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: CHANGES OF DIAZOTROPHIC COMMUNITIES IN RESPONSE TO CROPPING SYSTEMS IN A MOLLISOL OF NORTHEAST CHINA
Review round: 1
Reviewer: 4

Output:
Basic reporting: In the manuscript “Changes of diazotrophic communities in response to cropping systems in a Mollisol of Northeast China” Zou and colleagues assessed the abundance of nifH gene and their sequences in and Mollisol in China under different soybean/corn management. They found that the crop rotation changes the abundance and composition of diazotrophic community in the soil. It is not a great scientific novelty, but they prove their hypothesis applying a very robust and high-throughput approach.
Experimental design: Despite to be a very new experiment, only 5 years to crop rotation, they found interesting results showing that, even after a short time, the diazotrophic communities change dus to the crop management.
The molecular and statistical analysis were well done.
Some comments are given in the section “General comments” to improve he quality of your materials and methods description.
Validity of the findings: The findings support the hypothesis that the crop rotation changes the diazotrophic communities in agricultural soils and brings new results to Mollisols. Some comments are given to improve your discussion.
Your conclusions must be rewritten according to stated in the next section.
Additional comments: Introduction:
Your introduction is very straight. Congratulations.
But I suggest to add more citations, and proper comments, of studies with diazotrophs in your Mollisols, even those based in isolation and diversity assessment (if there are some paper).
Materials and methods
Line 85: The average temperature in your experimental site is 1.5°C?? Are you sure?
Lines 87-89: State that your treatments CSC and SCS have annual rotation of crops and that, at the moment of sampling you had, corn and soybean, respectively, in the sites.
Lines 105-112: Please, cite the methods for each soil analysis.
Lines 126-127: It is not clear how do you prepared you standard curve. In the paper referenced (Yao et al., 2017), this information is also not clear. Please, state it clearly. Did you used a cloned nifH gene? Or a known DNA concentration of a given diazotrophic bacteria?
Results and Discussion
Your results are very well presented. Here, I made some appointments in the results for the further discussion.
Lines 174-180: The highest abundance of nifH copies in the cropping sites where soybean were grown in the sampling time (SCS and SS) is expected for two reasons. 1) the increasing of indigenous Bradyrhizobium populations and 2) the less N fertilizers applied. You should address some comment about this fact in the discussion of this topic, increasing your discussion in the lines 255-268.
Table 3: Please, take a look in the letters of your average comparison test. Sometimes the “a” is higher and sometimes is lower.
Lines 212-222: As well as commented to the qPCR analysis, the abundance of Bradyrhizobium in soybean growing sites is expected. Please, address this discussion in the proper section.
Lines 224-235: Note that your differences in Adonis analysis is related to the crop in the m moment of the sampling (soybean not equal to corn).
Line 241: Do you think that 5 years of crop management is enough to change such stable soil features such as pH and Carbon? You answered below!
Lines 297-305: Some papers sowed that Bradyrhizobium is an active endophytic plant growth promoter associated to grasses, for example sugarcane and elephant-grass. May be in your corn fields this populations were increased due to the active colonization of the corn plants.
The papers above should you include this discussion in this point.
Videira et al. (2013) Culture-independent molecular approaches reveal a mostly unknown high diversity of active nitrogen-fixing bacteria associated with Pennisetum purpureum — a bioenergy crop. Plant Soil 373:737–754.
Rouws et al. (2014) Endophytic Bradyrhizobium spp. isolates from sugarcane obtained through different culture strategies. Environ Microbiol Rep 6:354–363.
Menezes Júnior et al. (2019) Occurrence of diverse Bradyrhizobium spp. in roots and rhizospheres of two commercial Brazilian sugarcane cultivars. Brazilian J Microbiol. 50: 759-767.
Conclusions:
You are repeating some results. I edited your text to try a better conclusion. Please, add some text straight like this.
“The diazotrophs abundance and community structures differed with cropping systems especially continuous corn cropping. The soil parameters played a critical role in shaping the diazotrophic communities, specially to the available phosphorus. The continuous cropping and rotation systems significantly altered the abundance, diversity and community structures of the diazotrophic community in black soils”.


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: CHANGES OF DIAZOTROPHIC COMMUNITIES IN RESPONSE TO CROPPING SYSTEMS IN A MOLLISOL OF NORTHEAST CHINA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript was most times clear but requires further editing before it be recommended for publishing.  Important and generally updated references are used but a few other references are required to substantiate key findings. Tables and figures are generally well presented but key editing is required so that the major audience of this is journal can understand the article.
Experimental design: The knowledge gap is well identified. The study is conducted with high technical standard and the methods are clearly explained.
Validity of the findings: The findings are valid. The key information has been provided. Conclusions  are well stated and linked to the original research questions.
Additional comments: Changes of diazotrophic communities in response to cropping systems in a Mollisol of Northeast China

General Comment

This is the second review of this manuscript. The general suggestion is that the authors should now focus on improving the discussion section organizing it in paragraphs rather than subsections. They should not discuss every single result but the key findings, considering the objectives of the study. Suggestions on how to improve the presentation of tables is also provided. English writing should be improved in all sections. The specific recommendations are listed below.

Specific Comments

In Table 1: please should indicate mean  ±  standard errors. CC, SS, CSC and SCS meaning should be either provided in the title or with different  symbols. As the symbol appear now “a” represents cropping systems and the statistically best treatment mean. The same advice applies for soil properties (TN, TC ….etc.).  Make sure to provide a legend for the statistical mean-ranking symbols below the table.
Table 3: Please, include (%) in the title after abundance.
Line 40: “Although the barriers of continuous corn cropping are not as serious as those of continuous soybean”. The meaning is not clear.
Line 50: “Approximately 110 Tg of N is input into terrestrial ecosystems annually through the BNF method (Gruber et al., 2008)”. It is not a good idea to use over ten years old estimates, especially when more recent calculations, for example Davies‐Barnard and Friedlingstein (2020), are available.
Line 91: please, change text so that it reads: “Commercial fertilizers urea, diammonium…”

Line 178: Please, change text so that it reads “The abundance of the nifH gene in the SCS treatment was 57.4%, 123.8% and 382.3% higher  compared …”

Lines 179 – 181: Please, change text so that it reads “Pearson’s correlation analysis showed that nifH gene abundance was significantly correlated with C/N (r = -0.541, P = 0.031), NO3--N (r = -0.602, P = 0.014), and AP (r = 0.597, P = 0.015) (Fig. 1B, C, D).”

Lines 183 – 184: Please, change text so that it reads “Shannon and Simpson indices had opposite results. CC treatment had the highest score with Shannon index but had the lowest score with Simpson  index.”
Line 193: Please, replace “99.08” with “99.91”.
Lines 194 – 195:  Please, change text so that it reads “… while the highest relative abundances of Betaproteobacteria and Deltaproteobacteria were only 1,37%  and 1.71%, respectively.”

Line 198: Please, help your readers by listing the orders either alphabetically or in the same sequence they appear in the table.
Line 202:  Please, change text so that it reads “Contrary to Rhizobiales, Rhodospirillales was significantly (P < 0.05) more abundant in the CC treatment.”
Line 207 – 209: Please, change text so that it reads “Contrasting with Rhodospirillaceae, Bradyrhizobiaceae was significantly less abundance in the CC than in the other treatments.”

Lines 242 – 243. “No variation of soil pH observed in this study might be ascribed into two reasons: one is the soil samples were collected from bulk soil not of rhizosphere soil which has more buffer effect against the changes of soil pH, another might be related…  ” Jagadamma et al. (2008), authors that you cited, also sampled bulk soil but observed differences in soil pH. I would delete bulk soil as one of the reasons.
Lines 249: Please, replace “which” with “and”.
Lines 251”Please, replace “increased” with “higher”.
Lines 254 – 255: “ … rotations including corn put more N fertilizer into the soil compared with the SS treatment, which can increase TN content in soil…”. Please, substantiate this affirmation with relevant and recent literature.
Line 256: “…may also be related to different fertilizer regimes between corn cropping and soybean cropping”. Please, elaborate.
Line 281: “This phenomenon may be related to the significant increase …”  Please, make sure this is consistent with the previous statement.
Line 295: “Bradyrhizobium is the most abundant genus of diazotrophs in long-term rotation systems”. This if a very important information. Please, provide an explanation, the possible reason for this.
Line 297: Please, replace “increased” “with “higher”.

Reference
Davies‐Barnard T, Friedlingstein P (2020). The global distribution of biological nitrogen fixation in terrestrial natural ecosystems. Global Biogeochemical Cycles 34 (3): e2019GB006387. DOI: 10.1029/2019GB006387


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: CHANGES OF DIAZOTROPHIC COMMUNITIES IN RESPONSE TO CROPPING SYSTEMS IN A MOLLISOL OF NORTHEAST CHINA
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have made sufficient changes to the manuscript.
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: CHANGES OF DIAZOTROPHIC COMMUNITIES IN RESPONSE TO CROPPING SYSTEMS IN A MOLLISOL OF NORTHEAST CHINA
Review round: 2
Reviewer: 3

Output:
Basic reporting: The manuscript written by Zou el al. reports the changes in the diazotrophic soil communities due to different crop/soil management. In my first review, the paper showed several gaps that should be filled to increase the paper quality and reach those needed to be published in PeerJ. Now, the quality of the paper increased a lot. I congratulate the authors for their commitment to the reviewer's comments and to the effort to increase the paper quality.
A single point. In line 363, remove the word “many”, since the paper cited studied only two non-legume crops (elephant-grass and sugar-cane).
Experimental design: The quality of experimental presentation increased a lot.
Validity of the findings: The data presentation and discussion is more clear now.
Additional comments: The paper can be accepted for publication.


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

Input:
Title: BEYOND LOOT BOXES: A VARIETY OF GAMBLING-LIKE PRACTICES IN VIDEO GAMES ARE LINKED TO BOTH PROBLEM GAMBLING AND DISORDERED GAMING
Review round: 1
Reviewer: 1

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: PeerJreview-Lootboxesandgambling

gambling and the purchase and/or use of loot boxes in video games. This has led to speculation about whether loot boxes are a new form of risky gambling-like activity that is appealing to problem gamblers. The paper also tentatively raises the question as to whether loot boxes might be a softer gateway product to engagement into gambling which, in turn, can lead to problem gambling.

I was generally impressed with the sample size and choice of measures in this paper. The data and results are publishable, but there were many aspects of the paper which need to be revised to make it a stronger paper.

The first problem is that the study is not really a prevalence study. Stratification might make the sample superficially resemble the age and gender, ethnicity (probably mostly British vs. the rest), but Profilic samples and any survey of this nature will be heavily skewed towards people who are more highly educated and who like to s This paper presents the findings of an online panel survey that investigates the association between various forms of gambling behaviour, gaming and particular aspects of gaming, e.g., the use of loot boxes. The sample is drawn from Prolific and uses a stratified sampling method to attempt to make the sample reflect some of the demographic characteristics of the general adult population in the UK.  A sample of over 1000 people was obtained and the study included a number of respectable and standardised measures using the PGSI and Lemmen’s measure of gaming disorder.

The main finding of the paper (which replicates other similar studies by the author) is that there is a statistically significant relationship between it at home and do surveys. It will also tend to attract people who have an interest in the topic. Thus, any estimates will be far higher than what might obtain in the general population. I regard with significant suspicion any study that reports a prevalence of problematic gaming above 2-3%. The same holds for any figure above 1-2% for problem gambling. You just don’t get this sort of figure in probability sampling. Lemmen’s measure also tends to score higher than the Petry measure. All references to prevalence should be strongly qualified or removed. It’s really a convenience sample.

A second problem is that the study does not really engage with selection and exposure effects. I find the ‘gate-way’ argument to be long stretch and found a lot of the argument in this paper to be too causative. Gambling and gaming are largely different classes of activity. Loot boxes might look like gambling in the context of video games, but they have nothing to do with gambling as we regularly know it. One does not gamble on loot boxes when one visits a high street betting outlet, visit a casino or when one gamblers on Bet365. A stronger argument is that this is merely a selection effect. The sorts of people who like to gamble ALSO like to play video games and, if you are really into video games, you tend to buy loot boxes. In other words, loot boxes are just another way to extract money from people who also like to gamble a bit more (probably young men). The two run in parallel. I’d take out all the linkage stuff which is unfounded and definitely not talk about ‘gate-ways’.

A bit more could be made of technological convergence- that people who go online to gamble or use their devices a lot also have a chance to play games as well.

A third problem is that the paper does not control for gender. I think the regression model needs to do this. Males will play more VGs, open more loot boxes and also gamble a lot more.

 The writing needs lots of editing to tighten up the argument.

Abstract:  This reports involvement almost like a prevalence study. Not what this sample should be used for.

You should not be using phrases like ‘gaming must urgently be investigated’. Far too rhetorical. This is not a call to action or activist paper. Needs to stay neutral and academic in style.

Line 37: I felt that this was over-stated too. Only the author is advancing this argument. The link and casual link is tenuous. Don’t talk about ‘concerns’ and build up the rhetoric to sell the paper. OK in a grant, but shouldn’t be in a paper. Line 38 is not a sentence.

Line 44 This paragraph needs to be reworked. Syntax seems odd.

Line 48 This needs to be revised ‘gate-way’ etc.

I found the listing off all the other activities in small paragraphs a bit choppy. Could this be put into a series of (a), (b), (c) indented paragraphs. Otherwise, feels disjointed.

-Very few people actually pay money to get extra credit on SCGs. The prevalence of ‘social casino spending’ is NOT high. It might be 1%.

-The logic in the line 107-115 section seems out to me. VGs and problem are parallel activities. I don’t think there’s much of a link of the nature described here.

Line 128 ‘to begin investigating’. Hasn’t the author published on this before?

Line 131: No it’s not a prevalence study

Line 331 sounds too much like prevalence; linked is too causal.

Iine 355  Not sure about the meaning of clinically significant effects.

Next paragraph: a lot of the differences could be due to sampling.

The conclusion starts to grapple with the causality issue, but this should have been said earlier (line 420 onwards)

Overall, a potentially useful set of data, but needs a bit more analysis of gender; a lot of editing and bit of restructuring of the argument; and more tempered conclusions about the causal relationship between activities.


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: BEYOND LOOT BOXES: A VARIETY OF GAMBLING-LIKE PRACTICES IN VIDEO GAMES ARE LINKED TO BOTH PROBLEM GAMBLING AND DISORDERED GAMING
Review round: 1
Reviewer: 2

Output:
Basic reporting: The present paper presents data concerning a survey investigating how gambling problems and disordered gaming are related to opening loot boxes, esports betting, real-money video gaming, token wagering, and social casino play. A representative sample of 1081 adults from the UK aged 18+ was recruited for the study.

Major issues:
Line 106: By which standard can it be said that an eta-squared of .04 is clinically significant. Please back this statement with clinically relevant empirical findings.

In the introduction the author uses the terms problem gambling and disordered gaming. These concepts should be defined.

Line 143. Although an initial sample of 1201 was recruited – what was the response rate – e.g. how many was approached – how many could not be reached and how many refused to participate?

Please place the description of the instruments before the description of sample characteristics, as the latter, e.g. gambling category, is based on the former.

Minor issues:
Line 37. The sentence starting with “gambling like transactions” seems to be missing something.
Line 140: Please explain what you mean by “self-reported remotely”
Line 161: Please don’t start a sentence with a number

Please also check that spaces between words are as they should be (e.g error in line 372, 389) and that the same font size is used allover.
Experimental design: In terms of the PGSI – please state that the scoring procedure deviates from what is most common – and please state what defined disordered gambling. Also, provide information about the Cronbach alpha for the scale.

Line 249. The acronym APA has not been introduced to the reader. Please state that the Internet Gaming Disorder Scale (use capital letters) is based in the diagnostic criteria for Internet Gaming Disorder found in the 5th edition of the Diagnostic and Statistical Manual for Mental Disorders. It seems (table 2) that you used continuous scores based on the IGDS in some of the analyses. Please provide information about this. Also provide information about the Kuder-Richardson-20 (equivalent to alpha for scales with dichotomized response alternatives).

Although the original sample was representative it might be different response rates in different demographic groups (e.g. typical lower response rates among males and young people). Did you adjust for the discrepancy between sample and the UK population in terms of important demographic variables?

Table 1: Explain the acronyms LLCI and ULCI.

What was the rationale for selecting just some potential variables as independent in the regression analyses? A better/or detailed justification is in order. I think you should consider including age and gender as independent variables.

Regarding the regression analyses: Did you check that there were no violations of the assumption of normality, linearity, multicollinearity and homoscedasticy?
Validity of the findings: Regarding the discussion: There does not have to be any causal link between study variables. The associations may for example reflect a common underlying third variable like age, impulsiveness, interest in gambling and gaming etc.

It is also possible that the common method bias may play a role as self-report data only, with a cross-sectional design, were collected – causing inflation in the estimated associations between study variables.

Please also comment on the fact that gaming related activities explained only 5.6% of the variance in problem gambling whereas they explained 21.7% of the variance in problem gaming. Hence, could it be argued that the convergence between gaming and gambling is not as large as suggested by the author? Please discuss this issue further.

Please also suggest some directions for future research (e.g. longitudinal studies).
Additional comments: No additional comments


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

Input:
Title: BEYOND LOOT BOXES: A VARIETY OF GAMBLING-LIKE PRACTICES IN VIDEO GAMES ARE LINKED TO BOTH PROBLEM GAMBLING AND DISORDERED GAMING
Review round: 1
Reviewer: 3

Output:
Basic reporting: English is not my first language, but to me, the manuscript appears to be written clearly and to the point. It succeeds at communicating the main research questions, findings and discussion, without using unnecessary jargon or overly complicated statistical analyses.

The tables are adequately labelled and explained, and raw data, as well as complete item descriptions, are supplied via the OSF repository.
Experimental design: The study appears adequately designed to address the research questions outlined in the “Summary” section of the introduction.

The investigation is conducted and presented using fairly standard statistical approaches, and I found the reporting adequate.

The survey items are included via the OSF repository, enabling anyone interested to replicate.
Validity of the findings: I am aware that novelty is not a requirement for PeerJ, but would still like to commend the author for collecting this data. The results should be useful for any party interested in the grey area between traditional gambling and video games. I do, however, have some specific issues that the author may want to consider.

1.	Representativity. Although it is not claimed that the sample is representative for the UK, steps taken to ensure representativity in terms of ethnicity, sex and age are mentioned. I am unfamiliar with the details of how Prolific Academic works, but am under the impression that it is a service where people self-select to partake in an online panel, where they can be invited to surveys and be paid for their time. I would like to see more details but suspect that the resulting sample cannot be regarded as representative of the UK 18+ population due to the nature of how PA service works (i.e., not all of UK are members of this panel and those who are might differ from those who are not). Also, information about response rates would be useful in this regard, if at all available.

2.	Incentives. Were participants paid, and if so, how much

3. 	Esports betting. I think a case could be made for categorizing this activity as a form of gambling rather than a “gambling-like” or “gambling-related” practice in video games (as it is described in the introduction). I think it would make more sense to group this with the 11 traditional forms of gambling – since this activity is gambling and usually offered by gambling companies rather than the video game industry. I would not, however, see this as a crucial change and would leave it to the author to decide which approach is most suitable. I do, however, believe that some clarification needs to be made. In the introduction, it is referred to as “a variety of gambling-like and gambling-related practices … in video games” (line 40), and then “gambling-like video game practices” (line 130). In the measurements section, however, it is referred to as “gaming-related forms of gambling and gambling-like behaviour” (line 205). I believe e-sports betting fits the latter description, but less so the first two. Regardless, please be consistent in how these concepts are described and referred to.

4.	Some of the introduced concepts are probably unfamiliar to a large proportion of the respondents. In particular, I believe many will have little intuitive understanding of what “Real money gaming” and “Token wagering” entails. For example, although I have a keen interest in video games, both academically and as a hobby, I am not entirely sure if I have taken part in token wagering even after reading the explanation provided in the survey (OSF repository). I believe some discussion around how this might have influenced the findings would be warranted.
Additional comments: 1. I would like to thank the author for conducting this exciting piece of research and believe it is a valuable contribution to the field. However, after checking the complete list of items provided in supplemental materials, I think there are several additional and relevant points that should be included in this manuscript. Including:

-Was there a relationship between problem gambling and disordered gaming?
-What was the proportion of purchasing loot boxes among people playing games that included these products?
-Data from the “what came first” items would be very interesting and relevant information.
-More information about the video game habits of responders could be provided

2. The proportion of respondents with problem gambling or disordered gaming appears to be quite high compared to other studies (I believe the UK Gambling Commission reported 0.7% of problem gamblers, compared to 2.2% in the present study). I think these numbers should be discussed. Could a lack of representativity be a part of the explanation? (see the previous comment under “Validity of the findings”).


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

Input:
Title: BEYOND LOOT BOXES: A VARIETY OF GAMBLING-LIKE PRACTICES IN VIDEO GAMES ARE LINKED TO BOTH PROBLEM GAMBLING AND DISORDERED GAMING
Review round: 2
Reviewer: 1

Output:
Basic reporting: Better
Experimental design: It's correlational.
Validity of the findings: A few comments below.
Additional comments: 2nd review
I thought that this version of the paper was much improved and provides a more balanced and logical coverage of the material.  However, there are still a few points which raise some concerns for me.

Abstract:  It IS a convenience or panel sample. No way that this sample was obtained by probability methods. All the references in the world re. value of stratified samples can be cited, but it still cannot be read as a prevalence study.  I still think this is misleading and not good scientific practice because journalists won’t know the difference.
Table 6:  Don’t like the word ‘influence’. Association better.
In the Discussion there is a reference to 18.5% of gaming behaviours being on par with other forms of gambling, but wouldn’t the fairer comparison be with the overall participation rate for gambling? I’d cite the most recent BPS and this will be much higher than 18.5% for the annual participation rate for any form of gambling?
The paragraph that starts on Line 512 I’d start off with the more plausible explanation in the abstract: PGs who play video games probably like loot boxes rather than loot boxes really having any influence on whether people take up gambling. The evidence barely shows this. There is really only the Molde et al (2020) that shows one cross-lagged effect and it’s small: .15 between problem gaming at Wave 1 and PGSI at Wave 2.
566-569:  I didn’t find this all that convincing. A lot of male PGs will gamble online in the UK, and they will ALSO gamble on land-based activities.  Yes, they have other land-based activities, but they spend a lot of time online and this would increase the likelihood of online gaming and loot boxes.
586 paragraph. It IS a convenience sample.
A broader Q: are LBs really a major problem in terms of harm? Is $20-30 per month (as found in the other papers) really a lot? It is enough to cause harm?
Luke Clark is singular? Their?  Luke is not identifying as non-binary as far as I know.
Just one comment on the rebuttal. Just because others are saying the same thing- doesn’t strengthen the argument. An example:  I have a lot of doubts about adolescent problem gambling prevalence figures (too high), but lots of us have got these results. It’s a reliable finding, but is it valid? The measures might be consistently misinterpreted by young people.


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: BEYOND LOOT BOXES: A VARIETY OF GAMBLING-LIKE PRACTICES IN VIDEO GAMES ARE LINKED TO BOTH PROBLEM GAMBLING AND DISORDERED GAMING
Review round: 2
Reviewer: 2

Output:
Basic reporting: I asked the authors to provide a definition of both problem and disordered gaming. Still, I cannot find that the latter (disordered gambling) has been done.

Also, the rebuttal latter is not acceptable. Either the authors should link (e.g. by line no) the answers to specific places in the manuscript or specifically in the rebuttal letter write how they have changed the ms. Just writing in the letter that this and this have been augmentet is quite arrogant, and make the job as reviewer much more time-consuming than neccessary.

Please use correct reference for the DSM-5 (line 353).
Experimental design: I asked the authors to say something about the response rate. They refuse do to so, and just repeat the number of participants. N and response rate is not the same thing. I also searched the whole ms for the expression "response rate" and it did not appear.
Validity of the findings: I probed the authors about the representativness of the sample. The authors claim that the sample was stratified, but use the term quata sampling instead - withouty defining the difference between stratified and quota sampling.

Also, even if a sample is stratified or based on quotas, different response rates in different quotas may make it non-representative. This was my point, unfortunately the authors have not adhered to this!

I asked also for a rationale for the independent variables. The authors just reply that age and gender now have been included. But thats not the same as providing a rationale.

I asked the authors about third variables (e.g. impulsivity) - but could not find anything explicitly about this in the revised ms.

Gaming-related behaviors explained only approximately 5% of the variance in problem gambling - instead of being humble the authors state: In fact, we would argue that it is instead the case that the relationship between gaming-related behaviours and disordered gaming is surprisingly large, and bears further study. So when did 5% become surprisingly large?
Additional comments: When you make a rebuttal letter you need to be much more specific - incorporate all changes in the letter - it is not sufficient to say that the ms has been augmented.


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: BEYOND LOOT BOXES: A VARIETY OF GAMBLING-LIKE PRACTICES IN VIDEO GAMES ARE LINKED TO BOTH PROBLEM GAMBLING AND DISORDERED GAMING
Review round: 3
Reviewer: 1

Output:
Basic reporting: See below
Experimental design: See below
Validity of the findings: See below
Additional comments: I am generally happy with the responses to this second revision. In particular, I am content with the sample being described as a purposive sample to avoid any confusion. It's a sensitive topic at the moment because there has recently been a 'prevalence' study conducted in Africa (gaming area) which has been pulled to pieces online (by Nick Brown, the research integrity blogger who may be on the warpath against the gaming area). Very important that any paper in this area is tempered and balanced in its conclusions.

I actually agree about the limitations relating to probability sample. I've recently seen some post-weights in prevalence studies which are enormous and questions could definitely be raised about whether these cases (young males in particular) are representative of the population. Using some quota sampling or stratification can be useful. This is a topic where I've had lengthy chats with Rob Williams who has just run the Canadian prevalence study. I think that researchers run into problems if you post an add (e.g., on Reddit or somewhere else online), get people to self-select/ opt-in and then try to make it sound like a population sample if the basic demographics look like the population (or are weighted to be the way). You never get past the response bias (respondent 'I do this because this is about people like me!'). Bob Ladouceur's paper on volunteer sample biases might be worth citing (in 1990s). You get over-estimates..... which I think is what you've found too for gambling vs. the BPS for some forms of gambling?

The best strategy may be to use probability method to identify your panel in advance (Rob's view). The HILDA sample in Aust is like this. Do all you can at the outset to get a panel via random methods and then try to fill in the gaps until you get a final cohort that requires as little weighting as possible. This means that you haven't just got people who have opted in or self-selected to take part because it's about gambling or LBs or gaming. In effect, everyone in the population has a chance to take part at the outset (probability method), but you then use other methods to make it representative.


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: BEYOND LOOT BOXES: A VARIETY OF GAMBLING-LIKE PRACTICES IN VIDEO GAMES ARE LINKED TO BOTH PROBLEM GAMBLING AND DISORDERED GAMING
Review round: 3
Reviewer: 2

Output:
Basic reporting: It would be in line with normal reporting also to insert a reference for APA in line 353 although it has been sited eralier.
Experimental design: Regarding response rate I still disagree with the author. Response rate is not the number who participated. Response rate is the proportion of those who who actually participated to the number of those who were invited (or who could participarte). I cannot see that his number is  provided. This number is important in all epidemiologoical research as it says something about the representativity of the sample. In the abstract it is stated that 1081 participated.

In the methods it is stated that an initial sample of 1201 was  collected. Further you state that sample was recruited for us by Prolific Academic. How did this take place? How did Prolific Academic recruit them - from where? How many did Prolific Academic ask in order to obtain the sample? Thats important issues to comment on and to address properly.
Validity of the findings: N/A
Additional comments: N/A


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

Input:
Title: IDENTIFICATION OF POTENTIAL BIOMARKERS AND THEIR CLINICAL SIGNIFICANCE IN GASTRIC CANCER USING BIOINFORMATICS ANALYSIS METHODS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is interesting study describing the identification of aberrant alternative splicing (AS) variants (32.166 in total) in 375 gastric cancer (GC) patients by using RNA-seq data and clinical information of STAD cohort from TCGA. Among them 2.042 AS events were significantly associated with patient survival. Also, pathway analysis showed that these genes play a critical role in regulating gastric cancer-related processes such as GTPase activity and PI3K-Akt signaling pathway.

1.	Major concern is that the results of this manuscript are based on in silico analysis. It is important that the authors confirm some of their results by RT-PCR with RNA extracted from normal and tumor samples.
2.	A number of altered AS variant were associated with CG. This is the case of CD44 exon V6; was this event also identified through analysis that the authors have performed fromTCGA?
3.	In the discussion, it would be useful for the reader to improve the role of ECT2 in gastric cancer.

Minor
1)	Abstract (line 24): Replace “indicated that these genes play a important role in regulating gastric” WITH “indicated that these genes play an important role in regulating gastric”.
2)	Result: Page 8 (line 106): the abbreviation ME is not explained before”.
Experimental design: N/A
Validity of the findings: see above
Additional comments: see 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: IDENTIFICATION OF POTENTIAL BIOMARKERS AND THEIR CLINICAL SIGNIFICANCE IN GASTRIC CANCER USING BIOINFORMATICS ANALYSIS METHODS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English language should be improved to ensure that an international audience can clearly understand your text.

The discussion must be written from scratch. The version provided cannot be considered a formal discussion of results.
Experimental design: The research question is interesting but the methodological/statistical analyses require additional and mandatory controls. To note, experimental validations of the proposed results are missing. This downgrade the tone of the results.
Validity of the findings: Impact and novelty not assessed.
Statistical analyses require additional and mandatory controls.
Additional comments: Before starting with my comments, I would say that the Figures that this Reviewer can access are of very low quality and this will severely impact on my comments/concerns.

The introduction needs some more detail about the importance, and the state of art, of the therapeutical potential of targeting splicing. I would suggest that you improve this part to provide more justification for your study. Lines 63-65 provides too much details for the scope of this paper.

The Materials and methods are synthetic and require more (mandtory) details that are missing.  For example, on line 83, the selection proposed by the authors is unclear. How do the authors define alternative spliced events? Line 91 did the authors correct the pvalues for multiple tests? Line 92, what kind of gene network analysis did they perform? Where are the details of GSEA analysis?

Similar to the previous section, also Results requires more details and explanations. First of all, how did the authors define AS events (line 98)? As I mentioned, Fig.1 is low quality and difficult to read. Detail on the univariate Cox model are needed. What is the stratification of samples that the authors used? Similar for the multivariate modelling (line 130)

Lines 122-127 are the obvious and not relevant.

Lines 160-170. Lauria et al (Nucleic Acids Research, 2019) have extensively shown that, when considering heterogeneous RNA-seq data (i.e. TCGA), GSEA is not the most appropriate method to perform gene set analysis. Therefore, the authors should comment on their choice of using GSEA for the analyses they performed. Details and justifications of this analysis are missing and they are required. The author never present the number of samples that they used in each analysis. This is a mandatory value to show, the reader must have the opportunity to understand whether statistical tests require or not correction for sample sizes. did the author check for this? Did they evaluate the statistical power of their results? All these analysis are missing. Justifications are required.

The Discussion is completely useless. Lines 178-245 are useless in the context of a “discussion” of the results of the paper. The only sentence that this Reviewer could link to a formal discussion of the results are lines233-234. Please write a proper discussion section where the author comments proactively on their results not on others’ results, and trash this one.

Please downgrade the tone of the conclusion section. This data must require an extensive experimental validations in the future to become real biomarkers.

The English language should be improved to ensure that an international audience can clearly understand your text. Some examples where the language could be improved include lines 80,81, 98 – the current phrasing makes comprehension difficult.


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

Input:
Title: IDENTIFICATION OF POTENTIAL BIOMARKERS AND THEIR CLINICAL SIGNIFICANCE IN GASTRIC CANCER USING BIOINFORMATICS ANALYSIS METHODS
Review round: 1
Reviewer: 3

Output:
Basic reporting: In this work, the authors used bioinformatics tools to analyze survival associated alternative splicing (AS) events in gastric cancer (GC) and identified the ECT2 gene as a potential biomarker.

Sufficient background information not provided as further elaborated in general comments for the authors.
Experimental design: The aim of the study is well-defined and the methods used are appropriate. However, methods are not described with sufficient details as further elaborated in general comments for the authors.
Validity of the findings: Overall conclusion are supported by the results shown. However, the impact and novelty of the findings are not assessed.
Additional comments: 1)	The introduction needs improvement as sufficient background covering previous bioinformatics work is not provided to understand the significance and novelty of the work. For example, lines 69-70, authors write “Integrating clinical information and large-scale of RNA-seq data to systematic analysis of AS at individual exon resolution in GC have been lacking.”. However, there has been previous bioinformatics work analyzing AS in GC (to mention a few, PMIDs: 30106437, 31857793, etc.). Consequently, the statement on line 247 “This is the most comprehensive study to assess GC predictors” should be changed accordingly.
2)	The discussion needs major improvement and should be re-written. A major part of the discussion (lines 179-232) is spent describing alternative splicing or its role in cancer in general. Authors do not provide any critical analysis and interpretation of their results neither they placed their findings in the context of published literature. For example, one of the main results of the paper is the identification of ECT2 as a potential biomarker. However, previous literature (PMID: 28493890) have also identified altered AS of ECT2 in GC. Neither this result is presented in the discussion nor its importance discussed in the context of published findings.
3)	Lines 243-245, “…This finding is inconsistent with previous studies that most survival-related AS events in ovarian cancer (Zhu et al. 2018). In contrast, GC showed a lower level of poor prognostic factors, but a higher level of favorable prognostic factors.…”. It is not clear what authors are trying to convey here? Moreover, what does it mean for a finding in GC data to be inconsistent with that in ovarian cancer?
4)	Methods are not sufficiently detailed. For example, authors must clearly mention the dataset being analyzed (TCGA-STAD in this case), and the number of tumor and normal samples considered. Also, details of the Cox regression and clinical covariates used should be added in the methods section. Furthermore, the details of the settings used in Oncomine analysis should be added.
5)	Lines 80-81, “…In RNA-seq data, splice in and splice out support splice in respectively. And the number of reads that occur with splice out.…”. It is not clear what authors are trying to convey here?
6)	Lines 82-83, “…The filter condition is sample percentages with PSI values≥75, average PSI values≥0.05(…”. The authors should expand on what the filtering condition is for? Also, how can PSI >= 75 if the value range is 0-1.
7)	Line 103, “…After strict filtering by survival, 32,129 AS events…”. What criteria are used for filtering?
8)	There are spelling mistakes at several places in the manuscript. For example, “A amd B” on line 145. Also, abbreviations are used without first defining it. For example, STAD, ACRG, and AS on lines 18, 20, and 22 respectively.


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

Input:
Title: IDENTIFICATION OF POTENTIAL BIOMARKERS AND THEIR CLINICAL SIGNIFICANCE IN GASTRIC CANCER USING BIOINFORMATICS ANALYSIS METHODS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors performed most of the work that I requested and the manuscript text has been modified according to the reviewers’ suggestions
Therefore, I have no additional reservation.


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

Input:
Title: IDENTIFICATION OF POTENTIAL BIOMARKERS AND THEIR CLINICAL SIGNIFICANCE IN GASTRIC CANCER USING BIOINFORMATICS ANALYSIS METHODS
Review round: 2
Reviewer: 2

Output:
Basic reporting: NA
Experimental design: NA
Validity of the findings: NA
Additional comments: Authors have addressed all my concerns. I recommend the article to be considered for publication.


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

Input:
Title: ESTABLISHMENT AND CHARACTERIZATION OF FANTAIL GOLDFISH FIN (FTGF) CELL LINE FROM GOLDFISH, CARASSIUS AURATUS FOR IN VITRO PROPAGATION OF CYPRINID HERPES VIRUS-2 (CYHV-2)
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is a clearly written and technically sound article about a new goldfish cell line in India. There are currently very few cell lines available for HVHN researches. Therefore, every new cell line that is sensitive to CyHV-2 will be warmly welcomed by the scientific community.
Experimental design: Although the introduction gives a good descriptive overview of currently available goldfish cell lines for CyHV-2 propagation, it would be better if the authors could also summarize these data in a short table with names and characteristics of these cell lines and references to the original description in literature.

Materials and Methods section:
Line30:
The manufacturer and lot number of L-15 should be added.
Line31:
The manufacturer and lot number of FBS should be added.
Line 124:
Please describe the trypsinization protocol in detail.
Validity of the findings: Results section:
Fig. 3B:
It looks as if the majority of the cells (60%) did not show a chromosome number of 2n=104 as described in the text, sth wrong with the x-axis markers?

Fig.6:
The scale bar of 6A is totally different with the other three, and I just can’t read the length of the bars of 6B~6D: they are too small and illegible.
Additional comments: Three items would contribute to the value of the paper:
	a quick test to determine if these cells can be transiently transfected with exogenous DNA, such as a lacZ reporter gene plasmid.
	basic Illumina RNAseq analysis of the cell line could provide a good insight into the nature of the cell line.
     Histopathological analysis and transmission electron microscopy results of virus-infected cells are appreciated.


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: ESTABLISHMENT AND CHARACTERIZATION OF FANTAIL GOLDFISH FIN (FTGF) CELL LINE FROM GOLDFISH, CARASSIUS AURATUS FOR IN VITRO PROPAGATION OF CYPRINID HERPES VIRUS-2 (CYHV-2)
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Arathi and colleagues describe a simple protocol for successful establishment and characterization of a permissive cell line through explant method and continous propagation of CyHV-2 with high viral titre using this cell line. CyHV-2 is an important virus as it causes high mortality in aquaculture. Cell lines are considered the gold standard for isolation and identification of viruses. The newly developed FtGF cell line will play significant role in future research on CyHV-2. It is a topic of interest to the researchers in the related areas but the paper needs very significant improvement before acceptance for publication. My detailed comments are as follows:
1. The presented MS has so many problems that including mainly language and other basic common errors, and the preparations lacks lot of information in methods and the same reflecting in their results and discussion. Due to the language issues, it is very hard to follow the complete MS. I suggest the authors to rewrite the whole manuscript with suggestions from the qualified scientific writing individuals.
2. Title: seems inappropriate, suggested to change it
3. Introduction: Most of the words seems irrelevant which makes difficult in understand search for similar and appropriate scientific word choices during revision. Some of the sentences are not even at the basic level of technical writing.
4. Materials and methods: In general, tiere is a lack of explanation of replicates and statistical methods used in the study.
5. Results:For every section start with brief introduction of experiment and explain your obtained results in details. You have to provide all your results that are obviously seen in your figures and the figures should be represented that you were describing in this Section.


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

Input:
Title: ESTABLISHMENT AND CHARACTERIZATION OF FANTAIL GOLDFISH FIN (FTGF) CELL LINE FROM GOLDFISH, CARASSIUS AURATUS FOR IN VITRO PROPAGATION OF CYPRINID HERPES VIRUS-2 (CYHV-2)
Review round: 1
Reviewer: 3

Output:
Basic reporting: The Authors clearly explain the steps used to develop the cell-line. This is an important step because growing cyprinid herpesviruses is still a big Challenge in fish research. The back ground is Clear and sufficiently explains the problem faced With Current culture of CyHV-2. The literature cited and References are update.

Tabls and figures used are Clear although it could have been good if the Authors had shown the gross pathology in the challenged fish apart from the histology which they have shown
Experimental design: The experimental design is well explained on how the explants were collected and cultured. They explain the different temperatures used as well as the FBS concentration used. The CPE is Clear in the infected monoloyaer while the presence of virions is well explained by Electron microscopy.
Validity of the findings: the conclusions are well supported by the data provided.
Additional comments: This is an important contribution to the culture of CyHV-2


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: ESTABLISHMENT AND CHARACTERIZATION OF FANTAIL GOLDFISH FIN (FTGF) CELL LINE FROM GOLDFISH, CARASSIUS AURATUS FOR IN VITRO PROPAGATION OF CYPRINID HERPES VIRUS-2 (CYHV-2)
Review round: 1
Reviewer: 4

Output:
Basic reporting: There are some discrepancies between the graphs and description in the text: the temperatures in which the cells were incubated are different: text gives 15°C, 20°C, 28°C, 30°C, 37°C. fig2b: 20°C, 24°C, 28°C, 35°C, 37°C. Please check and correct.
Experimental design: Authors developed the cells line (characterised it somehow) and used it for propagation of the CyHV-2 with which they were able to infect the fish. This in not how the usability of the cells should be shown: interestingly the cells seems to be highly susceptible to the virus (produce high titor) therefore diagnostic use would be better.

Cell could be used also to show the temperature range for virus replication.

Non of the graphs description presents how many replicates were used.

No statistical analysis was done to compare the cell growth speed.
Validity of the findings: Please see point 2.
Additional comments: The paper has very classical approach to the development of the new cells for culturing CyHV-2. The virus could threaten food security in several countries. In my opinion authors missed the opportunity to use the cells in some useful and innovative way: e.g. comparing the diagnostic value of the cells to the PCR the paper would be definitely worth publishing. The infection with cyprinid herpesviruses is difficult to diagnose using the cell (many cells fails reliably diagnose even the acute infections) and perhaps the new cell line developed by would change this.


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: ESTABLISHMENT AND CHARACTERIZATION OF FANTAIL GOLDFISH FIN (FTGF) CELL LINE FROM GOLDFISH, CARASSIUS AURATUS FOR IN VITRO PROPAGATION OF CYPRINID HERPES VIRUS-2 (CYHV-2)
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comments
Experimental design: No comments
Validity of the findings: No comments
Additional comments: Authors answered my comment satisfactory. Still diagnostic use of the cells would be better approach especially as Authors performed experimental infection from which well-defined material could be used in estimating diagnostic usability of the cells. I find that this was a missed opportunity from the authors.


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

Input:
Title: THE VALIDITY OF ACTIVITY TRACKERS IS AFFECTED BY WALKING SPEED: THE CRITERION VALIDITY OF GARMIN VIVOSMART® HR AND STEPWATCH™ 3 FOR MEASURING STEPS AT VARIOUS WALKING SPEEDS UNDER CONTROLLED CONDITIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This paper compares step counts from two wearable accelerometers (Garmin Vivosmart HR and StepWatch 3) with observer-counted steps during treadmill walking. The trial was repeated six times, each with a different walking speed (1.6–5.6 km/h). I found the paper interesting to read and it is reasonably well written. There are several small errors with wording or sentence structure throughout. While these errors are small, they collectively reduce the quality of the work. I suggest a thorough proofread.

The field of physical behaviour measurement has evolved rapidly over the last several years. I find it odd that the authors do not acknowledge any recent research – including research which specifically examines the validity of the two devices assessed in this study. After a quick search, I see 5+ papers published in the last four years examining the validity/reliability of these two wearables. This paper seems as if it was written four years ago and not updated (given the most recent reference is 4 years old). I suggest the authors reposition the rationale considering recent evidence (i.e. position it such the rationale for replication is clear).

The layout of the paper and results tables are presented well. A small suggestion would be to drop Table 3 and simply add an additional column to Table 2. For the figures, there is duplication (i.e. same data are shown in Figures 5, 6, and 7). I would suggest collapsing Figures 3–7 into two new figures: one for the Vivosmart and one for the StepWatch. Each figure would have 6 panels/facets for the different walking speeds. This would make it easier to visualise the mean difference and LOA for each device/speed combination.
Experimental design: The overall design seems reasonably sound (see some specific limitations below). I understand the general purpose of the study, but the written aim (at the end of the introduction) is not well articulated. This study “aims at creating a knowledge base…inform…enlighten…” is not a measurable aim. I suggest rephrasing this, so it is specific and measurable.

What you are primarily assessing in this study is the accuracy of the manufacturer’s step detection algorithm(s). One of the main issues with consumer-grade wearables is the activity detection algorithms are closed-source (and black box). This also means that the manufacturer could update these algorithms without the user knowing. I think one of the main study design ‘issues’ is the use of the Garmin Vivosmart HR. This device was discontinued in Q1 2016 (Garmin only produced it for ~6 months). It is very unlikely the step detection algorithm in the HR would be identical to the one used in the current Vivosmart 4. This issue has not been discussed.

The rationale for many of the design decisions (e.g. sample size, inclusion criteria, treadmill speeds) is justified ‘based on previous work’. This is fine, but I feel these decisions should also be rationalised within the context of the current study.

One of the main rationale points in the introduction is the slow walking speeds in elderly. The sample consisted of 30 healthy adults, which limits the generalisability to other populations, particularly elderly. Slow walking speeds alone do not account for the biomechanical differences in walking. Step frequency could be similar but stride length much shorter in elderly (for example). This limitation is not described (in fact, no limitations are presented in the discussion).

In my mind, accurate step detection consists of two parts: 1) the ability to detect steps, and 2) the ability to reject non-steps. The current study design only addresses the former, meaning you can only assess the sensitivity of each step detection algorithm and not the specificity. A protocol that includes things like shuffling, standing with movement, jumping, kicking a ball (ankle sensor), throwing (wrist sensor), etc. would be needed to demonstrate specificity.
Validity of the findings: All underlying data and analysis code have been provided, which is excellent, and makes the results reproducible. However, I feel the conclusions of the paper are somewhat overstated. Stating that ‘This study concludes that the Vivosmart and StepWatch are valid in detecting steps…’ is slightly misleading. The conclusions should be presented in the context of the limitations mentioned above, and the existing work which was not considered.
Additional comments: Overall the study was reasonably well-conducted, and the paper was enjoyable to read. The main issue is not recognising the limitations of the design, and not considering the (substantial) new evidence that has been produced over the last 4 years.


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 VALIDITY OF ACTIVITY TRACKERS IS AFFECTED BY WALKING SPEED: THE CRITERION VALIDITY OF GARMIN VIVOSMART® HR AND STEPWATCH™ 3 FOR MEASURING STEPS AT VARIOUS WALKING SPEEDS UNDER CONTROLLED CONDITIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. There are sections where the english can be improved. For example, in the first paragraph, the frequent use of “e.g” is incorrect.

Lines 61-74, the authors switch back and forth from past to present tense. This makes it confusing for the reader.

2. Line 4- Recent work by iMin lee found this 10,000 step recommendation to be misleading. The authors should update this section to highlight recent work done examining the validity of these recommendations.
Experimental design: The authors need to add additional information into the introduction explaining the the differences in the step watch vs Garmin. Wrist worn vs ankle worn. Another paragraph discussing the various locations and how they can impact device accuracy would benefit the reader.

The authors state that “ethical approval” was not required? But then the participants signed an informed consent? Was IRB approval needed or not? If not, why did they have the participants fill out the Par Q and other materials? Clarity here would be appreciated.
Validity of the findings: Well designed study.

I would like the authors to add more into the discussion as to how these devices could be used among a clinical setting, seeing as they introduced that possibility in their introduction.
Additional comments: I enjoyed reading this manuscript. I believe it offers value to the field and will assist future investigators determine whether these devices should be used in their research.

Grammar needs to be cleaned up.


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 VALIDITY OF ACTIVITY TRACKERS IS AFFECTED BY WALKING SPEED: THE CRITERION VALIDITY OF GARMIN VIVOSMART® HR AND STEPWATCH™ 3 FOR MEASURING STEPS AT VARIOUS WALKING SPEEDS UNDER CONTROLLED CONDITIONS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I'm happy with the authors' (substantial) revisions and rebuttal responses. The paper is much better than the initial manuscript, and probably warrants publication in PeerJ.


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 VALIDITY OF ACTIVITY TRACKERS IS AFFECTED BY WALKING SPEED: THE CRITERION VALIDITY OF GARMIN VIVOSMART® HR AND STEPWATCH™ 3 FOR MEASURING STEPS AT VARIOUS WALKING SPEEDS UNDER CONTROLLED CONDITIONS
Review round: 2
Reviewer: 2

Output:
Basic reporting: I'm satisfied with the authors' response to my suggestions.
Experimental design: I'm satisfied with the authors' response to my suggestions.
Validity of the findings: I'm satisfied with the authors' response to my suggestions.
Additional comments: Nice job responding to both reviewers' comments/edits. I look forward to seeing this in print.


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

Input:
Title: SIDEROPHORE AND INDOLIC ACID PRODUCTION BY PAENIBACILLUS TRITICISOLI BJ-18 AND THEIR PLANT GROWTH-PROMOTING AND ANTIMICROBE ABILITIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript has many apparent inconsistencies and needs a thorough revision. English grammar should be checked throughout the text. Honestly, it is difficult to judge the quality of the manuscript itself. The language and structure are poor. The introduction section is poorly organized. The novelty and objectives of this study are not clear. The materials and methods section is not accurately written. I feel like the authors have not revised the manuscript. There are even inconsistencies with the abbreviations, acronyms and culture media composition.
The hypothesis is not presented. I would request the authors to update the literature review. In short, I can not judge the scientific quality of this article because of the above-mentioned issues.
I found this manuscript really heavy to read.
Experimental design: From my point of view, there is a clear lack of novelty in this article. The simple description of some secondary metabolites itself does no support the publication of an article. Further experiments should be conducted in order to study some specific aspects of those siderophores. The discussion section is full of expression like "it is easy to speculate...", "it´s speculate..." "it´s deduce..."
I´d suggest the authors re-write this article. Also, please focus on specific objectives/hypotheses. This article is too descriptive and the discussion is based on speculations.
Validity of the findings: No comment
Additional comments: Please avoid contractions!
Some of the acronyms are defined twice in the text (e.g. LB). Please double check.

Line 46. What part of the plants? Please be more specific
Line 48. What do you mean by "concentration"?
Lines 51-52. You said that Paenibacillus spp. can suppress the growth of plant pathogens such as bacteria, fungi, and nematodes. Do you mean that all bacteria/fungi/nematodes are pathogens? Please be more specific. You might want to add the specific pathogenic species.
Line 56. There are more recent references for that matter.
Lines 57-60. You stated that Paenibacillus was isolated from wheat. Again, be more specific. Did you mean from the rhizosphere of wheat?
Lines 70-73. We might want to define what is a siderophore and its benefits for plants.
Line 74. Why and "so on". Are not all the siderophores important? Why did you highlight those 3 of 7?
Lines 75-77. This statement is too vague. Even more, you did not define secondary metabolites which are the goal of your paper.
Lines 93-95. The objective is not clear and the hypotheses are not presented. Also, what do you mean by "multiples roles"?

The Materials and Methods section is not clear. Further details must be added. For example in Line 124 (how? concentration?), Line 141 (culture media composition), Line 147 (Incubation time? concentration?. Also, you changed from imperative to past tense to present tenses throughout this section. Please, be consistent. I found it really heavy to read.

The discussion section is full of speculations. Again, the objective is not clear and therefore your conclusions are not informative.


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: SIDEROPHORE AND INDOLIC ACID PRODUCTION BY PAENIBACILLUS TRITICISOLI BJ-18 AND THEIR PLANT GROWTH-PROMOTING AND ANTIMICROBE ABILITIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: •	Abstract, Intro, Methods, Results, Discussion, Conclusions are not clear and ambiguous. A thorough check of spelling and English are required.
•	Figures are poorly labelled.
Experimental design: •	The NMR data provided for 1 and 4 are not clear.
•	Provide NMR data of 4, 5, and 6 and LC MS data for 1-6.
Validity of the findings: •	Raw data provided are insufficient to determine impact/novelty of research
Additional comments: Overall, a thorough check of English is required


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

Input:
Title: SIDEROPHORE AND INDOLIC ACID PRODUCTION BY PAENIBACILLUS TRITICISOLI BJ-18 AND THEIR PLANT GROWTH-PROMOTING AND ANTIMICROBE ABILITIES
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: This manuscript reported six secondary metabolites from the titled  bacteria, including a new siderophore which contained three fusarinine units, as well as a new paenibacillic acid A. However, the data in bioassays revealed that these compounds didn't show antimicrobial activity and cytotoxicity. Furthermore, I sugguest that the positive controls should be changed into powerful antibiotics against  Staphylococcus aureus and  Bacillus subtilis. The current data make no sense. Inaddition, the manuscirpt didn't provide full NMR data for compound 1, which is unacceptable for identification of a new conpund. I suggest that it should be declined.


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: SIDEROPHORE AND INDOLIC ACID PRODUCTION BY PAENIBACILLUS TRITICISOLI BJ-18 AND THEIR PLANT GROWTH-PROMOTING AND ANTIMICROBE ABILITIES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The article is clear and has sufficent references..
Experimental design: Yes the methods are sufficient.
Validity of the findings: Provide the annotated NMR spectra in the SI with the corresponding structure.
Additional comments: Comments:
Title: Siderophore and indolic acid production by Paenibacillus triticisoli BJ-18 and their plant growth-promoting and antimicrobe abilities
(pls refer to the no-markup line numbers)

Abstract
Line 25. Change “N2-fxing” to “N2-fixing”

Introduction
Line 56. Write “enzymes” in small case
Line 58. Change “referred as MoFe protein” to “referred to as MoFe protein”
Line 87. I think it is “Pirycularia oryzae” NOT “Piricularia oryzae”
Line 89. I think it is  Cryphonectria parasitica” NOT  “Cryphonectria parastitica”
Line 98. Insert “the” … Endophytic bacteria promote the growth…
Line 103. Change “inoculation with P. triticisoli BJ-18 significantly promote growth” to “inoculation with P. triticisoli BJ-18 significantly promotes the growth”

Materials and Methods
Line 145. I don’t understand this statement.
“The General Experimental Procedures optical rotations measurement was conducted on an Anton Paar MCP200 polarimeter (Anton Paar, Austria).”
Is “The General Experimental Procedures” a subheading?!
Line 152. Small case for “254 nm” wavelength. “UV at 254 nM”
Line 162. Use the term “seed culture” instead of “bacterial solution”
Lines 183-200. Indicate the mass error (∆) in ppm of all isolated compounds
I suggest that the numbers of the compounds are written in bold, such as compound 1, compound 2… to distinguish it from the rest of the numbers in the main text. It is very confusing.

Results
Line 270. Write ChemSpider as uppercase “S” not Chemspider
Lines 293-296. “Among the six compounds, compounds 1-3 were identified as fusarinines that were classical siderophores, while compounds 4-6 were identified as indolic acids (Fig. 2).”
Which ones are new? Among compounds 1-6? Better indicate here. I cannot follow the flow of discussion afterwards. Confusing.

Line 298. Compound 2 was a cyclic tripolymer which has three same submits (m/z 284.1366) and identified as desferritriacetylfusigen (Anke, 1977).
What do you mean submits?

The authors claimed that paenibacillic acid A and deshydroxyferritriacetyl fusigens are novel structures. Are they really novel? Or is it just a modification of known siderophores or indolic acids? If they have a completely new backbone, then the compounds are novel. Otherwise, just state as new compounds.

Line 367. Insert “the” … Effects of indolic acids compounds on the growth
Line 389. “Krber’s method” or  “Spearman-Karber’s method”? Check the spelling
Line 395. Add the letter “s”…compounds 1-3 are siderophores.
Line 440. To “inhibit” the growth instead of “inhabit”
Line 448. Which supplemental files? Be specific.
Supplementary Info
Annotate each NMR spectra in the SI and show the corresponding annotated structure of each metabolite.

Figure 2. Instead of letters below the structures, use numbers 1-6 in bold similar to the the main text.
Table 3. The table caption is “Antimicrobial activity of compounds 1-6” but there are no numbers indicated in the name of the compounds.
Write the numbers 1-6 in bold after the name of the compound. What is the meaning of the blank rows and columns? Check the spelling of Karber’s method. Write as “5 µM, 10 µM and 20 µM” instead of “5 uM, 10 uM and 20 uM”


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: ASSESSMENT OF FISH BIODIVERSITY IN FOUR KOREAN RIVERS USING ENVIRONMENTAL DNA METABARCODING
Review round: 1
Reviewer: 1

Output:
Basic reporting: attached
Experimental design: attached
Validity of the findings: attached
Additional comments: The manuscript clearly showing the potential of applying eDNA in Korean river. The one thing need to be improved the most is the English writing to meet the international audience


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: ASSESSMENT OF FISH BIODIVERSITY IN FOUR KOREAN RIVERS USING ENVIRONMENTAL DNA METABARCODING
Review round: 1
Reviewer: 2

Output:
Basic reporting: Authors present an interesting manuscript dealing with the use of eDNA techniques to assess species richness in four freshwater rivers of the Republic of Korea, presenting some interesting results which are potentially relevant to conservation and future studies on related subjects in Korea and other regions as well. As such, I congratulate the authors and recommend it for publication after major review based on the points below and in the suggestions, comments and remarks in the attached file. In my view, the main overall problem with the manuscript seems to be related to the proper use of English, which is often confusing and imprecise throughout the text. In the attached file, I made several suggestions and also indicate sections of the text that needs improvement. Even if my suggestions are accepted in the following version of the manuscript, it still might be interesting to the authors to evaluate the use of a professional scientific translation service, or to check on the possibility of having the text reviewed by a fellow native speaker. There are also some inconsistencies in the application of proper ecological/taxonomic/conservation terms, which also require review (again, I tried to indicate all I could in the attached file, but I probably missed something - please carefully re-check all the document). It might also be advisable for the authors to have the manuscript reviewed by someone more familiar with the proper use of terms from those areas of biological sciences. Please bear in mind that those criticisms are not meant to disqualify the paper, which indeed presents some interesting results. I congratulate the authors for the study conducted and for their effort to present it all in a nice, coherent, manuscript. This is an open review, and authors may contact me in case something in this review and/or in the attached file requires more clarification.
Experimental design: As far as I could comprehend (due to confusions of language used in the manuscript), experimental design has no flaws and falls in the scope of the journal.
Validity of the findings: Results seem to be overall consistent, and as far as I can see, underlying data was provided. There are, however, several questions raised in my review of the attached file in the discussion and conclusion that needs reviewing by the authors before the manuscript is accepted for publication. Some parts of the discussion are more speculative, but the degree to which they might be accepted in a likely upcoming version of the manuscript might depend more on the proper use of the language than its content. Please check the attached file for details.
Additional comments: Same as the basic reporting - Authors present an interesting manuscript dealing with the use of eDNA techniques to assess species richness in four freshwater rivers of the Republic of Korea, presenting some interesting results which are potentially relevant to conservation and future studies on related subjects in Korea and other regions as well. As such, I congratulate the authors and recommend it for publication after major review based on the points below and in the suggestions, comments and remarks in the attached file. In my view, the main overall problem with the manuscript seems to be related to the proper use of English, which is often confusing and imprecise throughout the text. In the attached file, I made several suggestions and also indicate sections of the text that needs improvement. Even if my suggestions are accepted in the following version of the manuscript, it still might be interesting to the authors to evaluate the use of a professional scientific translation service, or to check on the possibility of having the text reviewed by a fellow native speaker. There are also some inconsistencies in the application of proper ecological/taxonomic/conservation terms, which also require review (again, I tried to indicate all I could in the attached file, but I probably missed something - please carefully re-check all the document). It might also be advisable for the authors to have the manuscript reviewed by someone more familiar with the proper use of terms from those areas of biological sciences. Please bear in mind that those criticisms are not meant to disqualify the paper, which indeed presents some interesting results. I congratulate the authors for the study conducted and for their effort to present it all in a nice, coherent, manuscript. This is an open review, and authors may contact me in case something in this review and/or in the attached file requires more clarification.


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: ASSESSMENT OF FISH BIODIVERSITY IN FOUR KOREAN RIVERS USING ENVIRONMENTAL DNA METABARCODING
Review round: 2
Reviewer: 1

Output:
Basic reporting: Overall, the study meet the criteria for scientific paper to be publish in PeerJ. This study also has an novelty in the methods and results, and I am sure will be a huge addition for the eDNA study in Korea in general. Notes for the phylogenetic tree figures: if it possible, a better resolution for the figures will be perfect. The second note is: it will be informative if the authors can show the reader about the genbank accession number for the results, or may be the the link of repository for the data obtained from this study.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: ASSESSMENT OF FISH BIODIVERSITY IN FOUR KOREAN RIVERS USING ENVIRONMENTAL DNA METABARCODING
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript has improved substantially, and I truly congratulate the authors for their effort to present a much more coherent and easier to understand text. However, there is still a substantial number of phrases that need to be more properly clarified in terms of the English language, unfortunately. I made several suggestions throughout the text in the attachment file, but it is still my view that the manuscript would greatly benefit from a more thorough review from a fellow scientist with more skills in the use of the language. Also, following correct taxonomic procedures, it is necessary that species names authorities are indicated after the first mention of all species dealt with in the text. In addition, in my opinion, terms like "Japanese haplotype" or "Korean haplotype", etc, should be avoided in the text. The words Japanese and Korean (or Brazilian, for instance) typically refer to human beings, and of course that is not what the authors mean. Please try to use "haplotypes of (a particular species) identified in Japan", something like that, in order to avoid this confusion. Overall, I again congratulate the authors for their interesting paper. This is an open review - Fabio Di Dario.
Experimental design: As far as I can see, experimental design has no flaws and falls in the scope of the journal.
Validity of the findings: As in my previous review, I still think that results are overall consistent, and as far as I can see, underlying data was provided. However, there still are some questions raised in my review of the attached file in the discussion and conclusion that needs reviewing by the authors before the manuscript is accepted for publication. Please check the attached file for details.
Additional comments: Same as basic reporting - The manuscript has improved substantially, and I truly congratulate the authors for their effort to present a much more coherent and easier to understand text. However, there is still a substantial number of phrases that need to be more properly clarified in terms of the English language, unfortunately. I made several suggestions throughout the text in the attachment file, but it is still my view that the manuscript would greatly benefit from a more thorough review from a fellow scientist with more skills in the use of the language. Also, following correct taxonomic procedures, it is necessary that species names authorities are indicated after the first mention of all species dealt with in the text. In addition, in my opinion, terms like "Japanese haplotype" or "Korean haplotype", etc, should be avoided in the text. The words Japanese and Korean (or Brazilian, for instance) typically refer to human beings, and of course that is not what the authors mean. Please try to use "haplotypes of (a particular species) identified in Japan", something like that, in order to avoid this confusion. Overall, I again congratulate the authors for their interesting paper. This is an open review - Fabio Di Dario.


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: ASSESSMENT OF FISH BIODIVERSITY IN FOUR KOREAN RIVERS USING ENVIRONMENTAL DNA METABARCODING
Review round: 3
Reviewer: 1

Output:
Basic reporting: The manuscript improved significantly since the first rounds of revision, but major adjustments in terms of language are still necessary. In order to try to speed up the process of revision, I took the liberty of editing the .docx version of the manuscript that was uploaded by the authors in the system, using Microsoft Word's Track Changes. Please see the attached file for details (I had to save it as a .pdf since the system doesn't accept other types of files, but authors can contact me - didario@gmaill.com - if they want to have access to the .docx file). I also indicated in that file some sentences that need further clarification - I'd have suggested changes in them as well if I understood the idea, but that was not the case in some parts of the text. It's still my opinion that the manuscript would greatly benefit from a more thorough review by someone close to the authors more familiar with the English language (ideally a fellow scientist) before the next submission. It's my understanding that the manuscript is improving between subsequent resubmissions, but the pace in which the language is being updated is not exactly fast. However, the contents of the manuscript are, in my opinion, scientific sound, and the manuscript also seems to be relevant to the assessment of biodiversity in the Republic of Korea. It will eventually deserve publication, but more work on the language is definitely necessary.
Experimental design: Please check previous reviews.
Validity of the findings: Please check previous reviews.
Additional comments: Same as those in Basic reporting, adding that this is an open review (Fabio Di Dario) - The manuscript improved significantly since the first rounds of revision, but major adjustments in terms of language are still necessary. In order to try to speed up the process of revision, I took the liberty of editing the .docx version of the manuscript that was uploaded by the authors in the system, using Microsoft Word's Track Changes. Please see the attached file for details (I had to save it as a .pdf since the system doesn't accept other types of files, but authors can contact me - didario@gmaill.com - if they want to have access to the .docx file). I also indicated in that file some sentences that need further clarification - I'd have suggested changes in them as well if I understood the idea, but that was not the case in some parts of the text. It's still my opinion that the manuscript would greatly benefit from a more thorough review by someone close to the authors more familiar with the English language (ideally a fellow scientist) before the next submission. It's my understanding that the manuscript is improving between subsequent resubmissions, but the pace in which the language is being updated is not exactly fast. However, the contents of the manuscript are, in my opinion, scientific sound, and the manuscript also seems to be relevant to the assessment of biodiversity in the Republic of Korea. It will eventually deserve publication, but more work on the language is definitely necessary.


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: ASSESSMENT OF FISH BIODIVERSITY IN FOUR KOREAN RIVERS USING ENVIRONMENTAL DNA METABARCODING
Review round: 4
Reviewer: 1

Output:
Basic reporting: I am really glad to see that the manuscript improved substantially from the previous rounds of review. I congratulate the authors for presenting a much more concise, clearly written, and interesting manuscript that in my view should be published after just some minor adjustments are made in the text, as indicated in the attached pdf file. Given that the modifications I suggest are really small, I don't think it's necessary that the manuscript is sent again to a new round of reviews, as long as the adjustments are made. And again, this is an open review - Fabio Di Dario.
Experimental design: Same as previous rounds of review.
Validity of the findings: Same as previous rounds of review.
Additional comments: Same as basic reporting and previous rounds of review. Congratulations! Nice and relevant paper!


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

Input:
Title: A NOVEL HISTOZOIC MYXOSPOREAN, ENTEROMYXUM CAESIO N. SP., INFECTING THE REDBELLY YELLOWTAIL FUSILIER, CAESIO CUNING, WITH THE CREATION OF THE ENTEROMYXIDAE N. FAM., TO FORMALLY ACCOMMODATE THIS COMMERCIALLY IMPORTANT GENUS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Regarding the language, the manuscript is technically correct and clearly written and only a few minor mistakes have escaped the author’s corrections.
- L149-159: Units should be separated by one space from numbers.
- L168: Please, correct verb conjugation or tense.
- L272: Future tense should be replaced by present tense.
- Table 1: “Length Mean”, “Width Mean” and “Thickness Mean” should be replaced by “Mean Length”, “Mean Width” and “Mean Thickness”.
- Table 1: Spelling of “Scophthalmus maximus” should be corrected.

There are further minor errors regarding the content of the manuscript, which should also be reviewed.
- Table 1: Polar filament coils are not given in µm.
- Table 1: Means are missing for myxospore dimensions of E. leei in D. puntazzo.
- Figure 2: The authors claim to have found a new parasite species in the “glandular gastric tissue of the stomach”, but no host tissue or cells are recognizable in the image shown. A lower magnification image would help to locate the parasite stages in the organ.
- Figure 2: Does the insert belong to the Giemsa-stained histological section? It should.
- Figure 3: There is no “black scale bar”. Please correct.
- Figures: I would suggest maintaining a consistent scale bar format, i.e. indicating scale bar length either in the figure caption or in the image.
- References: Please, check reference order (e.g. Diamant A 1997; Diamant A, Lom J, Dyková I 1994; Diamant A 1998). Please, references of same authors and years should be identified by letters as different citations in the text and in the reference list (e.g. Freeman MA, Krismundsson Á 2015).
Experimental design: The first issue regarding the present experimental design concerns the method used to identify infected fish: field observation of wet mounts of stomach and intestinal scrapings. This method is a fast and easy one to determine the presence of Enteromyxum spp. only in advanced, high intensity infections. Pre-sporogonic stages are difficult to be identified in this way, and even more at initial infection stages when parasite stages are small or even unicellular. Furthermore, authors do not give any explanation why stomach and intestine were chosen for this, while esophagus and rectum were ignored, nor which intestinal segment (anterior, middle, posterior, entire?) was used.
Therefore, the presence of the parasite in the examined fish might have been underestimated, and thus, authors limited their sample size to just one infected fish.

There is a further issue, mainly, the authors are claiming to describe for the first time E. caesio n. sp. in redbelly yellowtail fusilier and they included in the manuscript TEM images of another parasite of the same genus.
Though images (Figure 4) show nicely the host-parasite interaction at cellular level in E. fugu-infected tiger puffer, as was previously demonstrated in E. leei-infected sea bream (Cuadrado et al. 2007), this is not relevant to the research work presented herein. The manuscript’s title also excludes the significance of this result by itself and its relevance for the E. caesio n. sp. description by omitting to mention it and it is a surprise for the reader when it shows up in the manuscript since it has no relationship with the main matter. My suggestion is to eliminate the results on E. fugu from the manuscript, since the aim of this work as it is now, is not the characterization of host-parasite interactions. These data would be very interesting for further research on fish-myxozoan interactions.

Materials & Methods
This section is lacking important details about the animals used and their sampling. Some of the missing information (e.g. fish numbers and size) is surprisingly provided later in the results section.
- L83: “Fish were bought… others were captured” is too ambiguous. Fish species, amount, size and if dead or alive should be indicated here. If captured fish were collected alive, authors should describe how the sacrifice was performed.
- In this regard, this reviewer ignores the legal issues of lethally sampling wild animals for scientific purposes in Saint Kitts and Nevis, but the authors do not mention in their manuscript any bioethics committee approval or adaptation of their methods in accordance to the local legislation. An ethics statement containing such information has to be provided in this section.
- L87: Authors declare to have taken images of fresh myxospores, but they do not show them. In addition to my previous comments on Figure 2, I would suggest to include a wet mount image of parasite stages (not only as an insert).
- L88-89: “Infected tissue samples…” Please, define which tissues. I would strongly suggest examining the entire digestive tract (from esophagus to rectum).
- L92: Again, authors should mention fish origin, number, size and method used for their sacrifice.
- L96: Authors should clarify in the text, which fish they used to obtain these intestinal scrapings.
- L149: “ A total of 8 fish…” Authors should indicate which fish species they are talking about since they worked with fusiliers and puffers.
Validity of the findings: The main drawback of the present work is that the entire research is based on the one and only infected fish sampled, due to the low infection prevalence found and to the small sample size.
I wonder how convenient and valid the taxonomic redistribution and the new species description are, which the authors suggest based on the SSU rDNA sequence they obtained from one single sample.
However, molecular phylogeny is not my area of expertise, and thus, I leave this matter up to other reviewers’ opinion or editorial decision.

There is a further important issue that needs to be improved by the authors. Histological samples should be taken immediately after death, but in the case of the fusilier’s samples tissues were fixed “some hours after being caught”. Though authors state that therefore, “reliable description of host tissue response was not possible”, they affirm, “disporous pseudoplasmodia were seen in the glandular part of the stomach wall (gastric glands)”. The main issue is that, at least from the present image (Figure 2), the cellular host response as well as the location of the parasite stages at tissue level is impossible, as I mentioned before. Only by providing histological images in which the tissue structure is discernible, the parasite presence in the gastric glands would be confirmed. Otherwise, histological samples have the same validity as fixed and stained tissue scrapings or squashes. Therefore, the obtaining of life, infected fish is crucial if authors aim to describe tissue location of the parasite. A further suggestion once this is achieved, is to include ultrastructural observations by TEM in their study.
Additional comments: The present results aim to define a new species within the Enteromyxum genus and the erection of a new taxonomic family in order to accommodate members of this genus separated from Gastromyxidae and Myxidiidae. Nevertheless, the quality and amount of samples used therefore should be improved in order to validate this very interesting and promising data, which seem still preliminary.