Additional experimental details are needed in the Materials and Methods section even if described previously. For ICSI, the manipulator system used. For RNA-seq, the Illumina kit used (75, 150, 300 bp?). I assume paired-end?
Validity of the findings: (5)	The word “clean” is not typically used to describe sequencing data after processing. A more appropriate term is “trimmed” or “processed.”

(6)	On Lines 1218-1220, the authors state: “In addition, it is well known that early embryos have the ability of correct the alterations in gene expression during the subsequent development.” Do you have a reference for this statement b/c as far as I know this has not been established.
Additional comments: In the revised version of their manuscript, Li and colleagues addressed some of my questions and concerns. However, there are still certain issues that need to be remedied.


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: VISIBLE IMPLANT ELASTOMER (VIE) SUCCESS IN EARLY LARVAL STAGES OF A TROPICAL AMPHIBIAN SPECIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: The study is well designed. However, it would have been much more interesting, and probably more similar to field conditions, if it would have included more than one VIE color and different sides of the tadpoles, simulating multi-marking in the field.
Validity of the findings: The main novelty of the study relays in the application of a well-known technique (VIE) to a new group of organisms, at a particularly small size. This on itself, is not a great novelty, but opens the field for conducting much more detailed studies on the ecology and behaviour of Dendrobatid frogs, which present many characteristics that make them especially interesting study subjects.
Additional comments: This is a nice methodological study that presents the results of VIE marking in Dendrobatids all across the larval development. Authors were able to mark individuals right after hatching and followed them until metamorphosis. The study reports the VIE marking of the smallest tadpole until now. The study presents a high rate of VIE retention that will allow to export this technique to field conditions in order to conduct detailed studies on the ecology and behaviour of these species.

Overall, this is an interesting study, with lot of potential to influence future work. I have only minor comments that will hopefully improve the manuscript.

Line 45: The range of marking techniques included here is a bit limited. I guess it should be good to include some of the more used methods for individual marking in wildlife e.g. metal and colour rings (birds), ear tags or collars (mammals)...

Line 48. Substitute "frogs" by "amphibians" in order to be a bit more general.

Line 63. Expand this comment. Individual identification is important not only for behavioural studies, but for studies on physiology, life-history, and ecology in general.

Lines 64-65. Expand this sentence also, considering the previous comment. Add more examples here.

Lines 67-68. As mentioned before, much more examples of widespread techniques including metal and colour rings in birds, ear tags and collars in mammals, GPS/radiotracking devices.

Line 80. Change “anurans” by “amphibians”, more general.

Line 86-89. Tadpoles are not especially plastic in the tropics. They are, overall, highly plastic in temperate regions too as many studies have shown (see studies by Gómez-Mestre, Laurila, Merilä…).

Line 92. I suggest to add a few examples about the interest of individual marks in amphibians, before moving to the specific case of poison frogs.

Lines 97-99. These lines are the classical ones you expect to find on the last paragraph of the Introduction. I short of understand the rationale of having something like this here. I suggest authors try to find a good way to move them from here to the end of the Intro, and simply introduce here the study species, or something similar.

Line 99. Change “is” by “are”.

Methods. I miss a more general description of what VIE are. For the ones we are familiar with VIE elastomers it is quite clear, but more general audiences would benefit for a more detailed description of the material, different types of elastomers (colours, possibility of UV detection…).

As a general methodological comment. Since the idea is to test this method for future use in the field, it would have been better to test combinations of colours, and sides of the tadpoles in order to generate a larger set of codes. Understanding the detectability and relocation of colour combinations is key for the use of this technique in the field.

Statistics. I am not proficient on Bayesian models, so I cannot assess this section properly.

Line 250. I think authors should be careful when commenting on the role of the methods for transport behaviour studies due to the really small sample size (n= 2). I suggest to say that the study “suggests” that VIE do not prevent, rather than using more absolute terms (“did not prevent”). Same for lines 288-289.

Line 301-302. Mortality during early developmental stages in amphibians is high, no doubt, but I guess here is probably due to sub-optimal conditions in the lab (feeding, probably) more than anything.

Lines 316-324. These results on the relocation of VIE are really interesting.

Figure 1. Not sure about this formula as figure 1.

Figure 4. “growth rate percent” is a rather unusual way to express growth rate in tadpoles. I suggest to change this trait to mg/day or something similar, more often used in other studies.


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: VISIBLE IMPLANT ELASTOMER (VIE) SUCCESS IN EARLY LARVAL STAGES OF A TROPICAL AMPHIBIAN SPECIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors report a study assessing a new anesthetization technique coupled with a trial of VIE label marking in early stage Dendrobates tinctorius.

As the authors point out, marking methods have been disproportionately focused on temperate species; so, it is great to see a study on a tropical species. This study is impressive in the small size of tadpoles that were marked. Methods for marking early stages are sorely needed but few methods exist. I applaud the author’s success at marking such small tadpoles (and for attempting to even mark embryos).

Overall, I found the writing easy to follow. Although I have some questions and suggestions about the methods/analysis, I thought the experimental design and their interpretation of the results were straight forward.

There are two main issues I have with the manuscript as written. First, the observational methods need to be detailed much more explicitly since the main results hinge on them. Second, the issue of marking through metamorphosis needs to be addressed formally. I detail these in subsequent sections.

I had a lot of fun reading this manuscript and appreciate the hard work that the authors put into this study.
Experimental design: In general, I think the experimental design is reasonable. However, I do have some important concerns.

My main criticism of the methods is that the main result (successful mark retention) is contingent on observation of the tag; however, there is not enough description of the observation method. For instance, did a single person observe at a time or did multiple people look for the mark to validate? Did the same person observe the larvae at each every time-point? Without knowing the answer, it is hard to know how confident we should be in a false negative—were false negatives the result of observer error or the mark physically migrating during development?

It is similarly difficult to know how confident we should be about positive observations. For instance, were observations conducted blind to control for bias? Were control animals also assessed? If so, it would be useful to report the false positive rate (e.g. in Supplemental Figure 5 it looks like there are other fluorescent particles that might lead to false positives).
Validity of the findings: First, I want to thank the authors for including very easy-to-follow code. I especially appreciated that they left their comments in the code, which made it easy to understand their justifications.

Overall, I think the stats are sensible, especially given their sample sizes. However, I think the authors need to provide more justification for why they are mixing inference frameworks. For instance, tag retention and survival could have been modeled with frequentist mixed models in the same way that growth rates were analyzed. Or, Bayesian methods could have been employed for all. I fully trust that the authors had good reasons for the analysis strategies they chose, but would like to know why.

A few specific points:

I would like to see model report tables for growth rates and survival somewhere (supplemental information is fine). This information is included for the mark retention analysis but not for the other models.

One minor, but potentially important issue in the statistics code is that, when comparing linear mixed models, model should be fit with maximum likelihood. The authors are comparing models fit with restricted maximum likelihood, which is invalid (see Zuur's 2009 book on mixed effect models). Also, note that there is no link function specified in the glm model, which means that it is fitting the same model as the lm model (this is apparent when comparing AIC values). I seriously doubt that this will influence the results, so I'm not too worried, but just a good practice to be aware of.

Throughout the manuscript, the authors mention the success of their technique for marking across metamorphosis. I realize that mortality precluded formal analysis through metamorphosis, but some reporting on mark retention in metamorphs needs to be included. Given the limited number of metamorphs, I'm not sure strong claims about marking success between lifestages are warranted.
Additional comments: Here are some comments about specific sections or lines in the text:

The introduction seems broad for the context of this study. I understand that the intent is to make the case that marking is useful in biological studies, but perhaps it would be better to anchor the motivation for this study more in amphibians rather than large mammals? There are lots of reasons herp folks need to mark individuals that are directly relevant.

Line 78-79: “long-term marker-based studies have not often been applied to animals with complex life cycles” is not necessarily true. Marked have been used on animals with complex life cycles, but only in adult stages. I think you mean that marking “across ontogenetic stages” has not been common.

Line 85-89: It sounds like you are making the argument that tagging success may differ because neotropical species are more plastic than temperate species, which, I don’t think is true, at least morphologically and physiologically.

Line 97-99: The last sentence seems out of place in this paragraph. Maybe it would fit better in the final paragraph of the introduction?

Line 103: Just a note: Andis (2018) tagged larvae earlier than stage 30 to assess mark retention within the larval stage, but only considered inter-stage marking through metamorphosis in larvae over stage 30.

Line 111: You cite Gosner (1960) here but the first reference to Gosner staging is in the prior paragraph.

Given the focus on the somewhat unique natural history of D. tinctorius (e.g. parental care, larval transport, etc.), it might be useful to include some natural history for the species in the “Study organism” section. At the very least, I would like to know how long the typical larval period is for this species in the wild. Also, since other larval marking techniques have been implicated in reduced fitness and increased predation, it would be good to know what relevant selection pressures D. tinc. larvae face in nature.

Lines 122-132: This paragraph switches between past and present tense quite a bit.

Line 163-164: Out of curiosity, does this mean that the elastomer is waning the whole time or is there some threshold after which it starts to diminish?

Line 184-188: This does not seem to make the result very applicable to field use since, in the wild, an undetected mark will be categorized as “unmarked” and would not be associated with prior or subsequent observations (since, in the field, individual ID is reliant on the mark, unlike the lab where individual ID is known regardless). Maybe a better way to present this information would be to report false negative rates (and false positive rates, if you can) over time. This would have the added benefit of illuminating any ontogenetic pattern (if, for instance, there is some stage in which integumentary development tends to mask the mark).

Line 240-262: Somewhere in the results the number of metamorphs and detection rate for metamorphs needs to be reported. (I recognize that mortality made it impossible to make statistical inferences about post-metamorphic retention, but the raw numbers can still be reported). It would also be useful to know at which week larvae tend to metamorph, since all the graphs and models deal with time in weeks rather than developmental stage.

Line 373-377: It might be worth discussing how fluorescent marks may impact survival in the wild. For instance Carlson and Langkilde (2015) found that a staining procedure made larvae more susceptible to predation. Given that amphibians have evolved specific color patterns for a reason, it seems reasonable to consider that altering that pattern very well may inhibit crypsis or aposematic signaling.


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: VISIBLE IMPLANT ELASTOMER (VIE) SUCCESS IN EARLY LARVAL STAGES OF A TROPICAL AMPHIBIAN SPECIES
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is clearly written and easy to read. The introduction and background are well-supported with references to the literature. The manuscript conforms to PeerJ’s structure, the figures are well-presented (although I think one may be unnecessary – see minor comments), and the raw data are supplied (see raw data check).
Experimental design: This is an original experimental study with a well-defined research question. There are a few aspects of the study and methods which I think need additional information (see comments) to be replicable but the vast majority of the information is present.
Validity of the findings: Conclusions are generally well stated and clearly supported.
Additional comments: The manuscript entitled “Visible implant elastomer (VIE) success in early larval stages of a tropical amphibian species” presents an experimental lab study. The authors marked larval frogs at an early developmental stage and tracked them across development to examine the persistence of the marking and to understand any effects of early marking on growth and survival by comparing with unmarked individuals. The authors found that marks persisted at a remarkably high rate for the first month with declining retention over time. However, some individuals retained their marks through metamorphosis. In addition, the authors found no effect on growth or survival as a result of the marking procedure. The study is well-written and the motivation is well-explained. This manuscript will appeal strongly to anyone interested in marking animals at early life stages, particularly researchers studying organisms with complex life histories. In addition, I thought the authors’ discussion of mark retention vs mark detection was very thoughtfully done and I’m glad they included it. Below, I detail my comments for the submitted manuscript.

-Persistence of marks and study of the animals over time:

Much of your introduction is framed around the idea that researchers are in need of a marking method that can be applied to an organism at a very early developmental stage and a small size that will persist through metamorphosis. I agree with this framing and feel your study can provide an important tool here. However, there were some important details missing from the manuscript that prevented me from feeling that I fully understood how well the marks persisted over time and how the animals were kept during that time period.

How long were the animals housed in the manner described at the end of the first paragraph of the methods? Your figures give data across a 12-week time span but it’s not written in the methods or results. In addition, Line 243 says that 3 months is approximately the amount of time that these animals took to reach metamorphosis and Line 283 says that this is the only study to follow individuals across metamorphosis but no mention is made of how the animals would be housed after metamorphosis. How long is time to metamorphosis, on average, for this species? Did you house post-metamorphic animals for the duration of this study? If so, how were they kept?

Did you follow all individuals all the way through metamorphosis? You indicate in several places that there was high mortality around metamorphosis and high juvenile mortality but how many individuals survived to metamorphosis? Past metamorphosis? And for how long? Much of the follow through to your introduction depends on your reader understanding that the marks can persist across metamorphosis. However, apart from telling us that they did so in the discussion, this information is not provided in the methods or results.

The raw data seems to indicate that animals were marked in October 2019 and some animals were followed through April 2020. Given that I am reading your manuscript in May 2020, it is completely reasonable that you stopped analyzing these animals at some point for this manuscript. However, please explicitly tell us the time span that the analyses here covered and the number of individuals surviving at the end of that time.

-Novel anesthetic:

You mention in the abstract and in the introduction that you are using a novel anesthetic, 2-PHE. I think it would be useful for your readers to give some additional information about it. You give us a brief explanation at the end of the introduction, lines 113-115 – that it does not need to be buffered and can be stored at room temperature which makes it advantageous for field work. This is helpful but I’d also like a bit of background. Has it been in use as an anesthetic previously in other organisms? What were its previous applications? Can it be handled safely and easily? Is it difficult to obtained?

For non-amphibian researchers, it might be useful to talk briefly about how amphibians are usually anesthetized and why you think 2-PHE is advantageous.

Also, on Line 136, tell us more specifically how many times the solution was used within a day? It doesn’t need to be exact as I can guess that the number of individuals that you tagged on given day varied. However, an idea (5, 10, 20?) would be useful to give the reader an understand of how many times the anesthetic can be used within a day, once prepared.

Minor comments:

-The reference list is duplicated within the Reference section. The repeat begins on Line 545.

-Figure 1 was unnecessary for me. I thought your verbal description of the statistical methods was very thorough and I understood how tag retention was marked in your dataset without it so I think you could remove it or move it to the supplement.


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 ABILITY OF THE GREEN PEACH APHID (MYZUS PERSICAE) TO PENETRATE MESH CROP COVERS USED TO PROTECT POTATO CROPS AGAINST TOMATO POTATO PSYLLID (BACTERICERA COCKERELLI)
Review round: 1
Reviewer: 1

Output:
Basic reporting: The study presented in this manuscript is a relatively small yet straightforward lab test.  However, there are some critical assumptions and a great deal of speculation included. For example, this study did not test the screens against TPP, but the authors did cite a study  by Merfield et al. (2015).  Interestingly, the Merfield study found TPP nymphs and adults underneath the row covers (see Fig 1 in Merfield et al.).  Thus, while TPP populations may have been reduced under the row covers, the impact on occurrence of Clas did not appear to be measured. Thus, the assumption that mesh covers will provide economic levels of CLas reduction may be premature. In addition, the authors used considerable space in the manuscript to describe how biocontrol agents might be used in conjunction with the screening to control GPA in the field.  However, no experiments were conducted to test their hypothesis that biocontrol agents can provide economic suppression under row covers in the field, so this discussion could be shortened.  In addition, it was not clear to me why adding biocontrol agents were the only options discussed.  Could the screens be treated with pesticides or an aphid repellent and achieve the same goal at less expense?  How about coloured screening?

The list of locations provided in the first sentence of the abstract suggests this is the range of the pest.  TPP is a pest in many other countries (Butler and Trumble 2012. Terrestrial Arthropod Reviews 5: 87–111).  I suggest the authors consider stating the pest is a problem on potatoes throughout most of Central America and all of North America, as well as New Zealand.

It would be useful if the authors compared their work to other studies where screens with different mesh sizes were tested against green peach aphids.  For example, Bethke et al. 1994. (Screens deny specific pests entry to greenhouses. California Agriculture. California Agriculture 48(3): 37-40) indicated the mesh sizes that prevented entry to green peach aphids.  Do their results agree with what was found in this study?  Is other literature available?

Some of the potato fields in NZ are quite large.  Is covering all of the rows with screening economically viable?  Or is this screening primarily being considered for seed potatoes?

Finally, the authors repeatedly point out that the interaction between aphid morph (alate and apterous) and the leaflet touching mesh or not was ‘nearly significant’.  They then indicate several times why this so important.  Finding importance or trends in non-significant data is questionable, and almost never justified.

Minor stuff:

Line 193: add ‘aphids’ after “apterous”.

In the Munyaneza references, the genus and species name of the TPP need to be in italics.
Experimental design: The experimental design is OK.  The statistics appear to have been conducted appropriately, but see the comment above on "nearly significant" findings.
Validity of the findings: see above
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 ABILITY OF THE GREEN PEACH APHID (MYZUS PERSICAE) TO PENETRATE MESH CROP COVERS USED TO PROTECT POTATO CROPS AGAINST TOMATO POTATO PSYLLID (BACTERICERA COCKERELLI)
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors evaluated the effectiveness of using insect-proof mesh screens as a barrier for green peach aphid, Myzus persicae and studied the effect of different mesh hole size, the stage of the green peach aphid that penetrates the mesh, to find whether or not leaves touching the underside of the mesh would increase insect feeding, and if adults can feed from outside of the mesh.
The manuscript is very well written with well laid hypotheses and good flow of information in the introduction. The authors have taken effort to consider various factors that would possibly affect the outcome of the study and taken necessary precautions at various stages of the study right from formulating the hypotheses to obtaining their final results keeping the reader informed.
Experimental design: Given the various factors involved in the study, the authors chose to do a factorial design. However, I find the authors have also mentioned about the randomized complete block design in line 133 and wish the authors explained about the 'so eight "controls"'.
Lines 127 to 131 in the methods sections is not very clear and may be rewritten for clarity.
Validity of the findings: The authors have done a great job reporting their findings. The findings are novel and informative. The authors also mentioned introducing bio-control agents under the mesh as a future scope of research.
In lines 184 and 185 the authors tried to explain a non-significant result. If results are non-significant there is no need to mention if the actual differences were small are large.
Additional comments: Some minor corrections are mentioned which do not detract the quality of the manuscript in any way.
Line 44 - delete 'the' glo
Line 52 - use quotation marks for the scientific name of the pathogen
Line 63 - delete 's' from covers and insert "to" reduce
Line 102 - potatoes under "the mesh cover"
Line 123 - cotton wool was "wrapped around" the petiole
Line 174 - large apterous "adults" ??
Line 201 - disperse "by" wind currents

The discussion is a little deviating presumably out of the scope of the listed objectives as to what mechanisms/cues the aphids adopt to land on the food source.
I have not checked the citations. So it may be good to verify the reference section.
Overall, the authors have done a great job presenting their research in a scientific manner that is acceptable to the standards of PeerJ. I therefore recommend publication of this manuscript in PerrJ following minor corrections.


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

Input:
Title: THE ABILITY OF THE GREEN PEACH APHID (MYZUS PERSICAE) TO PENETRATE MESH CROP COVERS USED TO PROTECT POTATO CROPS AGAINST TOMATO POTATO PSYLLID (BACTERICERA COCKERELLI)
Review round: 1
Reviewer: 3

Output:
Basic reporting: This paper details the use of Mesh crop covers on potatoes to protect against potato psyllids and additional challenges posed by aphids. They examine the different mesh sizes to manage the psyllid in potato. They further examined green peach aphid ability to penetrate different mesh hole sizes. Overall, this study is nicely done, and the methods seem sound. The analysis seems appropriate. The manuscript is well written with no revisions required.
Experimental design: Research methods were seem sound. The analysis was appropriate. The manuscript is well written with no revisions required.
Validity of the findings: The findings were 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: THE ABILITY OF THE GREEN PEACH APHID (MYZUS PERSICAE) TO PENETRATE MESH CROP COVERS USED TO PROTECT POTATO CROPS AGAINST TOMATO POTATO PSYLLID (BACTERICERA COCKERELLI)
Review round: 2
Reviewer: 1

Output:
Basic reporting: The revised manuscript has now included appropriate comparisons with previous literature.
Experimental design: no comment
Validity of the findings: This aspect of the manuscript is much improved.  The authors have reduced the speculation and focused the manuscript on what was accomplished.
Additional comments: This version of the manuscript is much improved.  The authors have adequately addressed all of my concerns, and I can recommend acceptance.


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

Input:
Title: OF POWER AND DESPAIR IN CETACEAN CONSERVATION: ESTIMATION AND DETECTION OF TREND IN ABUNDANCE WITH NOISY AND SHORT TIME-SERIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors describe their approach clearly and demonstrate impressive background reading with their literature citations.  The figures are well composed, and present a lot of information clearly and compactly.  Their are a few minor issues in English.  At line 126, for example, the plural of “scenario” in English is “scenarios” (despite its etymology!).
Experimental design: The authors motivate their simulations with a clear review of the difficulties of detecting trends in the populations of marine mammals.  They state clearly how they hope their simulations will improve the situation.  The paper would be strengthened if the authors connected their simulations to actual management goals.  For example, effect size (r) ranged between 0.99 and 0.5.  Is this range based on management goals within the EU framework?  Would a decline of 40% (r=0.6) in, say, 10 years be acceptable?  A technical point that I found puzzling was to generate the CVs from a uniform distribution (line 136). The precision of the estimates is related to the amount of sampling effort and to the technique (eg, line-transect vs mark-recapture), but it is under some control of the researchers, not a random variable.
Validity of the findings: Technically, the simulations were carried out as described.  The results are clearly stated, and both positive and negative aspects of the results discussed.

The structure of the simulations, however, could affect the validity of the findings.  The authors chose to simulate data and to carry out regressions in a way that did not conform to classical assumptions of linear regression.  Because of this, they find that the classical (unregularized) approach without priors produces biased estimates of trend, inconsistent type 1 error rates, and inaccurate coverage.  Their main conclusion that a weakly informative prior performs better than classical regression could be due to the structure of the simulations which caused the classical method to perform poorly.  The authors should address this issue.  If they believe that their non-standard regression procedures, including anchoring on the first point in the series, more accurately represents the way trend detection SHOULD be carried out with marine mammal data, they should explain why their non-standard approach is more appropriate.  The regression methods they used would not be the methods used by most researchers.
Additional comments: I have 3 general comments.

First, I am uncomfortable with the authors' endorsement of a method (regression with a weakly informative prior) that will tend to produce underestimates of the size of a decline (lines 401-3).  This is contrary to a precautionary approach, as the authors admit.  They attempt to put a positive spin on the underestimation, but in my experience saying that a decline is “at least as large as x%” is not effective.  The result will still be interpreted as a decline of x%.  The recommended non-precautionary approach is inconsistent with the authors’ call “for a more widespread and effectual application of the precautionary principle in conservation instruments” (line 435).

Second, the authors base their estimates of a decline on an estimate of abundance at each time period.  Thus, they consider that the information available (at each time) is a single number, the point estimate of abundance.  However, a point estimate is a summary statistic for the available data at each time, and a lot of information is lost by using only this single number.  Many papers have discussed the advantages of an integrated approach, which considers all the original data across all years within a single analysis.  Gerrodette (2011) showed the power of this method specifically for the detection of a change in the vaquita population.  The authors cited this paper, but did not implement or even discuss one of its main conclusions that would be relevant to their paper.

Third, “This paper hence deals with contesting a norm, not in challenging its validity but its current application” (line 383).  Thus, the simulations were carried out within the flawed framework of null hypothesis significance testing, a method of inference which has been widely criticized, especially in recent years.  Indeed, the authors present an excellent review of the issue.  However, they have chosen not to challenge the null hypothesis paradigm in this paper.  That is fine, but it means that the results of the paper will make, at most, an incremental improvement in the detection of trends in marine mammal populations, leaving all of us, as the title says, in a state of despair.  I wish the authors had challenged the validity of the current norm.  Perhaps they will in a future paper.  That would be, in my opinion, a more substantial contribution.


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: OF POWER AND DESPAIR IN CETACEAN CONSERVATION: ESTIMATION AND DETECTION OF TREND IN ABUNDANCE WITH NOISY AND SHORT TIME-SERIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: Basic reporting is well written. well cited, good structure. I have re-run much of the code and it seems well organised. I have not reproduced all results as the simulations take ~ 1 day each. However the authors did a really good job of their code and making it accessible.
Experimental design: A few comments on the text to improve clarity of the maths:

Data Assumptions:
In general please define parameters and subscripts before using them later on. For example t and r are both presented before their definitions.

Line 117: Please define the subscript ‘t’ early in the definitions section.  I assume it refers to discrete time intervals t= 1…T. Also, it might be useful to define your temporal sampling scheme as having a total of T sampling occasions evenly spaced at times t=1…T.  Perhaps defining the sampling scheme as the first assumption would clarify things.

Line 122, 123: I find the description of pt relative to r confusing. For instance in line 123, you express H0 as r=1.  Strictly speaking, If r^[(t-1)/(T-1)] is exponentiated, then H0 is only true when both t and T are 1.

Line 123:  This does not appear to be an assumption. I would specify it as a calculated parameter that arises from the assumptions.

Simulation Scenarii
Line 127:  As above, Equation (1) does not seem to make sense.  If r is the fraction of the original population remaining, then it should be expressed in terms of biomass or density.

Line 129: “… at time T >  1…”  do you mean t>1?

Line 143: “We varied the value of r (= eb )” here you provide another definition for r. Please clarify this in the definitions and equations above.

Estimation
Line 173: “The location parameter of the Cauchy distribution”

Line 176: “of the unknown trend parameter r  (b )”  this appears to be another definition for r.  Please make it obvious in the definitions if this is the case.

Case studies
Line 195: “We collected abundance or density estimates from published research or reports (Table 1).for density estimates from published research or reports (Table 1).”  Please say how many.
Validity of the findings: Findings are robust and the application to real-world data is good and relevant.
Additional comments: Very nice work. So good to see a good simulation  being used on real-world data.


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

Input:
Title: THE PHOTO-INHIBITION OF CAMPHOR LEAVES (CINNAMOMUM CAMPHORA L.) BY NACL STRESS BASED ON PHYSIOLOGICAL, CHLOROPLAST STRUCTURE AND COMPARATIVE PROTEOMIC ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The English of the manuscript must be improved because it is hard to understand overall.

The abbreviaton are not consistent through the manuscript. Some abbreviaton are not specificated like Caro (page 7 line 54) or PS1 and PS2 (page 8 line 67). Maybe a table with all the abbreviaton used could be useful.

Discussion's first section (page 22 line 383) is just a description of the results followed by a list of references.
Experimental design: No comment
Validity of the findings: What does biological replicate (page 17 lines 276 and 277) exactly mean?

Three replicate for the physiological analysis are not enough to obtain robust data.
Additional comments: The research question is well defined and the methods used fited the focus of the research.
However, the manuscript is not clear. The English must be improved and some sections should be written again to make them more understable.
Furthermore, the number of replicates seem insufficient for the variables measured.


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 PHOTO-INHIBITION OF CAMPHOR LEAVES (CINNAMOMUM CAMPHORA L.) BY NACL STRESS BASED ON PHYSIOLOGICAL, CHLOROPLAST STRUCTURE AND COMPARATIVE PROTEOMIC ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Grammar and language style should be improved, as several sentence are not correct, e.g.  “As a habitat of camphor seedlings, Jiangsu has about 15 km2 of its area along the coast is degraded…”(lines 86-90).

Many references deal with herbaceous species, mostly crops, not related taxonomically to .Cinnamomum camphora. I suggest to include only references on taxa of the same family or at least only on woody species.

Structure conforms to PeerJ standards.

Figures are relevant,of high quality, However, Figure 7 (A) (Photosynthesis pathway) and (B) (Photosynthesis antenna protein)  are not original, and the information they bring is not relevant for the MS. I suggestion their deletion or at least citation of their source in the legend.

Raw data are supplied..
Experimental design: Original research within the scope of the journal.
The aims of the work are well defined.
THE MS includes an extensive data set, with a diverse methodology, but the number of biological replicates is low.
Validity of the findings: Data are insufficient for a statistically sound treatemnt.
Conclusions are well stated, linked to the aim of the work.
Additional comments: The manuscript entitled "The photo-inhibition of camphor leaves (Cinnamomum camphora L.) by NaCl stress based on physiological, chloroplast structure and comparative proteomic analysis (#34911) " deals with a topic of interest. The authors have done a good and timely need research work. The article is well organized, starting from the abstract till conclusion and includes an extensive data set. The methods used are diverse and of high standard. However, my major concern is related to the number of biological replicates, just three for physiological analysis (the minimum for a statistic treatment of the data!) and only two for proteomics (insufficient for statistics!). There is no mention of the number of replicates for the ultra structure analysis.
Regarding the Introduction section, many of the references cited deal with herbaceous species, mostly crops, not related taxonomically to .Cinnamomum camphora. I suggest to include only references on taxa of the same family or at least only on woody species. Information on the of wild habitat of camphor tree and more details about the risk of secondary salinization in the area of origin of the seeds would be of interest, and important for justifying the aim of the study.
In Material and Methods section I could not find the number of seedlings submitted to treatments and details about the saline treatment (watering into the pots, into trays, etc…). Why the rather unusual concentration of 103.45 mM NaCl was chosen?
Results are well organized ad clearly presented and figures are of good quality. However, Figure 7 (A) (Photosynthesis pathway) and (B) (Photosynthesis antenna protein  are not original and in fact the information brought is not relevant. I suggestion their deletion or at least the citation of their source in the legend.
The Discussion should be focused olyon  related or woody species (as mentioned for the Introduction) and not on crops.
Grammar and language style should be improved, as several sentence are not correct, e.g.  “As a habitat of camphor seedlings, Jiangsu has about 15 km2 of its area along the coast is degraded…”(lines 86-90).


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 PHOTO-INHIBITION OF CAMPHOR LEAVES (CINNAMOMUM CAMPHORA L.) BY NACL STRESS BASED ON PHYSIOLOGICAL, CHLOROPLAST STRUCTURE AND COMPARATIVE PROTEOMIC ANALYSIS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The English used through out the work is of good level, however improvements can be made. I advice the authors to review the grammar used in this work, or even pass it by a native speaking scientist for corrections.

The work itself is of good standard, and has a lot of merit. However the authors did not highlight their main findings in a sufficient manner (the increase of MDA under stress and connecting it with their other findings in regards of the buildup of oxidative stress and its effects on the photosynthetic pigments).

The literature referenced could be improved especially in the discussion as well as in the introduction. I would highly recommend including some work from Pr. Tim Flowers or other leading scientists in the field of salt stress (maybe Pr. Shabala or Pr. Hasegawa), especially their most recent reviews.

The article is well structured and the figures are of excellent quality. The buildup of the work and results seem to be in good terms with the set hypothesis.
Experimental design: The presented work is with good terms of the aims and scope of this journal, and its stated hypothesis is clear and concise. The investigation of the salt stress effects on Cinnamomum camphora, a tree of commercial and economic value to the local economy of southeast China, makes this work relevant and holds merit.

The methods used are described in a very informative and sufficient manner, and the findings are presented in a good fashion. Though some more emphasis in discussing them could elevate the quality of this work.
Validity of the findings: The findings are novel and all in all make sense with the most recent findings in the field of abiotic stress, notably salt stress in trees. My only issue is that the discussion is not thoroughly discussing and highlighting some of the findings, with a lack of cited references.

The data is robust, and seems to be of good quality, on the other hand a two-way ANOVA could have increased the resolution of the statistical differences among treatments and within every treatment at different time points, combined with a student test. However the current form of used statistics (one way ANOVA) seems to be sufficient.

This work on young plantlets (less than one year old), and as such any findings and conclusions should state this and confine any speculations to this early stage of growth in the studied tree life.
Additional comments: I would suggest addressing the comments I have stated in my submitted revision of the manuscript, as well as seeking some help from a scientific translator or a native speaking scientist. The work is of good quality and holds merit, and would be a shame to associate it with anything but an excellent level of English language.


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: SILENCING OF THE ARK5 GENE REVERSES THE DRUG RESISTANCE OF MULTIDRUG-RESISTANT SGC7901/DDP GASTRIC CANCER CELLS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The language used in this article leaves much to be desired. There are several grammatical errors. The language and scientific terminology used are erroneous throughout the manuscript. The authors appear to exaggerate the significance of their findings without adequate scientific data (either from their own study or from available scientific literature) to validate them.

Examples of errors in language/grammar:
Line 26: “ARK5 gene is closely related to the multidrug resistance of gastric cancer cells in vitro and in vivo.” – Language needs to be revised. ARK5 gene could either “promote” or “stimulate” multidrug resistance of gastric cancer cells. The words “closely related” do not have inform the role of this gene in mediating drug resistance.

Line 48 “…satisfactory survival rate…” – what is a satisfactory survival rate? For any disease condition, the obvious satisfactory survival rate is ideally 100%. However, that’s not what we presently observe in cancer therapeutics. Please revise language by citing the current survival rate for gastric cancer.

Line 51: “…exposing to the one drug…” – the correct language here should be “exposing to one drug”.
Experimental design: 1. The cell lines used in this study were not authenticated. This substantially brings into question the validity of the findings of this study.

2. Only one set of cell lines (parental and multidrug resistant) were used in this study. The study findings should be confirmed in at least two other cell lines (parental and multidrug resistant) before they can be considered conclusive.

3. In line 172, the authors state, “A total of 6-week-old female NOD/SCID mice…”. The authors need to specify the total number of mice employed in this study.

4. In Figure 2, the western blot image is 1,2, 3 and 4. Please indicate what each of these numbers stand for in the figure legend.
Validity of the findings: The discussion of available scientific literature in this research and therapeutic area is inadequate. Discussion section of this manuscript needs to be revised.

The role of ARK5 in multidrug resistance of gastric cancer cells is not novel. However, the authors claim, "this study not only provides a new experimental basis for the key role of ARK5 in multidrug resistance of gastric cancer cells, but also provides a new target and new exploration direction...". This is not accurate. This study does not provide a "new experimental basis", "a new target", or "new exploration direction" in gastric cancer therapy. The authors repeatedly exaggerate the findings of their study in this manuscript.
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: SILENCING OF THE ARK5 GENE REVERSES THE DRUG RESISTANCE OF MULTIDRUG-RESISTANT SGC7901/DDP GASTRIC CANCER CELLS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is clear, written in professional english. The article structure seems to corroborate with the guidelines. Minor mistakes in sentence construction or typos do exist. I have pointed them and provided suggestions. The background seems to be concise and detailed. I have some suggestions which I have mentioned in my report.
Experimental design: Experimental design is sound and well described. The knowledge gap has been addressed and the experimental procedures have been mentioned with sufficient details.
Validity of the findings: Discussion and conclusion are robust, well stated. It is linked to the original research question and supported by the results.
Additional comments: Abstract

Line number 23: “Gastric cancer as…” should be replaced as “ Gastric cancer is…”

Introduction

Line number 43: Please do not start the abstract and the introduction with the same kind of sentence. I would suggest starting the introduction section of the article with a different sentence.

Line number 47-49: Consider breaking up the sentence into two. You can start the second sentence like : “ Besides chemotherapy resistance plays a crucial role in the failure of its treatment.”

Line number 49-52: Consider breaking up the sentence to improve clarity.

Line number 53: Replace “… provides an effective…” with “ ….providing an effective…”

Line number 60: Reference missing.

Line number 60: Consider breaking up the sentence to provide fluidity.

Line number 63: Consider modifying the existing line with: “ Previous studies indicated that AMPK which is a sensor of metabolism and a regulator of energy homeostasis has positive effects on promoting gastric cancer.”

Line number 66-68: Briefly mention what are the upstream and downstream targets of ARK5. Also give brief description of ARK5 pathway.

Line number 72: Add “…cells with high expression of ARK5…”

Line number 73: Replace “ studies” with “ cases”

Line number 74: Consider writing : “We propose that ARK5 may play a….”

Line number 78: Please rewrite this sentence. You can start like “ To investigate the molecular basis ….”

Line number 84: Use a separate sentence. Consider writing “ We also carried out….”

Materials and Methods:

Line 95: Line should be “ …cultured in plates containing the culture…..”.Mention if any antibiotics were used.

Line 162: Mention why this particular concentration of the drug was used for this experiment.

Line 172: “ A total of 10, 6 week old mice…..”. Please mention 10 here.

Line 139: sentence should be “…EP tube after resuspension and centrifugation….”

Results:

Figure 1: Consider writing a Figure caption : “ARK5 expression levels in parental and MDR cell lines”.
Please mention that it is a baseline expression level experiment.
What is the molecular weight of ARK5? Please add a molecular weight marker on the left-hand side and an arrow to denote the protein on the right-side panel of the figure.

Figure 2: Please add a molecular weight marker on the left-hand side and an arrow to denote the protein on the right-side panel of the figure.

Please add that the SGC7901/DDP-shARK5 expression is comparable or almost similar to that of the baseline expression of the parental SGC7901 cell line.

Line 235: Please specify Fig 4 A and B

Figure 4: What was used as a control experiment- DMSO or complete media? Please mention the drug concentrations for each of the drugs that were used in this experiment in the figure legend.

Line number 244: Specify the Fig5 as Fig 5A and 5B.

Figure 5: What was used as a control experiment- DMSO or complete media? Please mention the drug concentrations for each of the drugs that were used in this experiment.

Why the apoptosis rates of SGC7901/DDP in ADR is higher than that of DDP or 5FU drugs? Why more cells are undergoing apoptosis in ADR even though ARK5 is highly expressed? Please clarify this issue.

Figure 6: Mention if DMSO or complete media was used in control experiment in the figure legend?

Figure 7: Mention the name of the drug(s) used in this experiment in the figure legend. Also mention the concentration of the drug(s) in the figure legend.

Figure 7B: The body weight of the SGC7901 parental mice seems to be going down after 20 days. Any reason for that?

Line 258: Please specify the Figure 7 as Figure 7A, 7B …

Line 259: Why only 5-FU was used in this experiment? Why other chemotherapeutic drugs were not tested should be mentioned clearly.

Conclusion:

Line 315: Replace “fin” with “in”


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: SILENCING OF THE ARK5 GENE REVERSES THE DRUG RESISTANCE OF MULTIDRUG-RESISTANT SGC7901/DDP GASTRIC CANCER CELLS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The article is generally well written, however it needs to be corrections for language and grammar. The references for a study are missing and in addition more literature related to this manuscript needs to be included (as mentioned in comments to author). There are few issues with figure and the required modifications are mentioned in  comments to author.
Experimental design: The research question is well defined and relevant to the field. Few experiments needs to be modified for better analysis of the results.
The methods are described fairly, however I have suggested few modifications to improve the manuscript.
Validity of the findings: Although the importance of ARK5 has been reported in lung cancer, but it is a novel study for gastric cancer, which has its own importance.

It seems inclusion of some data related to ABC transporter might shed more light in this study.
Additional comments: Dear Author,

The manuscript entitled “Silencing of ARK5 gene reverses the drug resistance of
multidrug resistant gastric cancer cells SGC7901/DDP” has covered an impact aspect in the field of cancer cell treatment. It has shown the role of ARK5 gene in multidrug resistance (MDR) in gastric cancer and the effect of ARK5 silencing on cancer cell survival.
Overall the manuscript is well written and study well designed, however there are many issues with the scientific rationale and experimental strategies, which need to be considered in order to improve the manuscript.

1.	This manuscript has mentioned about ABC (ATP-binding cassette) family, the P-glycoprotein as a factor associated with MDR in various cancers. However, the study focuses on the role of AMP-activated protein kinase (AMPK). Is there any known association between these two pathways and how these might interplay to induce MDR?
2.	The authors cited the finding of some latest study as “latest study found that the down-regulation of the expression of ABC transporter could not completely reverse the multidrug resistance of tumor cells”. The reference for this particular study is missing.
3.	It should be mentioned in which cancer the ABC transporter was downregulated and insufficient to reverse MDR. The relevance of that particular cancer with gastric cancer must be included, in order to justify the need to study for alternative mechanism in gastric cancer for MDR.
4.	The rationale for selecting ARK5 for studying MDR specifically in gastric cancer must be explained. If any specific role of ARK5 in gastric cancer is known that must be included.
5.	Possible mechanism of action for ARK5 in regulating MDR in gastric cancer in context of possible downstream molecules might be important for this study.
6.	This study predicts the possible role of p53, however the expression levels of p53 have not been estimated.
7.	Based on the effect of ARK5 silencing on in vitro adriamycin accumulation and retention assay in treated cells the expression levels of ABC transporter must be analyzed to understand the possible link between these two pathways.
8.	The bases for selecting different drug concentration in cell viability assay must be explained.
9.	Ideally, the viability assay are done over a period of time like 3-5 days, however it does not seem to have been done likewise. The rationale for choosing a different strategy needs to be included in methods.
10.	 In methodology section, under apoptosis assay it must be made clear whether or not the cells in supernatant were included.
11.	 Change the axis title “relative expression level of proteins” to “relative expression level of ARK5” in figure 1 and 2.
12.	The western blots for ARK5 in figure 1 and 2 for SGC7901 / DDP and SGC7901 are not consistent, although these are under similar conditions. In addition, there are two bands for ARK5 in figure 2.
13.	The manuscript needs to be corrected for English language and 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: SILENCING OF THE ARK5 GENE REVERSES THE DRUG RESISTANCE OF MULTIDRUG-RESISTANT SGC7901/DDP GASTRIC CANCER CELLS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have revised the manuscript substantially. The revised manuscript is well written.
Experimental design: The authors have revised the Materials and Methods section of the manuscript to address all my comments and concerns.
Validity of the findings: The authors have revised the manuscript to address my previous concerns. No additional comments.
Additional comments: No additional comments


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

Input:
Title: SILENCING OF THE ARK5 GENE REVERSES THE DRUG RESISTANCE OF MULTIDRUG-RESISTANT SGC7901/DDP GASTRIC CANCER CELLS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The article is clear, written in professional english. The article structure seems to corroborate with the guidelines. The authors have addressed all the queries I have raised before.
Experimental design: Experimental design is sound and well described. The authors have incorporated all the details required.
Validity of the findings: Validity of the findings have been represented in a clear and concise manner. Based on the reviewers' suggestions they have modified the discussion which has strengthened the impact of this article.
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: SILENCING OF THE ARK5 GENE REVERSES THE DRUG RESISTANCE OF MULTIDRUG-RESISTANT SGC7901/DDP GASTRIC CANCER CELLS
Review round: 2
Reviewer: 3

Output:
Basic reporting: The manuscript has been modified considerably to improve the data interpretation and better undersatnding of the results.
Experimental design: Few experiments those required further clarification and better description of the methods has been achieved.
Validity of the findings: The results and findings in this study have an important scope in this field and seem to be fairly derived.
Additional comments: The manuscript has been improved sufficiently with inclusion of more relevant literature and figures have been also improved.


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: A NOTE ON THE EFFECTS OF EPIDEMIC FORECASTS ON EPIDEMIC DYNAMICS
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: REVIEW REPORT on
A note on the effects of epidemic forecasts on epidemic dynamics (#48416)

The paper considers an important anecdote on the epidemic dynamics modelling. It examines in what way the epidemic forecasts effect epidemic dynamics via some adaptation on the traditional SIR model. In general, I found the paper appropriate and useful for scholars studying in this research area. Also it fits well to the scope of PeerJ journal. I have only some small points to be corrected before publication. They are as follows:
•	In line number 70, in the first equation of the traditional SIR model, “dT" should be “dt” as in other two equations.
•	Regarding the Question (2), authors use an infection peak equation. Where this equation come from?
•	Authors should briefly provide how they perform transformations from classical SIR to case 1 and case 2. It is beneficial to add what tools they benefit and in what platforms they solve the mathematical problems.


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: A NOTE ON THE EFFECTS OF EPIDEMIC FORECASTS ON EPIDEMIC DYNAMICS
Review round: 1
Reviewer: 2

Output:
Basic reporting: No Comment.
Experimental design: No Comment.
Validity of the findings: No Comment.
Additional comments: In this work, the authors examine how a coupled human system works using a model of viral infection – the susceptible-infected-removed (SIR) model – when the model is used in a forecasting context. Human behavior is modified by making the contact rate responsive to other dynamics, including forecasts, of the SIR system, so that there is two-way feedback between the forecast and the infection dynamics. Results show that a faster rate of response by a population to system dynamics or forecasts leads to a significant decline in peak infections. This is an interesting contribution to the existing literature, but the paper suffers from several shortcomings listed in the following comments.
-	The paper should be checked by a native.
-	A Conclusion section should be added.
-	The abstract’s text should be revised and improved.
-	The introduction should be updated by recent researches.
-	The novelty and contribution should be clearly bolded.
-	In Introduction, the authors should consider some works about different forecasting methods containing regression models and stationary and non-stationary time series models that can be applied to forecast and model different epidemic datasets. For example, they should cite “A novel method to detect almost cyclostationary structure”, “On the detection and estimation of the simple harmonizable processes”, “Periodically correlated modeling by means of the periodograms asymptotic distributions”, “Goodness of fit test for almost cyclostationary processes”, “A new method to compare the spectral densities of two independent periodically correlated time series”, “Testing the difference between spectral densities of two independent periodically correlated (cyclostationary) time series models”, “On the asymptotic distribution for the periodograms of almost periodically correlated (cyclostationary) processes”, “Testing the difference between two independent regression models”, “Testing the equality of two independent regression models”, “On comparing two dependent linear and nonlinear regression models”, “On comparing and classifying several independent linear and non-linear regression models with symmetric errors”, “On comparing, classifying and clustering several dependent regression models”.
-	Colure figures are suggested.
-	It’s better to suggest some subjects for future works.
Best regards,


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: A NOTE ON THE EFFECTS OF EPIDEMIC FORECASTS ON EPIDEMIC DYNAMICS
Review round: 2
Reviewer: 1

Output:
Basic reporting: No Comment
Experimental design: No Comment
Validity of the findings: No Comment
Additional comments: Authors made a careful revision against my comments.

Therfore, I recommend acceptance of this useful note for PeerJ with its revised version.

Best regards.


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: A NOTE ON THE EFFECTS OF EPIDEMIC FORECASTS ON EPIDEMIC DYNAMICS
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The title of your article is "A note on the effects of epidemic forecasts on epidemic dynamics".
The readers should know which methods can apply to forecast the epidemic datasets.
These methods are divided in two parts: regression methods and time series methods.
You should say about these techniques.


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: HIGH ANTIBODY TITRES INDUCED BY PROTEIN SUBUNIT VACCINES USING MYCOBACTERIUM ULCERANS ANTIGENS HSP18 AND MUL_3720 WITH A TLR-2 AGONIST FAIL TO PROTECT AGAINST BURULI ULCER IN MICE
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment, except for the fact that the research group recently published work with the same lipid adjuvant R4Pam2Cys in the context of cellular immune protection in a murine animal model that was not cited in the current work (Infect Immun 2020;88(3):e00753-19) and I think it would be better to make that citation, to clarify that the adjuvant was used earlier by this group; the references made to this adjuvant are probably less relevant than their own work.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: well written report; well illustrated
minor:
check line 181 µ in stead of u; line 302 signle, not plural: . .  There is


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

Input:
Title: HIGH ANTIBODY TITRES INDUCED BY PROTEIN SUBUNIT VACCINES USING MYCOBACTERIUM ULCERANS ANTIGENS HSP18 AND MUL_3720 WITH A TLR-2 AGONIST FAIL TO PROTECT AGAINST BURULI ULCER IN MICE
Review round: 1
Reviewer: 2

Output:
Basic reporting: Major issue:
The introduction and background does not give enough information on relevant topics the reader needs to know to understand the rationale of this study. Instead, the introduction dwells too much on basic BU knowledge and on the situation in Australia.
For example: Lines 52-56 is on BU transmission speculation (not relevant to understand this study) but the important information is only given in Line 71.

Not enough background on the reasoning for the choice of proteins is given.
(Line 84-86 does not have a reference where you could read up as an interested audience)
Not enough information on the reasoning for the choice of Adjuvant is given.
Information on why Antibodies could be of importance for protection against M. ulcerans infection and why this study looks at them is not given at all.

Relevant literature is partially missing. For example:
-	Line 61 should additionally cite:
PLoS Negl Trop Dis. 2013 Jun 13;7(6):e2252. doi: 10.1371/journal.pntd.0002252. Print 2013. Geographic distribution, age pattern and sites of lesions in a cohort of Buruli ulcer patients from the Mapé Basin of Cameroon.
-	Lines 363-370: Reference that shows that these people exist is necessary.
-	Line 372-373: Reference missing
-	Line 228: Reference 40 is not sufficient to show that MUL3720 is immunogenic

The Title of the manuscript could be improved, it only describes part of the study that is described (the positive aspect) and fails to mention that the vaccine is not protective.

Minor issues:
The Figures are mostly clear and well described. In Figure 3 E and F the titres for BCG could be removed to make the figure less busy. Those titres are baseline/background as can be seen in Figure 3 A-D, no need to plot them again. All information of Figure 6 is already present in Figure 3. This could be presented in a less repetitive way.

Most of the Article is easy to read and to follow. A few details should be changed:
Line 11: Affiliation is incomplete
Line 71: What do you mean with protective treatment? A vaccine?
Line 384: replace “to” with “than”
Line 229: Type on mice at the end of line
Experimental design: Major issue:
Overall, the article mostly reports data that have already been published by other authors in:
PLoS Negl Trop Dis. 2016 Feb 5;10(2):e0004431. doi: 10.1371/journal.pntd.0004431. eCollection 2016 Feb.
Vaccination with the Surface Proteins MUL_2232 and MUL_3720 of Mycobacterium ulcerans Induces Antibodies but Fails to Provide Protection against Buruli Ulcer.

Albeit with a different adjuvant and in a tail-infection model instead of the more commonly used foot pad model, but without clearly mentioning the above publication and without giving a rationale why the authors took the same two antigens again (Hsp18=MUL2232), combined with a different adjuvant and repeated mouse experiments, without explicitly addressing/discussing the similarities/differences between the two studies (not even in the Discussion).

Considering the above publication, I would question the knowledge gap filled (it is not identified in the text) with the study and the ethical standards (to some degree).

The methods are generally descried appropriately. However, there could be improvements, for example:
-	The timing of the immunizations and subsequent infection is unclear. From the M&M part it seems there were 14days between the second immunization and the challenge and all mice were sacrificed by day 75 (35 days of infection max.). However, Line 315 indicates day 75 corresponds to day 30 of infection? What is it?
-	Line 105: Where does that strain come from? (Reference?)
-	Line 206/207: 5 ug of protein per well? Per plate?
-	Line 217 following: How were the Antibody titres calculated?
-	Line 395-396: If LPS-testing was done it should be mentioned in the M&M
-	How did you determine IgG types? (different secondary Ab’s? If so, you should give the references)

Although the four stages of the disease progression in the tail model for BU are illustrated, it remains unclear whether there was a grading system used and at what point animals were sacrificed. This could be made more clear. Did you assess daily? And if so, where are the raw data for that?
Validity of the findings: I think the findings of the study are valid and meaningful, if not entirely novel. (See comment in Section 2).

Underlying data have been provided to some degree but not fully to enable for example to reproduce the figures or for example re-calculate the antibody tires (the method how they were calculated is also missing) and repeat statistical analyses.

Conclusions are clearly 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: HIGH ANTIBODY TITRES INDUCED BY PROTEIN SUBUNIT VACCINES USING MYCOBACTERIUM ULCERANS ANTIGENS HSP18 AND MUL_3720 WITH A TLR-2 AGONIST FAIL TO PROTECT AGAINST BURULI ULCER IN MICE
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is well written from the abstract to the discussion. The authors have provided the background of the previous studies, the aim of this study and they have cited the previous research properly.
Experimental design: This study is meaningful and has impact on the vaccine design against Buruli ulcer. The authors have designed and described the methods in a systematic way which is enough for the replication of this work.
Validity of the findings: The authors have validated their work in a systematic way and they have mentioned the limitation of their study and they have cited the literature in a appropriate place. the finding has been validated by the statistical methods and they have considered different statistical significance level for the validation their study.

The conclusion is well proved by the experimental setup and have great significance for the vaccine design.
Additional comments: Manuscript is well designed and written in a systematic way . So, I will recommend to accept 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: DESCRIPTIVE PROFILE OF RISK FACTORS FOR CARDIOVASCULAR DISEASES USING WHO STEP WISE APPROACH IN MADHYA PRADESH
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript uses a standard technique (WHO) and questionnaire for the conduct of the study, However I would like to see a few things added:
1. A flow chart describing the methodology.
2. A comparison with the NFHS data with regard to behavioral risk factors.
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: DESCRIPTIVE PROFILE OF RISK FACTORS FOR CARDIOVASCULAR DISEASES USING WHO STEP WISE APPROACH IN MADHYA PRADESH
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is well written, clearly articulated, and self contained with relevant results for the stated hypothesis. There are a few minor edits that I have outlined below for the authors to clarify/rectify -
- Line 41 - DALYs- expand DALYs
- Line 64 - 'clusters' to 'cluster'
- Line 73 - This sentence is unclear. Suggest to rephrase this sentence for clarity.
- Line 78 - This sentence is unclear. Suggest to rephrase for clarity.
- Line 88 - expand KISH and add a reference
- Line 89 - This sentence is unclear. Suggest to rephrase for clarity.
- Line 95 - Sentence is incomplete
- Line 100 - The make and manufacturer of the instruments used to measure height, weight and blood pressure need to be mentioned.
- Line 108 - It is unclear if a resurvey involved a systematic resampling of the sampled individuals or it was random. Also, it is unclear if there is a fixed number per cluster that was resampled. Would be good to mention this in the manuscript.
- Line 115 - The sentence needs to be corrected for grammar.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: No comments


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

Input:
Title: DESCRIPTIVE PROFILE OF RISK FACTORS FOR CARDIOVASCULAR DISEASES USING WHO STEP WISE APPROACH IN MADHYA PRADESH
Review round: 1
Reviewer: 3

Output:
Basic reporting: The abstract seems quite lengthy. The authors may trim it to retain only the vital information/summary of the project.

Line 41: What is DALY?

Line 95: The sentence seems to end abruptly.

Table 3: The authors may add info about total men/women participating at each stage of the survey. For Per capita income, the SD is quite high – this is perhaps due to the presence of data from rural & urban centres in this study. Maybe the authors can present the data for rural/urban separately to have more focussed stats.

Figure 2: The data has been represented by innovative means, but the figure legend does not clearly provide all the information. The authors must ensure that the legends are more detailed. Similarly, Table 5 has some abbreviations which are not explained in the legends or in another part of manuscript (for example, TR, AS,..)

The authors must also ensure the use of standard terms throughout the paper – Hypertension is mentioned as HTN in the tables and HT in the figures. It would be better to use a single identifier.

Line 194: Fourth or third from Haryana? If so, which is the other state? The statement is not very clear. Kindly clarify. It contradicts with Line 196.

Line 227: Table 4 or Table 5? Table 5 has not been cited inline in the manuscript
Experimental design: The study design has been planned well, in accordance to the WHO-STEPS protocols and it is laudable that the data has been collected in a paper-free manner, eliminating a lot of possible errors during the collection and consolidation phases.
Validity of the findings: While the study design is good, the authors could try to make the maximum use of the collected data to check on the trends in NCDs in age-group related manner as well.

At present, Table 4 provides data pertaining to the total population, as well as male/female numbers. It would be interesting to see how the trends are in each age group – whether there is a significant burden in a particular age-group for a particular trait, so to speak. The authors may even choose to provide this data in the supplementary section.

The authors mention that the smoking index has dropped drastically in MP since the data from 10 years ago – however, the previous study was a state-wide survey, as against the current study in only 10 districts, which only 30% representation in the Urban sector – perhaps, this might be a part of the reason for the drop in numbers?
Additional comments: This manuscript by Kokane and colleagues outlines the risk factors for cardiovascular diseases in the state of Madhya Pradesh in India, using WHO STEP guidelines. The work done is extensive and well presented. However, there are a few parameters which the authors may further fine-tune as suggested here.

The manuscript would benefit from being reviewed completely by a native English speaker or a language editor. There are multiple instances where there is a lack of connectivity/flow and coherence.

The table headings seem to indicate all tables as Table-1: the authors may kindly fix this issue (refer pages 25,27,29,32…)

Table 2 may be provided as supplementary material.


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: GEOGRAPHIC POPULATION GENETIC STRUCTURE AND DIVERSITY OF SOPHORA MOORCROFTIANA BASED ON GENOTYPING-BY-SEQUENCING (GBS)
Review round: 1
Reviewer: 1

Output:
Basic reporting: A. Important concerns

1. The introduction section lacks background information on the use of molecular markers on Sophorora moorcroftiana or related species.

2. Lines 100-108, This sounds more like your methodology! Delete it.

3. The raw data is not available online through the link provide by the authors. I suggest that you check it.

B. Minor concerns

1. I suggest that you add geography location on ADMIXTURE Figure 3

2. Provide legend  color for ancestors according to ADMIXTURE Figure 3

3. Lines 121,124, avoid to start a sentence with abbreviation and number.

4. English language is understandable but I am not an English native speaker and I cannot judge if it needs correction.
Experimental design: A. Important concerns

1. Provide a climate description of the sampling locations

2. Line 145, why did you combine the two paired end reads?

B. Minor concerns

1. How did you choose the sampling locations?

2. Line 166,  It is GCTA-PCA and provide reference as recommended here: http://cnsgenomics.com/software/gcta/#PCA

3. Lines 154 and 162, rewrite these parameters without abbreviations and by removing the two dashes.

4. Line 144, provide your python script as additional file.
Validity of the findings: A. Important concerns

1. Phylogenetic analysis

With GBS data you cannot construct species trees or gene trees because phylogenetics models have not been developed to take into account for example the amount of hybridization, incomplete lineage found at the intraspecific level. I suggest that you read this discussion (https://groups.google.com/forum/#!topic/raxml/1oSo4__nbms) where the author of RaxML is embarrassed with the use of RaxML for population genetics sampling. Therefore NJ-Tree is more powerful compared to RaxML.

2. Denovo assembly and snps calling on stacks

There are many missing information regarding the parameters and output results of stacks (SNPs data analysis section lines 146-176). First of all, the authors didn’t provide the version of stacks used and the parameters for denovo assembly using ustacks, cstacks, etc are missing. Secondly, I don’t understand why you used other software (PGDSpider_cli, VCFtools) to convert vcf format to phylogenetic format and for Hw, Ho, He, Fst calculations whereas all that can be provided by stacks (http://catchenlab.life.illinois.edu/stacks/comp/populations.php) ? Finally, the authors didn’t provide statistics for denovo assembly (Number of loci, number of snp, the depth,..). I suggest that you add statistics summary results of stacks as additional files.

3. Ploidy level

What is the ploidy level of the samples used in this study? Did you estimate the ploidy level of the samples used in this study?
Additional comments: The manuscript submitted is very useful for Sophorora moorcroftiana conservation. However, the authors omitted information about data analysis and without all these information this study seems not repeatable and I suggest that the author use the NJ-tree instead of RaxML for the phylogenetic tree. So, if the authors fully address all the points that I raised and provide the missing elements regarding data analysis, I encourage the authors to submit the revised version of the manuscript.


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

Input:
Title: GEOGRAPHIC POPULATION GENETIC STRUCTURE AND DIVERSITY OF SOPHORA MOORCROFTIANA BASED ON GENOTYPING-BY-SEQUENCING (GBS)
Review round: 1
Reviewer: 2

Output:
Basic reporting: Major comments:
1. According to the journal policies, raw data should be made available in a public repository such as NCBI SRA to ensure reproducibility of the analysis. I think in this case it may be worth to also make available the VCF file with raw SNP genotype calls provided by Stacks.

Minor comments
1. Some basic statistics should be reported as supplementary material, including the total reads per sample, the number of raw and filtered SNPs, distributions of minor allele frequency and observed heterozygosity both overall and within populations, percentage of SNPs genotyped per individual and percentage of heterozygous genotype calls per individual. All this information would allow to make much more informed filtering decisions.

2. In the admixture analysis (Figure 3) it would be much more informative if all k values from K=2 to at least K=7 are presented. I personally do not find useful the test to identify an optimum K value. On the other hand, presenting the progression of the clustering across k values provides information on the importance and stability of each cluster. I would make figure 3A supplementary to accommodate the results of the clustering with other K values. Please also organize the populations from 1 to 15. I do not see any rationale for the current organization of populations in figure 3.

3. About the structure of the document, I would move part of the current text of the discussion to the appropriate results section to provide readers with a better context of each result. The current results text looks just as a statement of the observed numbers and then in the discussion (especially the first three paragraphs) I had to keep looking back at the results. I think this change will also help the discussion to be more focused on the overall description of the evolution of the species and the importance of the obtained results. I would also remove the conclusions section because it just looks like a summary of the discussion.

4. I do not think it is worth to make and present separate regressions of diversity indexes with longitude and altitude given that these two values have a negative correlation of 0.93. The linear regression between longitude and altitude could be presented in one side of figure 1 and then only one of the variables could be related with diversity indexes.
Experimental design: Major comments:
1. The average observed heterozygosities are surprisingly high (far above 0.5 for all populations). This indicates that there should be an important number of variants for which all samples within populations are heterozygous. Biologically, the only explanation for this phenomenon would be high ploidy. Although this does not seem to be the case for this species, if this is the case, then variant genotyping should be modified to take into account this phenomenon and estimate allele dosages per genotype. More likely, heterozygosity rates could be inflated by false heterozygous calls produced by the stacks variant caller and lack of filtering of repetitive regions. Because the analysis is made de-novo (it looks like a reference genome for the species is not available), the repetitive regions are not known. One quick alternative to filter these variants is to perform a relative distance based filtering, for example keeping only one variant for each stack. An explicit filtering of maximum observed heterozygosity across the 225 individuals could also be performed to remove variants with abnormally high heterozygosity values. Please calculate the distributions of global expected and observed heterozygosity across the entire population. Given the observed population structure, observed heterozygosity should be generally smaller than expected heterozygosity due to the Wahlund effect.

2. The number of sampled individuals per population is a bit low to support most of the results presented in the manuscript. Moreover, if a maximum of 80% missing data per SNP is being allowed (as indicated in the methods section), the number of genotyped individuals per population can be too low to perform accurate estimations of population level statistics. For a given SNP, it could even be the case that some population have only one or even zero individuals genotyped (this could also explain the inflated observed heterozygosities). This issue can severely skew all results presented in the manuscript. Filtering for population structure analysis should be generally conservative. Normally, I recommend to have less than 5% of missing data overall.

3. If the population structure remains stable after improving the filtering process, my recommendation would be to make correlations of genetic diversity with longitude and latitude, and the analysis of gene flow considering only 4 groups: 1) populations 1-6, 2) populations 7 and 8, 3) populations 9,10 and 11, and 4) populations 12, 14 and 15. This way, each group would have enough genotyped individuals for accurate estimation of population level statistics. Instead of a linear regression, simple tests of difference between means could be used to identify significant differences in diversity values between these groups.
Validity of the findings: Minor comments:
1. I do not understand why throughout the manuscript populations 7 and 8 are split across two groups. In all clustering analyses these two populations appear as clustering together.
Additional comments: The manuscript describes a population genetic analysis of the perennial leguminous shrub Sophora moorcroftiana in 15 different high altitude locations across the Yarlung Zangbo River and three of its affluents. The study is interesting, relevant for population genetics and generally well conducted. The manuscript is clearly written and has good English quality. Major and minor comments are included in the relevant sections above.


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

Input:
Title: GEOGRAPHIC POPULATION GENETIC STRUCTURE AND DIVERSITY OF SOPHORA MOORCROFTIANA BASED ON GENOTYPING-BY-SEQUENCING (GBS)
Review round: 1
Reviewer: 3

Output:
Basic reporting: It is an interesting study. I think the topic of the paper is of interest to the audience of the journal. However, the manuscript in its present form has several weaknesses.
1.	Τhe authors have to improve the language. The manuscript has to be edited by a native English speaker or a professional.
2.	The abstract is very general especial the results.
3.	A part of the introduction is very general and it is not related to the present study.
4. The authors have to rewrite the conclusions which more or less are replication of the results.
Experimental design: The research is well orginised. However, the authors have to give more information about the sampling sites.
Validity of the findings: no comment
Additional comments: Some of my suggestions are indicated  in accompanying document