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

Input:
Title: DIFFERENTIAL RESPONSE TO STRESS IN OSTREA LURIDA AS MEASURED BY GENE EXPRESSION
Review round: 1
Reviewer: 1

Output:
Basic reporting: BASIC REPORTING

Major considerations

The manuscript is generally well-written and the language clear and generally unambiguous (specific problems are addressed further in this review.) There are some minor typos and occasional grammatical errors that can easily be caught in revision. The structure of the manuscript conforms to the PeerJ standard.
The Intro & background give the reader context and are well-referenced. However at times this section seems to delve in to what may be too much detail given the rather general scope of PeerJ.

The hypothesis is well-constructed and supported based on the background information, but commentary on reproductive cycle specifics may not be required based on the exact gene expression data collected and the discussion later in the paper.

The authors set out to test the hypothesis that one geographically separate population may have a more pronounced stress response than the others and mention relation to the Baldwin effect and reproductive performance in the discussion. There is mention of local efforts to restore O. lurida and reproductive performance is obviously a consideration for such efforts. For the generalist reader more explicitly mentioning these models and their relevance to our understanding of biology in general as well as their relation and application to restoration efforts may help the reader better understand the intentions and relevance of the research and the rationale for the general research design. Indeed, the rationale is in the manuscript, and understandable to the reader, but scattered and not emphasized in an obvious way. Currently the authors are under-selling the value of their work and relevance. A bit of reorganization and focus on greater biological questions at hand or potential application of the knowledge would remedy this.

The figures are all relevant and generally well-presented. The few concerns I do have are mentioned below.

Minor considerations

It would help if the reader is referred to Figure 1 in the introduction describing the different populations rather than in the Material and Methods section.
Experimental design: EXPERIMENTAL DESIGN

The specific experimental design directly tests the hypothesis that oysters from Dabob Bay will demonstrate a more pronounced response to stress via changes in gene expression compared to oysters from other geographic locations. This is done through comparison of mRNA levels of a battery of different O. lurida genes in animals taken from three different geographic locations that were placed under temperature or mechanical stress. The mRNAs studied were predicted to be involved in stress response based on sequence similarity to genes with well described functions in other systems.

The methods with respect to the biochemistry are described in sufficient detail for replication by investigators experienced with RT-qPCR although minor clarifications are required.

The data analysis, however, would benefit if the authors were more explicit with how changes in gene expression were calculated and why. This is especially true with respect to what a sample (n=1) represents in the data sets. It would be appreciated if the authors would clarify the following:

Major considerations

Line 117-127: Would the authors please clarify if tissue was pooled from animals. Explicit descriptions of what one data point represents would assist with interpretation of data and assessment of its robustness. Further, were technical replicates performed, or was only one RT-qPCR reaction performed per unique animal per gene?

Clearly “stating n= X animals per condition” for example in the results section or in the figure legend would make this crystal clear to the reader.

Line 174: This table is useful, but please comment on how gene function was inferred from sequence. Is this from GO terms based on closest known homolog? Is this manual or semi-automated curation? Additionally, are there potential paralogues for these genes and why was one picked over another? The authors even suggest that the HSP70 transcript they chose may not actually be the true HSP70-like protein in other organisms given the lack of transcript change in response to heat stress.
In the last paragraph of the introduction the authors state “A suite of genes was selected based on their predicted function (gene regulation, immune response, and growth).” Binning the genes in to these categories in the table helps connect the experimental design to the hypothesis and may make discussion of the results easier.

Line 187-201: Even though Real Time Quantitative PCR as a commercially available tool has been around nearly longer than some of the youngest current grad students have been alive, data analysis and reporting is still inconsistent between groups and multiple methods for absolute quantitation based on normal curves or relative levels to a reference gene are in use. Statistical analysis of these data represents even more of a “Wild West” situation.

The authors reference the Schmittgen and Livak, 2008 paper. Schmittgen and Livak offers a number of different analysis methods. The fold change=2−ΔΔCt method where 2−ΔΔCt = [(Ctgene of interest - Ctactin)treatment -  (Ctgene of interest - Ctactin)control] is often used by other groups and could also be appropriate for this type of data as best I can tell. Please explicitly state why the normalized mRNA levels are calculated as ΔCt= (target Ct – actin Ct) as well as why a logΔCt were used for statistical analysis. In the results section the y-axis is labeled as the data being plotted at a ΔCt value. The statistical calculations being performed on logΔCt value but the graphs showing just the ΔCt can be a bit confusing. It’s not so much that there’s an inherent problem with how the data were analyzed, but rather just being clear why it was done this way. Overall, the treatment of the data as ΔCt values, given the way the experiments were designed and run, is still better than doing fold-change calculations and statistics with the fold-change without efficiency correction and linearity controls as many do. Kudos to the authors for avoiding this pitfall.

Line 204-207: These data are mentioned in the discussion but it’s unclear from the introduction as to why these experiments were done in the first place. What direct relevance does this have on the rest of the data and or with respect to how this work falls within the greater body of the literature?

Minor considerations

Line 126-127: Was the tissue simply stored in RNAzol or was it lysed in RNAzol and one of the intermediary steps used for storage at -80oC? The manufacturer’s protocol has a number of convenient stop points but storage of whole tissue in RNAzol is not in any protocols I’ve seen or performed myself.

Line 148-149: Please comment on which primers were used to detect genomic DNA via PCR. Were primers targeted to intronic or mRNA non-coding regions? Or were primers generating an amplicon spanning an exon junction used?

Line 152-157. Awkward phrasing.

Line 161: Reference renders and links strangely in PDF. This may be something the editor needs to deal with.

Lines 159-168: Please comment on whether the priming sites for the primers were known to contain SNPs that may have affected hybridization efficiency. This should be available since the Authors’ lab appears to have made its own transcriptome and have access to raw sequencing reads. Since the authors are not using highly inbred populations and these populations are originating from different geographical locations where some degree of genetic drift, founder effects, selective pressures, etc. may have generated SNPs between groups there may be variability in the mRNA sequences. A single SNP in a primer binding sequence is enough to dramatically affect replication efficiency and the downstream relative abundance calculations. Even within highly inbred lab strains of fruit flies or C. elegans SNPs are observed within a lab’s own stocks and between labs- even in protein coding sequence or regions of high evolutionary conservation!
Validity of the findings: VALIDITY OF THE FINDINGS

Specific comments on the Results section

If n=8 animals as the methods suggest then the data appear to be generally robust, statistically sound, and controlled. No concerns there except for those raised above in terms of data representation and explicit statement of what n=1 constitutes. Interpretation of two-way ANOVAs can be tricky, but I think the authors have done a generally good job here. Lack of significant difference is also noted and is relevant to the hypothesis and the discussion of the results.

What may make the results section easier to interpret for a more generalist reader is if full gene names were given as they are in the discussion. Possibly the table currently in the methods section could be split up in to one table just for primer design and another put in to results for gene name and inferred function from sequence similarity or existing literature.

Specific comments on the Discussion section

In general, connecting changes in mRNA level of the battery of genes studied with existing literature is well done. The inferences are sane and based in literature findings without speculation that raises any flags. However, many of these genes have been very well-studied in model genetic organisms like mouse, Drosophila or C. elegans. It may be worth citing some of that literature as well. In some places the authors already do this. Are there potentially more overarching biological phenomena you’re observing here that are worth noting?

Given some of the literature cited in the discussion, I get the feeling that some of the mRNA targets were chosen based on what was already known in other marine organisms. That’s an understandable way to pick targets. If that’s true, maybe this should be brought up in the methods or results section when discussing what targets were chosen and why. This would also help with proposing gene function from sequence similarity arguments.

Lines 226-237: Again, the discussion on mortality seems disconnected from the specific hypothesis of this study. More needs to be done to connect this to the rest of the paper.

Lines 258-267: The lack of change in the HSP70 orthologue is a bit worrisome. In many model systems the HSP70 transcriptional response should be in the timeframe of the experiments performed in this manuscript (minutes to hours.) There is some speculation as to isoform specific responses. From the transcriptome available, are different isoforms of this gene observed? Do the RT-qPCR primers target all or only a subset of them?

Specific comments on the Conclusion section

The authors conclude that the Oyster Bay population appears to have the greater ability to effectively respond to stress. This runs opposite to their initial hypothesis that oysters from Dabob Bay will demonstrate a more pronounced response to stress via changes in gene expression.

Lines 303-312: Conclusions are supported by the data - well done.

Lines 319-335: Maybe a little too speculative. The experiments done here don’t really directly test these very general models. Although, this discussion does put the results in a greater context. Depending on how the authors restructure the introduction some of this section could be put in the introduction.
Perhaps the style of writing and papers I’m used to reading in my field is different, but front-loading this in the introduction would make the general relevance and aims of this study much more clear. The line:
“For long term restoration of O. lurida populations in Puget Sound, understanding the phenotypic plasticity of individual populations will help determine proper supplementation procedures for existing and historic habitats,” succinctly sells me the rational for the research and experimental design.
Additional comments: Overall Reviewer’s Report

The hypothesis proposed is well-developed and the experiments to test the hypothesis are well designed and directly test the hypothesis. The authors’ discussion and conclusion are grounded in the data for the most part and made relevant with respect to the existing state of the field. Overall this work is of good quality on the raw science side of things. However, lack of clarity and consistency in some parts obfuscates the interpretation of the data as well as the general intentions and objectives of the work. These can be solved with revisions to the text of the manuscript. No additional experiments are required other than some low level bioinformatics with existing tools. In the following sections I have made recommendations where clarification or reorganization would fix these problems. I would happily re-review the manuscript upon revision.


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

Input:
Title: DIFFERENTIAL RESPONSE TO STRESS IN OSTREA LURIDA AS MEASURED BY GENE EXPRESSION
Review round: 1
Reviewer: 2

Output:
Basic reporting: This research compares patterns of expression of selected genes in response to acute heat and mechanical stress in 3 populations of Olympia oysters.  These populations were selected based on a previous study showing differences in performance in the field, as reported in a different manuscript (a preprint in PeerJ).

The research reported in this manuscript shows differences in gene expression in response to acute stress between populations, confirming that oysters from these locations show differences not only on performance, but also at the level of gene expression.   These results have relevance for the restoration and aquaculture industries, indicating that more work should be done to evaluate the mechanisms behind those differences.
Experimental design: The experimental design is adequate, although the stressors used were very acute, resulting in 100% mortality of exposed oysters.  The authors should indicate if those are consistent with stress happening in the field.
Validity of the findings: The research is well presented in general and the results are valid. I have attached a version of the manuscript with specific comments and suggestions.  My major suggestion is to provide a more focused discussion that clearly differentiates between findings (differences in gene expression between populations in response to stress) and speculations regarding mechanisms behind those differences.  The authors should also express that some caution should be taken into interpreting the relevance of these differences, since there was no difference between populations on their ability to survive these stressors (all oysters from all populations that were stressed died).  Finally, I recommend that authors clearly indicate where the research should go next, and how that research will inform breeding in support of restoration efforts.
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: DIFFERENTIAL RESPONSE TO STRESS IN OSTREA LURIDA AS MEASURED BY GENE EXPRESSION
Review round: 2
Reviewer: 1

Output:
Basic reporting: Pass: The authors have addressed all my comments to review satisfactorily.  I consider that the manuscript it acceptable for publication.
Experimental design: Pass: The authors have addressed all my comments to review satisfactorily.  I consider that the manuscript it acceptable for publication.
Validity of the findings: Pass: The authors have addressed all my comments to review satisfactorily.  I consider that the manuscript it acceptable for publication.
Additional comments: The authors have addressed all my comments to review satisfactorily.  I consider that the manuscript it acceptable 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: COMPARATIVE SPIGOT ONTOGENY ACROSS THE SPIDER TREE OF LIFE
Review round: 1
Reviewer: 1

Output:
Basic reporting: The language is a bit verbose and very passive. The mss conveys a LOT of detail which is very important but diluted by the excesses. The following are examples: it is interesting that;  e.g., we found this to be the case; we also found that the most likely state; it is possible that; we determined that; it is possible that; This means that; it would be helpful; we found that; we do know that; it is possible; etc. etc.

Recheck spelling of all family names. The addition of the initials of a species author seemed excessive and if it is a journal demand then it is mostly unnecessary. In only few cases are the author's initial needed, eg. L. Koch vs C.L. Koch, O.P-Cambridge vbs F.O.P, Cambridge. In a number of cases, e.g. line 420, 525, references are needed. Unless the information is provided new here, cite a source.
Also, ease off on the conjecture, e.g. line 524, 574.
Experimental design: The science is EXCELLENT; the depth and  rigour of the analyses is more than adequate and the questions posed/tested great! This is a very important work that MUST be published.
Be careful with new "terms", like flankers and webless. Either put it in quotes are explain it. Please think about people for whom English is NOT their first language, e.g. l. 425 "hold steady" won't translate.
line. 517, must end in ', respectively"
Also, "sit and wait"  works but "sit and pursue" seems contradictory, are these standard, if so, cite the original usage.l. 451. [We also observed snip!]... on PLS of both species" Which sexes?
Validity of the findings: The findings are totally consistent, very valuable. However, the conclusions are useless, they should resemble something like the abstract.
Additional comments: Really, it could do with a powerful tightening of language and probably a better structure of presentation of that amazing and valuable information you have.


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: COMPARATIVE SPIGOT ONTOGENY ACROSS THE SPIDER TREE OF LIFE
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is well written in general, the references and context are well covered.

I think the title is a bit “overscoping” - it is supposedly about ontogeny in spiders, but the ontogenetic content is presented on a specific clade of Araneomorphae (Entelegynae). I know that journals and authors usually benefit from that :)
Experimental design: 295 al., 2016). We used a maximum likelihood method with a model of the weighted rate matrix of
296 substitutions for these spigots (Table 6).
-- How was made the distinction between "absent" and "loss" in the scored species (data)?

Rates in Table 6 need an explanation. (Same with Table s2). It seems very strange that only a few scattered and rather derived taxa lack any MS-Fl-PsF, and yet the ancestral state is inferred as "absent". This probably comes from the rate matrix, which needs a justification. (Otherwise the interpretation of lines 388-390 is dubious.)
Validity of the findings: Figure 4: Where the identification of Pseudoflagelliform for Phyxelida comes from?  I suspect it is arbitrary, since no histology was made (all were labeled as MS in Griswold et al. 2005).

384 gland spigots on the PLS (Table S1, Townley & Tillinghast, 2009). The predominately green
385 (Loss of MS spigot) clade was the RTA clade,
-- but green = "MS"

386 blue/green (Pseudoflagelliform or Modified, respectively)
-- but blue = "none"

340 ... In both species, a larger spigot was tentatively
341 identified as a ‘modified spigot’ (see Griswold et al., 2005: 61; character 96), with a potential
342 pre-modified spigot observed in the penultimate female stage of D. tenebrosus. These made no
343 other appearance in the ontogeny of both species spinning fields (Table 1, Figs. 7, 8).
-- This is strange, as there is no nubbin of a modified spigot (MS) in the male. According to my experience, MS spigots leave a nubbin in male (Ramirez 2014: Table 5).
The potential MS of Hogna is worth illustrating, since no MS was ever reported for lycosids.
This finding is later downplayed in the Discussion.
Additional comments: Minor edits:

73 2016). The former “Orbiculariae” Deinopoidea (cribellate orb builders) are now sister to the
74 RTA clade (includes wolf spiders and jumping spiders) rather than to the Araneoidea (sticky-silk
75 orb weavers) and Deinopoidea may not even be monophyletic (Garrison et al., 2016; Wheeler et
76 al., 2016).

-- I would say "closer" rather than "sister", because these relationships are unstable, and the Deinopoiea are not monophyletic, according to those studies.

Table 2 needs some explanation in the legend.

-- revise English:
99 & Opell, 2002; Blackledge et al., 2009; Pechmann et al., 2010). It is possible the higher
100 fecundity trends observed in orb-weavers and non-web builders compared to cribellate silk users

-- revise English:
163 taxa, particularly the singular, fiber producing spigot (MS, FL, PF) the on the PLS?

107 ... Most
108 of the Araneomorphae spiders possesses five types of spigots with another two appearing in
...
111 ... These are ...
116 ... 4) aciniform gland spigots (AC) on the PMS and 5)
117 aciniform gland spigots on the PLS that produce silk used in prey wrapping and lining egg sacs,
118 as well as the sheet portions in non-orb webs; and 6) cylindrical (=tubuliform) gland spigots
119 (CY) on the PMS and 7) cylindrical gland spigots on the PLS which are female specific and
120 produce fibers that form the egg sac (Fig.1, and see Fig. 1 in Garb, 2013).
-- items (4-5) and (6-7) sound like of the same type.

130 paracribellar spigots on the PMS. However, in T. perfuga, the modified spigot is flanked by two
131 smaller, unknown spigots whose function is currently undetermined (R.E. Alfaro, unpublished
132 data).
-- you can use Griswold et al. (2005) as a reference for this in T. radiata and many others.

291 N.I. Platnick (1987; Hypochilidae), Kukulcania hibernalis (N.M. Hentz, 1842; Filistadidae),
-- Filistatidae
292 Thaida peculiaris F. Karsch (1880),
-- ... (1880, Austrochilidae),

292 ... Megadictyna thilenii F. Dahl (1906; Nicodamidae)
-- Megadictynidae
293 ... The Nicodamidae are sister
-- Nicodamoidea


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: COMPARATIVE SPIGOT ONTOGENY ACROSS THE SPIDER TREE OF LIFE
Review round: 2
Reviewer: 1

Output:
Basic reporting: The english is excellent, the references complete correct and through, and figures appropriately labelled. I had no complaint with the original submission but this is slightly improved.
Experimental design: The scope and aims are well defined and answered. The methods are well designed and thoroughly documented. Excellent work!
Validity of the findings: Although the results may not be earthshaking to some, to me they are really valuable and open new paths for future investigation. It is truly great work! Congratulations to the authors!
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: PREDICTION OF LIFE STRESS ON ATHLETES’ BURNOUT: THE DUAL ROLE OF PERCEIVED STRESS
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1. The English language is communicable. However, there are many grammar mistakes and typos to be corrected.
2. The references are generally up-to-dated. There are several APA related mistakes to be corrected in your reference list.
3. The articles structure, figs, and tables are in a good shape. Raw data are provided.
4. The corresponding results are presented to address the proposed hypotheses. The proposed hypotheses should be back up with relevant literature.
Experimental design: 1. This is not an intervention/experiment. A cross-sectional survey design was used. The cross-sectional data, as acknowledged by the authors, limit the inferences of their research findings.
2. I am not sure why it is necessary to examine the relationships among life stress, perceived stress and burnout. In particular, life stress and perceived stress are highly correlated or closely related to each other. Without a doubt, perceived stress will mediate the relationship between life stress and burnout. It may be also possible (to test the alternative model) that life stress is a mediator between perceived stress and burnout.
3. Some background information about athletes' levels (classifications) in Taiwan may be provided under the "Participants" section.
4. The overall internal reliability of ABQ can be included.
5. Baron and Kenny (1986)'s approach to examine mediation effects has been criticized (Zhao, X., Lynch Jr, J. G., & Chen, Q. (2010). Reconsidering Baron and Kenny: Myths and truths about mediation analysis. Journal of Consumer Research, 37(2), 197-206.). Please use either bootstrapping or Sobel test to check the mediation effects.
6. How the interaction terms were created in your moderation analyses?
Validity of the findings: 1. If you take my comments about your mediation analyses, please update the findings of mediation analysis accordingly.
2. I am not sure why other demographic items were not controlled (entered as co-variates) in your regression analyses? Have you checked whether they were correlated to your major study variables?
3. Please state if your findings support your hypotheses or not.
Additional comments: 1. Line 77-78: Is it necessary to include these references here?
2. Line 115-118: Include the page number if you used a direct quo here?
3. Line 167-182: Your literature review is weak in developing your hypotheses. Consider citing relevant research to back up your hypotheses.
4. Line 194: You don't need the "plus and minus" symbol here?
5. Line 207: Do you mean "measures"? Measurement refers to the data collection process.
6. Line 285: You need a reference here.
7. Line 292: Replace "Beta" with its symbol.
8. Line 342-383: I would suggest you to cut down this section as they are not directly related to your findings.
9. Line 385-390: It is good to go beyond your findings to make a conclusion. However, it does not read like a conclusion here.
10. Table 1: Demographic items can be included. Put your notes under the table.


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: PREDICTION OF LIFE STRESS ON ATHLETES’ BURNOUT: THE DUAL ROLE OF PERCEIVED STRESS
Review round: 1
Reviewer: 2

Output:
Basic reporting: See my suggestions on the Discussion Section about some detailed comments. Other sections are fine.
Experimental design: Research questions and hypotheses are well presented. Data analysis conducted in a good way.
Validity of the findings: Data is robust and statistically sound.
Additional comments: A well-conducted study and the steps of data analysis are conducted in a nice way. I have some suggestions for authors to consider that may improve their manuscript.

1. Lines 76-80: For the abstract, I would like to suggest the authors make it simple and only mention the aim of the study like “Building on the Smith (1986) cognitive-affective model of athletic burnout, the current study examine the relationship….”

2. Line 85: For the abstract, I would like to suggest not say BUT “but “counter-stress” component of perceived stress moderated”, use the word “while” or “and”.

3. Lines 188-189: Please remove the double quotation marks from “perceived distress” and “counter stress” or use the acronyms like PD and CS to represent the key words if not that many.

4. Line 325, I suggest authors remove the subheading of “Theoretical contributions/implications” from the manuscript. It is obvious in the discussion section.

5. Line 325, in the first paragraph of discussion, the authors mention: “The preliminary results provide several implications as follow…” Then paragraph starts like first second. My suggestion is that (a) in the first paragraph briefly revisit the main findings of the current study and remove the last sentence; (b) Do not say first, further, further. Just start with the topic sentence will be fine.

6. Lines 326-383: Switch the order of applied implications with limitations and future directions.

7. Lines 342-383: In the applied implications, I noticed that the findings of the current study is not been talked that much. However, I suggest the authors could think a little bit more to link each of the applied implication suggestions with the current findings. Just need an angle to cut in.

8. Lines 386-387: I suggest the conclusion can be rephrased, currently it reads like part of the suggestion rather than a conclusion of the whole study. It should be a highly summarized findings of the current study.


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

Input:
Title: PREDICTION OF LIFE STRESS ON ATHLETES’ BURNOUT: THE DUAL ROLE OF PERCEIVED STRESS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Simple English, easy to understand.
In the questionnaire uploaded, suggesting to add English version together with Mandarin language so that other researcher could read the questionnaires.
Experimental design: Sufficient statistical analyses according to the research questions stated. Objectives achieved.
Validity of the findings: The data is valid and statistically sound.
Additional comments: I would like to congratulate the authors for conducting such an exciting study, looking on the other aspect of Smith's model on the perceived stress among college student athletes.

I have attached some edits, and repeat them here:

Abstract:
Reference: Lundquist, & Wagnasson, 2013.
Two separate hierarchical regression analyses found that the….
Line 90 – Delete “On the one hand”, suggesting “The college student-athletes are
ordinary students;…..
Line 93 – Delete “On the other hand”, suggesting use “Besides”
Line 98 – arrange the reference alphabetically.
Line 127 – suggest to use “the forth stage”…
Line 129 – suggest to use “Many studies had adopted Smith (1986) …
Line 160 – suggest it omitted some information…
Line 171 – no researcher attempted to adopts …
Line 184 – building on the above literature,
Line 185 – the two components of
Discussion, paragraph 2. – Further, we found several findings in the mediating
and moderating …. For example, on the mediating effect of…
Please add more information on the finding in the Discussion section.


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

Input:
Title: PREDICTION OF LIFE STRESS ON ATHLETES’ BURNOUT: THE DUAL ROLE OF PERCEIVED STRESS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Line 171: incomplete citation (Barbosa-Leiker et al., 201?)
Experimental design: NIL
Validity of the findings: NIL
Additional comments: NIL


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: PREDICTION OF LIFE STRESS ON ATHLETES’ BURNOUT: THE DUAL ROLE OF PERCEIVED STRESS
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: Thanks for your revision. I have no further comments.


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

Input:
Title: THE RELATIONSHIPS BETWEEN EXERCISE AND AFFECTIVE STATES: A NATURALISTIC, LONGITUDINAL STUDY OF RECREATIONAL RUNNERS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript “The relationships between exercise and affective states: A naturalistic, longitudinal study of recreational runners is clear written, with relevant figures and tables, which are of high quality and well labelled and described. The structure conforms to PeerJ standards except the abstract because headings are missing.
Nevertheless, there are some issues, which should be improved. My most important issue is with the theoretical background in the section introduction and some missing information about statistical analysis.
Introduction:
Experimental design: The introduction needs more detail (references and theoretical assumptions/backgrounds) and I would restructure it in putting some information in the method section. If you add these backgrounds, you could improve your argumentation and strengthen the relevance of your research question.
Validity of the findings: data is robust and conclusion are well stated
Additional comments: In this section, I listed some comments for each section (if necessary.
Introduction:
Line 29: These references are very old, try to find current evidence of the association between exercise and affective states.
Line 53: Your intention is to analyse possible interactions. You mentioned this issue in line 53, for instance. Here, the argumentation would become stronger if you provide theoretical assumptions about these possible interactions (e.g. dual mode theory). Furthermore, you assume that valence and arousal reduce progressively as time passes. You also should add references for this assumption, for instance: Wichers et al. (2011). A time-lagged momentary assessment study on daily life physical activity and affect. Health Psychology
Line 55 (et seq.): You mentioned that the association between exercise and affective states should be assessed in naturalistic studies. There are a lots of studies analysing the association between physical activity and affect in daily life and you should define in more detail your scientific contribution (see for instance the reviews of: Kanning, M., Ebner-Priemer, U., & Schlicht, W. (2013). How to investigate within-subject associations between physical activity and momentary affective states in everyday life: A position statement based on a literature overview. Frontiers in Psychology Movement Science and Sport Psychology; Liao, Y., Shonkoff, E. T., & Dunton, G. (2015). The acute relationships between affect, physical feeling states, and physical activity in daily life: A review of current evidence. Frontiers in Psychology Movement Science and Sport Psychology.
Line 67 (et seq.): In my opinion you should explain in more detail the theoretical background of affective states (e.g. circumplex model, Russel, because you measure valence and energetic arousal)
Line 70 (et seq.): This detailed information about your study belong in the section “Methods”, not in the section “introduction”

Data analysis:
Line 153: You should add information about the within- and between person variance (intraclass coefficient) and how you have centred the variables. To support understanding you could add the equations of the models.

Results:
How many missings do you have in your affect measures. You measured affect four times a day over six weeks (4measures x 42days = 168 measurement points). In average, how many affect measures did you achieve per person?
Line 180 (et seq.): It is correct to abbreviate standard deviation with s.d.?
Discussion:
You should discuss in the limitation section that you assessed physical activity with self-reports. The activity data depends on what the participant enters into the platform. You are not able to control if the entry about time, distance and intensity of the run is correct.


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 RELATIONSHIPS BETWEEN EXERCISE AND AFFECTIVE STATES: A NATURALISTIC, LONGITUDINAL STUDY OF RECREATIONAL RUNNERS
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: Clear specification of inclusion and exclusion criteria (health status, motivations, etc.).
Specify the average age of the female and male samples.
Clear formulation of the main purpose of the research.
Validity of the findings: Specify the limits, strengths  and perspectives of the research.
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: WEEKEND EFFECT IN UPPER GASTROINTESTINAL BLEEDING: A SYSTEMATIC REVIEW AND META-ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Report written using a good standard of English. Concise and clear use of language to convey the intended message effectively. Use of professional help for manuscript editing noted and commendable.
Seven figures and one table included in the article.
Figure 1 shows flowchart of selecting eligible studies.
Figure 2 shows Forest plot of OR for total mortality (30-day and in-hospital mortality) due to UGIB during weekend versus weekday.
Figure 3 shows Forest plot of OR for in-hospital mortality only.
Figure 4 shows Forest plot of OR for in-hospital mortality in a subset of patients with UGIB due to variceal bleed.
Figure 5 shows Forest plot of OR for in-hospital mortality in a subset of patients with UGIB due to non-variceal bleed.
Figure 6 shows Forest plot of mean time to endoscopy during weekday versus weekend.
Figure 7 shows Funnel plot assessing bias in study.
Table 1 summarises the pertinent points from the selected studies.
All included figures and tables relevant to the reporting of the study and included appropriately.
Of note, there is a mention of another table labelled S2 which is supposed to list the search strategy, which has been omitted from the reviewers copy of the article.
Experimental design: Study was well designed and follows the expected standards for systematic review and meta-analyses.
Study aims to assess the effect of weekend admissions on UGIB and the resulting outcome of mortality, which is a poorly understood phenomenon and is not widely reported.
Study question framed clearly and subsequent study reporting clearly focuses on addressing the study question.

Methodology reported well, in a clearly reproducible manner. However, ‘Table S2’ which supposedly lists the search strategy was not included in the manuscript for reviewer.

The standard steps for performing systematic review was followed e.g. searching and screening by 2 different reviewers separately and the involvement of a third reviewer to resolve any disagreement.
The major databases were comprehensively searched for without any language restrictions. Articles published in abstract only form or in non-peer review journals were excluded. There was also no mention of searches performed in the ‘grey literature’. However, although limiting the search to only peer reviewed journals from the major databases may restrict the number of studies that can be included, it may partly ensure that only higher quality studies with complete data are included.
Validity of the findings: Data reported in a clear and relevant manner with selected Forest plots of interest included.
Study findings were discussed in a fair manner, considering the effect of heterogeneity where relevant.
 The conclusion is well stated and addresses the study question.
Given the small number of studies meeting the inclusion criteria for this systematic review and the specific issues highlighted by the authors e.g. use of non-standardized definition for weekend, use of different primary end-points and reporting of time to endoscopy only in some studies; this study raises more questions than answers.
More robust well-designed prospective studies need to conducted in multiple centres worldwide to examine the weekend effect on UGIB admission to provide more conclusive findings.
Overall the effort of the authors in conducting and reporting this study is commendable.
Additional comments: Sound methodology and clear reporting of results.
Good discussion with explanations for observed results backed by current evidence.
Meets the required standard for publishing.


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

Input:
Title: WEEKEND EFFECT IN UPPER GASTROINTESTINAL BLEEDING: A SYSTEMATIC REVIEW AND META-ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Comment 1.	Please check grammatical error. For example, line 207 ” admission showed significantly shorted times”
Experimental design: no comment
Validity of the findings: Comment 2.	There were only seven studies shown in figure 5 for the increased in-hospital mortality due to non-variceal bleeding in weekend. And with eight studies recruited in figure 6 showing time to endoscopy during weekday is shorter. Yet, only two studies overlapped in these two figures (Wu, 2014 and Youn, 2012). So, whether longer time to endoscopy (figure 6) may explain the increased in-hospital mortality due to non-variceal bleeding (figure 5) may be a question. Because the initial settings of patients were different. Ideally, It would be better if all the recruited studies are the same in figure 5 and figure 6.
Additional comments: Comment 3.	Line 227 to line 228, I don’t think the different definition of weekend from recruited studies will affect the result. As long as there is a mention that lacking of manpower during the day which was defined as weekend. Are there any explanation of the definition of their weekend (for instance, less manpower in the subspecialty during the defined weekend) in the quoted articles?


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: WEEKEND EFFECT IN UPPER GASTROINTESTINAL BLEEDING: A SYSTEMATIC REVIEW AND META-ANALYSIS
Review round: 1
Reviewer: 3

Output:
Basic reporting: I would like to applaud the author for registering their protocol PROSPERO and following the PRISMA guideline. Many systematic reviews suffer from poor reporting and inadequate search methodologies. I was pleasantly surprised to see this systematic review was well reported and there was no major concern regarding the search strategies.

a few minor suggestions:
1) When reporting the databases, it is important to report the interface and the coverage for each database [line 25 and 26]. Searching the same database via different interfaces can lead to different results. For example, EMBSE is available via OVID or embase.com. CINHAL is available via EBSCO, OVID, and ProQuest.

2) It is recommended to report the number of articles you retrieved per line of search query.

3) It is also a best practice to report any software you use for screening and/or citation management
Experimental design: 1) In PubMed search, Line#6 and Line #17, there are typos: admission tim to admission time. I hope these were mistakes in reporting and not the actual search strategy.

2) In PubMed search, remove extra line (line #16) from the search. Otherwise, it might be misleading or confusing to the readers.

A general comment: Databases are very structured and are meant to be searched by controlled vocabularies or index term.  It might be important to use key mesh terms in your search. For example, Time-to-treatment or Time Factors could be very good mesh terms to add to your search. To increase the search sensitivity.
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: ISOLATION, IDENTIFICATION, AND PATHOLOGICAL EFFECTS OF BEACH SAND BACTERIAL EXTRACT ON HUMAN SKIN KERATINOCYTES IN VITRO
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript provides sufficient information in introduction section. However, there are few suggestions for the improvement of the manuscript.

I suggest the author to ask a native English speaker to revise the manuscript for spelling and grammar mistakes. The author needs to carefully recheck the manuscript, and the missing references should be added. Additionally, author should add the latest references.

Regarding title of the manuscript, I recommend the author to change it. Title should clearly represent the inside message.
Experimental design: The study is looking original with well-defined research questions. It fills a knowledge gap of isolation, identification, and health hazards of beach bacteria. It comprehensively and rigorously describes the pathological effects and underlying mechanisms of this strain.
However, the manuscript lacks details in many methods used, which seems difficult to be repeated by other reserachers.
How did the author control fungal growth?  Because fungal spores can easily grow on growth medium.
It is not clear whether each experiment was independently repeated three times, or the experiments were conducted once using three test samples.Please clarify it.
Validity of the findings: Findings in the present manuscript are looking good. Figures should be saved in high resolution format, and figure legends should be improved by adding sufficient information.
Additional comments: Results section should be written scientifically including the aim and outcomes of the experiments. If possible, add some references in results section too.


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: ISOLATION, IDENTIFICATION, AND PATHOLOGICAL EFFECTS OF BEACH SAND BACTERIAL EXTRACT ON HUMAN SKIN KERATINOCYTES IN VITRO
Review round: 1
Reviewer: 2

Output:
Basic reporting: The aims and approach presented in the manuscript, entitled “Isolation, Identification, and Pathological Effects of Beach Sand Bacteria on Human Skin Keratinocytes” represent a relevant perspective for human skin infection on the beaches. The manuscript overall well written.
Experimental design: Overall the experimental design is good enough
Validity of the findings: Majority of the findings are novel and there is sufficient novelty in the current manuscript.
Additional comments: However, the manuscript has some important revision that need to be addressed.
I suggest minor revision
1.	(Line 56-60) The following sentence should be changed with suitable one. “For example, Heaney and colleagues have reported that, following visits to both freshwater and marine beaches in different parts of the USA in 2003-2005, the incidence of diarrhea, vomiting, and nausea increased in visitors.
2.	Figure 5.  If possible mentioned the IC50 valve in the results.
3.	(Line 61) The skin is the largest organ in body and provides the primary barrier against bacterial infections. Change to …..primary barrier against various infectious agents.
4.	(Line 98-100) Change, the soil samples… to sand samples were collected from the seaside area.
5.	(Line 103) Kang et al., 2015 change to italic Kang et al., 2015
6.	(Line 131) 100 mg/ mL streptomycin change to 100 mg/mL streptomycin
7.	(Line 206-208) An understanding of the pathological microbiome is necessary to understand microbes pathological to humans, and to enable the development of successful therapeutic approaches for their cure. Change this sentence to “An understanding of the human pathological microbiome is necessary to enable the development of successful therapeutic approaches for their cure”.
8.	(Line 235) Change the “P. stutzeri” to’ FSRS”
9.	Figures should be saved in high quality.
10.	The legends should be improved for better understanding.


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: ISOLATION, IDENTIFICATION, AND PATHOLOGICAL EFFECTS OF BEACH SAND BACTERIAL EXTRACT ON HUMAN SKIN KERATINOCYTES IN VITRO
Review round: 1
Reviewer: 3

Output:
Basic reporting: The English of the manuscript is enough clear throughout the text.
Experimental design: The research is with in the scope of the journal and method adopted for investigation is technical and scientific.
Validity of the findings: conclusion is supporting the results and hypothesis of the manuscript.
Additional comments: This is an interesting work; the authors warn the people visiting beaches for luxury purposes to be harmful. Although there are interesting data presented in the manuscript, the article would need minor revisions before it could be accepted for publication.  The authors may need to address the following suggestions for improving the quality of the paper

1.	The authors did not mention the lethal dose or the IC50 concentration of the extract tested in cell proliferation data
2.	(Line 57) remove “following” and visit to “visitors”
3.	(Line 58) change “There have also been” to “There are several other similar reports”
4.	Figure 2-1: cluster should be defined
5.	Figure 4: IC50 value should be mentioned in the result if possible percentage of cell proliferation”
6.	(Line 244) remove “as” after could
7.	Please make sure that all the references are in the format of the journal.


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: ISOLATION, IDENTIFICATION, AND PATHOLOGICAL EFFECTS OF BEACH SAND BACTERIAL EXTRACT ON HUMAN SKIN KERATINOCYTES IN VITRO
Review round: 2
Reviewer: 1

Output:
Basic reporting: Well explained; having brief background.
Experimental design: Novel idea, with good outcomes.
Validity of the findings: Authentic.
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: ISOLATION, IDENTIFICATION, AND PATHOLOGICAL EFFECTS OF BEACH SAND BACTERIAL EXTRACT ON HUMAN SKIN KERATINOCYTES IN VITRO
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript was revised appropriately and I would recommend for publication in PeerJ.
Experimental design: The Experimental part was designed properly and written in a proper way.
Validity of the findings: The manuscript was revised appropriately and I would recommend for publication in PeerJ.
Additional comments: The manuscript was revised appropriately and I would recommend for 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: EFFECTS OF THE VISUAL-FEEDBACK-BASED FORCE PLATFORM TRAINING WITH FUNCTIONAL ELECTRIC STIMULATION ON THE BALANCE AND PREVENTION OF FALLS IN OLDER ADULTS: A RANDOMIZED CONTROLLED TRIAL
Review round: 1
Reviewer: 1

Output:
Basic reporting: While the manuscript adheres to a professional structure, there needs to be more precision with professional English, most notably in the introduction and discussion.
For example – linking words related to a fall or falls: line 35 (insert A before fall), line 41 (change to balance deficits are one of the known risk factors for falls), line 45 (insert to before stand), line 46-48 (needs re-wording), line 229 (reached instead of reach).

There is sufficient background within the introduction but further context could be included in the discussion regarding electrical stimulation.
Experimental design: Methods explained in appropriate detail allowing for replication.
It should be made clearer that the control group only undertook single leg balance training and did not receive further training or electrical stimulation.
Pictures of standing position/visual feedback provided may help to illustrate intervention protocols.
Effect sizes should also be included in table 2.

Further information on falls and associated injuries would be also be valuable.
Validity of the findings: Overall the data is robust and controlled.

Were improvements in balance and reductions in falls due to the balance training or the electrical stimulation? Or a combination?I think the authors might be overstating the potential impact of electrical stimulation without strong links/justification. As improvements in strength were not measured it is difficult to say how much of an impact the electrical stimulation training had. It is more likely that the visual feedback balance training (together with the single leg balance training) provided most of the improvements in balance and associated reduction in falls. Whilst there are links to improvements in strength and possibly function with electrical stimulation, these associations have not been strongly argued.The authors could expand on the reasons for this improvement (associated with balance training and visual feedback) in more detail as it is a very interesting and important finding.

In the conclusion – few adverse events (line 277). Does this mean there were some adverse events during training/testing – please clarify.
Additional comments: I commend the authors on conducting this well powered balance intervention in older adults. The force platform based visual feedback is certainly an interesting area that has great potential for improving balance and potentially reducing falls in older adults.


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

Input:
Title: EFFECTS OF THE VISUAL-FEEDBACK-BASED FORCE PLATFORM TRAINING WITH FUNCTIONAL ELECTRIC STIMULATION ON THE BALANCE AND PREVENTION OF FALLS IN OLDER ADULTS: A RANDOMIZED CONTROLLED TRIAL
Review round: 1
Reviewer: 2

Output:
Basic reporting: •	Manuscript was sent to me UNBLINDED.
•	Raw data was provided.
•	Manuscript was very clearly written and easy to follow
Experimental design: •	Research question was well defined, relevant and meaningful.  The results fill the gap of how platform and electrical stimulus training can improved measures of balance and reduce falls.
•	Methods described in detail
•	Reported with rigorous RCT methods
Validity of the findings: •	This research seems to be valid for bridging the gap between platform and electrical stimulus training.  On a side note, it would be interesting to see how effective each individual intervention (platform, stimulation & single leg balance) was.
•	Data is robust, statistically sound and controlled.
•	Conclusions were well stated
Additional comments: •	Overall the paper is very well written, easy to follow and methodologically sound.
•	I would speculate that perhaps a Repeated Measures ANOVA may be a more appropriate statistical method to use, but the description of the two-way factorial ANOVA sounds sufficient.


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

Input:
Title: EFFECTS OF THE VISUAL-FEEDBACK-BASED FORCE PLATFORM TRAINING WITH FUNCTIONAL ELECTRIC STIMULATION ON THE BALANCE AND PREVENTION OF FALLS IN OLDER ADULTS: A RANDOMIZED CONTROLLED TRIAL
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: The authors have clarified their findings in the results and discussion. This clarification was aided by the edits made to the methods.
Additional comments: Thank you for making the suggested changes to the manuscript. The paper now flows better and the discussion and conclusions are now better supported.
Congratulations on completing such an interesting and well powered study.


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

Input:
Title: A DIAGNOSTIC MODEL FOR MINIMAL CHANGE DISEASE BASED ON BIOLOGICAL PARAMETERS
Review round: 1
Reviewer: 1

Output:
Basic reporting: While manuscript is written in easy-to-read English language, it can be made more structured and of professional quality by substantially improving syntax errors, e.g....

*   Excessive use of abbreviations. Group them when applicable in abstract and under results section, e.g., type of proteins measured, immunoglobulins, etc. and suggest brief explanation on their relevance in MCD.
*   Provide specific examples instead of using keywords, e.g., "certain risk" (line 34), "widely used" or "mathematical model" (line 35), etc.
Experimental design: 1. My first major issue is with rationale (line 56-66) for development of mathematical model which is to avert invasive nature of the renal biopsy in patients with NS. Definitive diagnosis of MCD requires renal biopsy but it warrants electron microscopic examination as renal parenchyma looks almost normal under light microscopy. However, patients, mostly children with NS secondary to MCD have very typical clinical presentation with marked dependent edema, weight gain with growth retardation along with marked albumin secretion in urine and decrease in serum albumin. Unlike other causes of NS, MCD disease have usually normal renal function and lacks autoimmune dysfunction. Hence, present clinical guidelines rarely requires renal biopsy for diagnostic purpose and biopsy is usually reserved for rare cases (1-5%) where patient do not respond to steroids (majority does) or MCD is seen in adults (rarely seen). Since authors aim to provide their method for diagnostic purpose, it would help to have strong rationale to promote its clinical application. Such non-invasive approach, if accurate enough can be of much value for smaller fraction (< 1-5%) of cases where diagnosis is challenging, e.g., adult cases with hypertension, autoimmune disorders, etc. However, for larger cohort of patients of MCD are children (80-90%) and I have concerns and seek authors view on how their method provide additional value to existing clinical guidelines.

2. Another important issue is with methodology for developing risk prediction model. While t-test and logistic regression based model should work in MCD given a typical (a few features with less variance) patient presentation as described earlier, most biochemical parameters that Zhu et al. used (table 1) are part of standard or extended panel of investigations carried out by a physician for a patient presenting with NS. In that case, it would be useful to see how authors' model perform with and without including baseline biochemical indicators, e.g., total protein, albumin, creatinine, and lipid profile. Also, unlike reported ROC statistics in training set of 798 cases, it is equally important to report ROC statistics in cross-validation set (split from training set of 798 cases) if not the gold-standard, test or held-out set which is independent from the training set.

3. Under ROC statistics results, it is useful to provide ROC statistics for combined model using all 13 parameters and perform stepwise forward or backward selection in estimating coefficients of each of 13 parameters to predict which of parameters are of high vs low predictive value.

References:

1. Kumar, Abbas, Aster. Robbins Pathologic Basis of Disease. 9th edition (2015)
2. Mansur & Batuman. Minimal-Change Disease. 6/30/2017 http://emedicine.medscape.com/article/243348-overview
Validity of the findings: 1. With biomarker based prediction models, it is reasonable to not expect high level of accuracy and hence, authors reporting of sensitivity and specificity in range of 70-80% seems fair. However, I suggest authors can improve discussion section by adding a note on prioritizing sensitivity or specificity depending of trade-off between diagnosis false positive cases versus omitting true positive cases, respectively.

2. In discussion section, authors show total cholesterol and thrombin time as risk factors with highest predicted probability. Although these are known risk factors and often used in secondary panel of investigation to evaluate patients with NS, it would be useful to know if predicted probability of these markers remains high also among cases (from their training set) where diagnosis of MCD was challenging. If so, this can be an important finding to let physician weigh more on values of these two indices over others for the challenging case.
Additional comments: Zhu et al. have presented a logistic regression based diagnostic approach for minimal change disease (MCD), a common pathological feature of nephrotic syndrome (NS) in children. Authors have provided detailed supplemental data and description on their rationale, statistical methods and visualization of prediction results using standard ROC plots. After reading the manuscript in totality, I find following several issues (under three sections) which I suggest authors to address before it is being considered for second review.


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 DIAGNOSTIC MODEL FOR MINIMAL CHANGE DISEASE BASED ON BIOLOGICAL PARAMETERS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is clearly presented
Experimental design: the article is mathematical model for kidney disease in particular Minimal Change Disease. It is retrospective in nature. the data set is appropriate
Validity of the findings: The validity of the findings may not transcend to other ethnic groups
The authors note that HTN and diabetes were elevated in the MCD group. this does not appear to be the case in the table.
It the best of my reading the ROC was not applied to a subset of individuals not included in the original analysis
It is not clear if individuals entering the study were on other treatment for example therapy for HTN or hyperlipidemia
Additional comments: I believe that the study is very interesting but would find it hard to generalize this to other population subsets


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 DIAGNOSTIC MODEL FOR MINIMAL CHANGE DISEASE BASED ON BIOLOGICAL PARAMETERS
Review round: 2
Reviewer: 1

Output:
Basic reporting: None
Experimental design: None
Validity of the findings: None
Additional comments: I thank authors for having detailed look on their manuscript and addressing most of my comments. With respect to experimental design, given case population is all adults and issues with availability of renal biopsy setup in hospitals, it is reasonable to have computational based predictions to provide some level of support in addition to biochemical indices in cases with diagnostic dilemma. Accordingly, prediction model by authors may complement current diagnostic approach for MCD in adults. As prediction models are inherently prone for overfitting and resulting inaccuracy, I wish authors to follow up with at least cross-validated model if not the model performing better (arbitrary value of AUC ~>0.75 or more) based on actual validation set. Such statistical rigor on top of their approach to include biochemical indices in their existing prediction model should yield reliable non-invasive approach for adult patients with MCD.


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 DIAGNOSTIC MODEL FOR MINIMAL CHANGE DISEASE BASED ON BIOLOGICAL PARAMETERS
Review round: 2
Reviewer: 2

Output:
Basic reporting: appropriate
Experimental design: the authors have addressed concerns
Validity of the findings: the authors have addressed concerns
Additional comments: the authors have addressed concerns


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

Input:
Title: RISK FACTORS FOR OSTEOPOROSIS IN MALE PATIENTS WITH CHRONIC OBSTRUCTIVE PULMONARY DISEASE IN TAIWAN
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: 1) In Table 1, basic characteristics of controls should also be presented for comparison with COPD patients. In order to conclude that osteoporosis is more prevalent in COPD patients, data should be adjusted for various confounders including smoking status, renal function, the presence or absence of diabetes, and so on. It is also important for the authors to exclude COPD in control subjects by spirometric test.
2) According to the authors, controls included family members of COPD patients. But exactly how did the authors recruit these subjects? And how many were family members of COPD patients?
Validity of the findings: 3) In Table 2, mean values of CRP and proportion of subjects with CRP higher than 5.0 should be shown in each group.
4) In Table 2, eGFR should be included. If the authors considered creatinine as an indicator of muscle mass, how could they be sure that renal function was the same?
5) And in Table 5, important factors missing here, such as systemic steroid use, current smoking, respiratory function and so on, should also be included. Whether or not CRP contribution to osteoporosis was dependent on respiratory dysfunction is particularly important, because higher CRP is apparently associated with impaired respiratory function in Table 4.
6) In Table 4 smoking status in high CRP group (1/8, 1/8, 1/8?) seems wrong and should be corrected. And correct information should be used to analyze determinants of osteoporosis (See comment 4)).
7) Serum levels of 25-hydroxyvitamin D should be measured. Vitamin D deficiency is a well-known risk factor of COPD-associated osteoporosis.
Additional comments: COPD-associated osteoporosis is common, but its precise prevalence as well as its mechanism remains largely unknown. Thus, the subject is important. The manuscript is generally well written. However, some critical information is lacking, and statistical analysis seems incomplete. Comparison between control and COPD subjects is far from conclusive because of the small sample size and the way of subject recruitment.


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

Input:
Title: RISK FACTORS FOR OSTEOPOROSIS IN MALE PATIENTS WITH CHRONIC OBSTRUCTIVE PULMONARY DISEASE IN TAIWAN
Review round: 1
Reviewer: 2

Output:
Basic reporting: Clear, unambiguous, professional English language used throughout - yes

Discussion - Findings very well discussed
Specific comments
Lines
183-188
“However, the prevalence of osteoporosis in our study was lower than the 40% reported in a recent study also conducted in Chiayi, Taiwan (Lin 185 et al., 2015). This may be explained by differences in subject enrollment and osteoporosis. That study enrolled male and female COPD patients and defined osteoporosis as a BMD T-score <−2.5 or the presence of thoracolumbar vertebral compression fracture on radiography.” → Very good to put the difference in prevalence in perspective.
Financial support and sponsorship, conflicts of interest and acknowledgments indicated.

Intro & background to show context. Literature well referenced & relevant
General comments
Reasons for study well argued; referencing correct and up-to-date
Specific comments
Lines
50-52
“It affects >5% of the population (Centers for Disease Control and Prevention, 2012) and is predicted to become the third leading cause of death in the world by 2020 (Murray & Lopez, 1997).” → Which population, world-wide?
72-74
“Therefore, this study primarily aimed to investigate risk factors for osteoporosis, particularly whether increased CRP is a predictor for osteoporosis development in male Taiwanese COPD patients.” → Why did you specifically choose “high-sensitivity CRP”? Better predicator of osteoporosis than IL-6?

Structure conforms to PeerJ standards, discipline norm → yes

Figures and relevant, high quality, well labeled & described
Specific comments
Table 2
a, b, and c (indicating the different statistical tests) are confusing, one expects that they would indicate significance → Suggest that the description (or indication) of what test was used for which analysis being put in the “Statistical analysis” section – lines 133-135, and significance being highlighted in the table.
Table 3
See comment on Table 2
Table 4
See comment on Table 2
Are these still the COPD patients?

Raw data supplied, but as I don’t use SPSS, I can’t read the file. Can you please provide the data in an Excel file?
Experimental design: Original primary research within Scope of the journal → yes

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

Rigorous investigation performed to a high technical & ethical standard
General comments
•	In- and exclusion criteria provided
•	Written informed consent
o	Possible to see an English version of the consent form?
•	IRB# provided
Specific comments
Lines
79
“It enrolled male COPD patients aged ≥45 years from……”
AND
83-85
“Subjects who were willing to undergo anthropometric measurements ….” → How and by whom were the COPD patients recruited?
93-107
Description of the “Questionnaire interview and medical records review” → Would recommend putting the information in a table – would make reading it much easier.
120-121
“Lung examination: Chest radiography and spirometry data were interpreted by a chest physician.” → Not possible to get more than one chest physician to interpret the CxR? Was he/she a radiologist?

Methods described with sufficient detail & information to replicate → yes
Validity of the findings: Validity of findings
Rationale & benefit to literature is clearly stated

Data is robust, statistically sound, & controlled
Data analysis – correct
Results – well described
Specific comments
Lines
161-163
“The high hs-CRP group tended to have lower BMI, although there were no significant differences in age, smoking habits, and history of acute exacerbation (Table 4).” → Suggest moving this up to the previous paragraph – keep the hs-CRP information together.
159-160
“CRP showed strong associations with COPD severity and pulmonary function parameters (including COPD GOLD stage, FEV1, FEV1% predicted, FVC, FVC% predicted, FEV1/FVC, and FEF 25–75%) and steroid use.” → Can you differentiate the significance according to the different GOLD stages?
Additional comments: Conclusions are well stated, linked to original research question & limited to supporting results → yes

General comments → Very well written manuscript


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

Input:
Title: RISK FACTORS FOR OSTEOPOROSIS IN MALE PATIENTS WITH CHRONIC OBSTRUCTIVE PULMONARY DISEASE IN TAIWAN
Review round: 2
Reviewer: 1

Output:
Basic reporting: good
Experimental design: no comment
Validity of the findings: Conclusions are now well stated and supported by the results.
Additional comments: Revision has been well done.


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

Input:
Title: RISK FACTORS FOR OSTEOPOROSIS IN MALE PATIENTS WITH CHRONIC OBSTRUCTIVE PULMONARY DISEASE IN TAIWAN
Review round: 2
Reviewer: 2

Output:
Basic reporting: Lines 50-52
“It affects >5% of the population (Centers for Disease Control and Prevention, 2012) and is predicted to become the third leading cause of death in the world by 2020 (Murray & Lopez, 1997).” → Which population, world-wide?
Reply:
Much thanks for the reviewer’s carefully reading. The population meant the people in the United States. It seemed not suitable to put this statement here since it was not world representative. We have deleted it in the text and in the reference. (Page 4 # line 51-2; Page 20 # line 343-4)
Comment from reviewer: Addressed√

Lines 72-74
“Therefore, this study primarily aimed to investigate risk factors for osteoporosis, particularly whether increased CRP is a predictor for osteoporosis development in male Taiwanese COPD patients.” → Why did you specifically choose “high-sensitivity CRP”? Better predicator of osteoporosis than IL-6?
Reply:
We have chosen CRP because it was a common, cheap and quite available test in our hospital. To clarify this, we have addressed it in Page 5 # line 75-6.
Comment from reviewer: I don’t see the clarification.
Experimental design: Raw data supplied, but as I don’t use SPSS, I can’t read the file. Can you please provide the data in an Excel file?
Reply:
OK, we will upload the raw data in Excel file.
Comment from reviewer: Addressed√.

The study enrolled Taiwanese people, therefore, there was no English version of informed consent. We have translated the inform consent form into English for the reviewer.
Comment from reviewer: Addressed√.

Lines 79
“It enrolled male COPD patients aged ≥45 years from……”
AND
Lines 83-85
“Subjects who were willing to undergo anthropometric measurements ….” → How and by whom were the COPD patients recruited?
Reply:
I ask my several colleagues, who are also chest physicians to help us to enroll the out-patient COPD patients in chest clinic in Chiayi Chang Gung Memorial Hospital. If the patient had the interests to know more about the project, the chest physicians including me, would call the research assistant to help to explain the project more completely. If the patients agreed to participate the project, they would be asked to read and sign the inform consents.
Comment from reviewer: Addressed√.

Lines 120-121
“Lung examination: Chest radiography and spirometry data were interpreted by a chest physician.” → Not possible to get more than one chest physician to interpret the CxR? Was he/she a radiologist?
Reply:
Chest radiography and spirometry data were interpreted only by me. I am a chest physician but not a radiologist. However, in Taiwan, interpretation of chest X-ray is essential for becoming a qualified chest physician.
Comment from reviewer: Addressed√.
Validity of the findings: Tables
Table 1	Looking good
Table 2	Control (36)	COPD (59)
	L-spine BMD (35)	L-spine BMD (58)
		Hip BMD (57)
		Chronic hepatitis (58)
	If you weren’t able to measure these, mention that at the bottom of the table
Table 3	Variables: “B”?
Table 4	hs-CRP – put in either the mean ± SD or the %, it doesn’t read well with both in
Table 5	Looking good
Table 6	Looking good
Table 7	Variables: “B”?

Specific comments
Table 2
a, b, and c (indicating the different statistical tests) are confusing, one expects that they would indicate significance → Suggest that the description (or indication) of what test was used for which analysis being put in the “Statistical analysis” section – lines 133-135, and significance being highlighted in the table.
Reply:
We have highlighted all significant value in bold in all tables according to the reviewer’s suggestion.
We have explained the rules of statistical analysis in the “Data Analysis” section (Page 9 # line 135-42).. But we think the analysis process is complicated. It is very difficult to descript it without such labeling in each table. Therefore, we still kept them in each table and we think the reader would not be confused after highlighting each significant value in “bold”.
Table 3
See comment on Table 2
Reply:
The same reply as in Table 2.
Table 4
See comment on Table 2
Are these still the COPD patients?
Reply:
The same reply as in Table 2. Table 4 was the comparison of “COPD patients” with low or high CRP. To avoid be confused by the reader, we have added the word “COPD” in the title of the table. (see Table 5, changed from Table 4)
Comment from reviewer: Addressed√.

Lines 159-160
“CRP showed strong associations with COPD severity and pulmonary function parameters (including COPD GOLD stage, FEV1, FEV1% predicted, FVC, FVC% predicted, FEV1/FVC, and FEF 25–75%) and steroid use.” → Can you differentiate the significance according to the different GOLD stages?
Reply:
Because the case number in this study is small. Further analysis with cases divided into GOLD stage I-IV to compare the pulmonary function parameters difference between low and high hs-CRP revealed loss of most of the significance possibly due to insufficient statistical power. However, the tendency towards lower pulmonary function parameters associated with increased hs-CRP seemed still existed. The results were as following (Tables provided).
Comment from reviewer: Addressed√.
Additional comments: Lines 161-163
“The high hs-CRP group tended to have lower BMI, although there were no significant differences in age, smoking habits, and history of acute exacerbation (Table 4).” → Suggest moving this up to the previous paragraph – keep the hs-CRP information together.
Reply:
We have moved it up to the previous paragraph according to the reviewer’s suggestions. (Page 11 # line 169-73)
Comment from reviewer: Addressed√.


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

Input:
Title: FASTVIROMEEXPLORER: A PIPELINE FOR VIRUS AND PHAGE IDENTIFICATION AND ABUNDANCE PROFILING IN METAGENOMICS DATA
Review round: 1
Reviewer: 1

Output:
Basic reporting: I found the language in the manuscript to be sometimes a bit inaccurate and/or awkward, and I believe this should be improved so that the whole manuscript is entirely clear even for non-specialist readers (see examples below)
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In this manuscript, the authors present a new tool (named FastViromeExplorer) destined to help detect and quantify known viruses in metagenome dataset. This tool is using a relatively recent approach (kallisto) to map metagenomic reads to a viral database, and then add a set of cutoffs to identify false positives (usually viral genomes which recruit reads only on a portion of the genome due to a repeat / shared region). The whole pipeline is provided as a stand-alone tool.
The main caveat of this tool is that it relies on databases so can’t provide any virus discovery. Given the state of the viral databases, this limitation should be made very clear as early as in the abstract (note that this can be presented as a trade-off with speed though, since any pipeline involving de novo assembly and identification of novel viral genomes would likely be much more computationally demanding and longer). As of now, the only mention of this bias is in the Conclusion section l. 354 “, the limitation of FastViromeExplorer is that it cannot identify a virus or phage if a similar sequence is not present in the reference database.”, which I consider is not enough for a non-specialist reader to understand how big/small of a problem this reliance on databases can be for different use case scenarios. The authors could also test / illustrate this database bias by generating simulated metagenomes including new viral genomes published since they created their database, and observe the maximum “distance” at which FastViromeExplorer is able to identify these viruses (i.e. how different from the database genomes a new virus genome can be), although I don’t see this additional benchmark as absolutely essential. More generally however, I would ask the authors to provide a much more detail description of which type of study would benefit from FastViromeExplorer (e.g. known pathogen detection) and which should use the more “standard” pipeline relying on de novo assembly (e.g. viral diversity exploration).
My other main concern is with the expected coverage computed as part of the mapping cutoffs, and calculated using a Poisson law. This doesn’t take into account biases linked to the PCR amplification of sequencing libraries, usually required for all/most “low input” cases, i.e. ng of input material, which is still relatively frequent for example in clinical setups (see e.g. PMID 23663384). In these PCR-amplified libraries, the coverage of a genome is not uniform (and less so when the number of PCR cycles increases), which means that the expected coverage cutoff used could wrongly exclude a number of “true” viral detections. Although I suspect there is no magic trick to solve this, I believe the authors should discuss these cases, and provide guidelines for how to use FastViromeExplorer when the user would not expect a uniform coverage.
Finally, I found the language in the manuscript to be sometimes a bit inaccurate and/or awkward, and I believe this should be improved so that the whole manuscript is entirely clear even for non-specialist readers (see examples below).

Additional comments:

l.23: “In a healthy human body there are estimated to be” feels a bit awkward to me, please rephrase.

l. 25-26: “diabetes […], and depression […] and cancer” Please remove the first “and” or reword the sentence.

l. 31: “composition and behavior of environmental microbiomes”. I am not sure a microbiome can have a “behavior”, maybe “metabolism” or “host interaction” could be more appropriate ?

l. 41: “annotating the taxonomy” should be “annotating genes for taxonomy” or “taxonomically affiliating”, but I don’t believe it is correct to say one “annotate” a “taxonomy”.

l. 56-58: “However, read assembly can be […] annotation.” Although I agree with the authors about the current challenges of assembling metagenomes, it would be great it they could add one or two references to back this claim.

l. 95: “30 million paired-end RNA-seq reads for the human transcriptome” shouldn’t it be “from a” human transcriptome “sample” rather than “for the human transcriptome” ?

l. 207: “through analyzing metagenomics data” should be “through the analysis of metagenomic data”.

l. 254: “is only one time process” should probably be modified to something like “is computed only once”.

l. 270: “As viruses are known to have fast mutation rates, Blastn and its variants (e.g., Blastp) are considered the “gold standard” approach to annotate viral sequences in metagenomic data but very time-consuming, having similar performance yet running much faster is highly desired for an annotation tool.” I disagree with this statement, and am not convinced it needs to be made at this point in the manuscript. First, the authors are comparing read mapping method, and correctly using blastn as an alternative to their tool, which has no real link to the high mutation rate observed in some viruses. On the other hand, blastp is used to annotate novel viruses (i.e. de novo assembly of viral genomes, usually only distantly related to viruses in the databases, and for which the only level at which sequence similarity can sometimes be observed is on the amino acid sequences). This is an entirely other approach, hence I believe referencing blastp in the context of this manuscript is misleading. Overall, I would suggest the authors remove this sentence entirely.

l. 285-287: The number of viruses identified seems very low, and I believe it would be helpful if the authors could add one sentence with the expected number of viruses in these samples, so that a reader can appreciate how well a database-relying approach using NCBI RefSeq worked in this case.

l. 310: Should provide information about the number of viral contigs identified with the larger DB (as it can be seen as a proxy for the number of viruses detected).

l. 311: “Therefore, the host information of the mVCs can be used to examine the annotation result.” I believe the authors should specify here how many sequences are associated with a host in this larger database and how these were derived. As it stands now, a reader could think that the two databases (RefSeq and IMG/VR) are equivalent in terms of host information, which would be misleading.

l. 317: “or or” should be “or”

l. 339: “Blastn” should be “blastn”

l. 350: “can annotate both” I disagree with the use of “annotate”, FastViromeExplorer does not provide any annotation, but instead can detect / quantify known viruses. This comment apply throughout the manuscript, hence variations of “annotate / annotation” should be modified elsewhere too when relevant (e.g. 352).


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: FASTVIROMEEXPLORER: A PIPELINE FOR VIRUS AND PHAGE IDENTIFICATION AND ABUNDANCE PROFILING IN METAGENOMICS DATA
Review round: 1
Reviewer: 2

Output:
Basic reporting: I am happy with the writing style.
Experimental design: Good. I am happy with most of it other that the fact that this should have been tested with the full genbank viral database and not just the RefSeq as a database.
Validity of the findings: This method will be useful for people looking at known viruses in metagenomic datasets
Additional comments: The MS by Tithi et al describes a new rapid approach to identifying known viruses from metagenomics datasets. The approach relies on a reference viral database that is well curated e.g. GenBank or even the JGI viral metagenomics dataset and kallisto for rapid mapping of the reads. The output from the pipeline developed for FastViromeExplore provides a list of viruses that is sorted based on abundance. The latter is calculated based on number of reads.

I think this is a great tool for people who want to look for ‘known’ viruses in their dataset. However, I do see some major issues with the use of the word virome in the context of this software.
1)	The tool described here is not a virome explore but a virus explore. The pipeline is heavily reliant on a nucleotide database. Over and over various studies in the last 10 years have shown that we have barely scratched the surface of viral nucleotide sequence space. Hence most of the virus discovery relives on protein sequence space.
2)	The abstract “Identifying viruses and phages in metagenomic data has important implications in improving human health” is a bit odd. First of all phages are viruses and second identifying known viruses is not necessarily going to improve human health – it will trigger a response for clinicians and epidemiologists. Also with the approach you’ll only identify known viruses that may be emerging.
3)	I am not sure refseq on it’s own is a good enough set of sequence for testing this tool. I would have tested in on the whole GenBank viral DB which includes variant. Roughly the RefSeq sequences represent a centroid in a 10% sequence variantion sequence space at a genome level. So it is likely that some reads may not be assigned to the correct “taxa” as it is missing from the database. Also you make a comment about in silico chimaeras during de novo assemblies. Read mapping to reference genomes also suppers from the same issue – that is assigned of reads to a wrong taxa. So you may want to address this.
4)	Line 24-25: Please check this “Studies have shown that there are connections between human gut microbiome (viruses and bacteria) and diseases such as diabetes…” but as far as I am aware these studies have only looked at the role of bacteria not viruses.

I like this paper and think it has merit for publication in PeerJ. I also think that the authors may want to take a step back note that this tool is only for identifying viruses based on nucleotide sequences in the compiled database. I would change the name of the tool to FastVirusExplore a the word virome is totally misleading and this tool does not at all enable virome exploration.


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: DNA BARCODE-BASED SURVEY OF TRICHOPTERA IN THE CROOKED RIVER REVEALS THREE NEW SPECIES RECORDS FOR BRITISH COLUMBIA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Introduction
Main Issues.

This work could be an important baseline for future monitoring and impact assessment. However, the way is written is more like a report and not a scientific article. There is not a clear question or hypothesis presented to be evaluated. There has been growing literature on this topic presenting great information of species list of Ephemeroptera, Trichoptera, and Plecoptera in Northern Canada and this study can be used to compare the species richness with other regions in Canada (look for Yukon and Northwest Territories EPT records in Cordero et al. 2016). Based on previous species distributions studies: how are your contributions relevant? Are the new records you present unexpected? What is the novelty: altitude, latitude? Put your study in a scientific context and not only in a report format. As well, more support in your introduction is necessary, there are many concepts or facts that are not cited

minor issues:

line 29: what portion of the COI? be concise, refer to the subunit I. Also this information needs a citation.

line 42: Support this statement. How do you know this area is true Arctic? mind that in Northern Canada, latitude alone is not an indicator of a geographic area. Recent studies have addressed this regional classification.
Experimental design: Main Issue:

The paper does not follow the format of a scientific article but looks more like a report. The research question is not stated and hence no gap in the knowledge is addressed. The contribution is not relevant the way it is presented. It needs revision about the goal of the article.

specific issues: Methods.

What is the importance of using both morphological and molecular identification analysis? Could you solve any problem presented by morphological analysis? Did you apply a sampling protocol in the different sites?
Validity of the findings: Main Issues:
The use of BOLD or other database is very useful as an initial reference. However, assumptions or conclusion made from direct comparison or the match percentage is not very strong evidence. There are many mistakes in the registered species names in the databases and the only way to confirm your findings is to put your individuals in a phylogenetic tree and see real differences. Your initial idea is good but the way how you support your findings is not enough, even to claim for first records, much less for claiming new species records. Be careful with conclusions based on arbitrary 2%

line 140: If there are no other studies how you found only three new records if all the other records should be new too? You need to dig deeper in the literature since there are good records from the 1980's for places like Yukon that are far more Isolated, I can't imagine a good amount of literature for aquatic insects in British Columbia, considering the number of studies made on altitudinal gradients in the Rocky Mountains.
Additional comments: I think the authors should be encouraged to re-think this paper. I always find very valuable information about diversity but this information should be put in a scientific context to increase the relevance of the paper as well as the journal quality. I would recommend to accept the manuscript if they address two main issues:
1. Question or hypothesis.
2. Comparison of data by the use of a phylogenetic analysis.


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: DNA BARCODE-BASED SURVEY OF TRICHOPTERA IN THE CROOKED RIVER REVEALS THREE NEW SPECIES RECORDS FOR BRITISH COLUMBIA
Review round: 1
Reviewer: 2

Output:
Basic reporting: This paper makes a very good contribution to the knowledge of the caddisfly fauna of an interesting river in an interesting ecosystem.  The methods of analysis are relatively new to trichopterogy and represent one of the first attempts to use established DNA databases as a means of identifying local caddisfly biodiversity.  However, I think the paper needs better justification as to why this method is better than the “traditional” method of collecting adult caddisflies with light traps or by netting or other means.  Trichoptera species taxonomy is based on male genitalia (almost all holotypes described in the 20th century and now are males).  Larvae are secondarily associated with males through rearing, use of pharate adults, or by COI sequence match.  The authors should explain why they collected larvae only, or at least mention the value of adults in caddisfly taxonomy.  They may have valid reasons - adult taxonomy is specialized, adults do not fly to light in cool weather, adults may fly in from other watersheds, etc.  If the objective was to show that collecting larvae only was sufficient to assess the biodiversity of a stream, they should state that.  It would be interesting to compare the results of a strictly larval survey with adult collecting only although I realize that this is beyond the scope of the paper.  However, as presented the objectives of the paper are not expressed in context to standard caddisfly survey methods nor is it indicated their relative value.

In addition, the three new records are not placed in any context of the known distribution of the species.  For example, C. harwoodi is largely an eastern North American species, so its presence in BC is surprising.  This should be at least mentioned, if not discussed.  Without knowing otherwise, one might conclude that the vouchers of these sequences in BOLD are misidentified.

I found only a few typos grammatical issues in the paper.  I am still under the assumption that data are plural, but in some places it is used as a singular noun.  Also, the authors use dashes quite a bit to separate clauses.  Perhaps it is more appropriate to use commas or semicolons.

I did not find the larval photographs to be informative, although I think photographs of vouchers are required for submission to BOLD.  Do these photographs match known, published descriptions of these species>

I especially find the photographs of the adult Lepidostoma to be uninformative.  I can only tell that it is a Lepidostoma and it appears to be a female.  There is no indication of how this specimen was obtained.
Experimental design: see above
Validity of the findings: see above
Additional comments: see above