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: CHARACTERIZATION OF THE MICROBIOME OF THE INVASIVE ASIAN TOAD IN MADAGASCAR ACROSS THE EXPANSION RANGE AND COMPARISON WITH A NATIVE CO-OCCURRING SPECIES
Review round: 1
Reviewer: 3

Output:
Basic reporting: This manuscript was generally well-written. There are a few instances of grammatical errors, which I have noted in the general comments. The figures are aesthetically pleasing and well-designed, though I have some comments on them below.
Experimental design: The methods and results sections of this manuscript seem more complicated than they need to be, mainly due to the separation of the data into three datasets analyzed with different rarefaction levels. It is unclear whether all these different rarefaction levels are necessary. If you run all of the analyses with just the lowest level of rarefaction that you use, will you get the same results? With just one large dataset, you could still separate out the two sample types (skin, gut), or even run the stats with all of the data and use posthoc tests to detect finer scale patterns.

There are some aspects of the methods that need more detail (see below).
Validity of the findings: no comment
Additional comments: This manuscript described skin and gut bacterial communities on a non-native toad from three sites in eastern Madagascar, comparing them with those on a native frog in the area. The paper is generally well-written and tries to put microbial patterns into the context of the toad invasion. Understanding the role of microbiomes in the spread of non-native hosts is important, and this paper provides an interesting comparison between a native and non-native frog. I have a couple larger comments on the manuscript and its organization, followed by more minor edits needed. Some of these comments are merely suggestions, which I think would help the flow and focus of your paper.

Hypothesis: Can you elaborate on why you hypothesized higher richness/diversity on the non-native toad? The references you cited in the discussion don’t support/lead to this hypothesis. It’s difficult to make predictions here based on previous research, since there hasn’t been much, but even if you added something to your hypothesis like, “because it would be comprised of a mix of microbes from the toad’s native and introduced habitats,” it would help the reader to follow your train of thought. It’s not clear how the factors you listed as causing this predicted pattern (representing distinct ecology from the other species investigated) tie into your hypothesis. Having different microhabitat preferences and different behaviors don’t inherently make toads have more diverse skin bacteria. Furthermore, it’s interesting that you analyzed the interaction between species and site in your permanova. If you hypothesized that the communities would differ between the species but not at all sites, or not in a similar way among the sites (because of the length of time the toads have been at the sites), that would also be interesting to note with the hypothesis.

Methods/Results: The methods and results sections of this manuscript seem more complicated than they need to be, mainly due to the separation of the data into three datasets analyzed with different rarefaction levels. It is unclear whether all these different rarefaction levels are necessary. If you run all of the analyses with just the lowest level of rarefaction that you use, will you get the same results? With just one large dataset, you could still separate out the two sample types (skin, gut), or even run the stats with all of the data and use posthoc tests to detect finer scale patterns.

If you simplify the larger dataset (use one instead of three), you could also subsequently simplify the results section. While patterns in skin and gut microbes should be addressed separately, there’s no clear reason why sex differences need their own section except due to your use of a separate dataset for sex analyses. For each type of analysis of the skin dataset, you could follow the results of species and site with whether or not the two sexes of toads differed. Condensing in this manner would dramatically help the flow of the results section. I also think that the results that are less discussed (LEfSe and KEGG pathways) could be given less space in the main manuscript.

Throughout the manuscript, there are places where providing some reasoning behind your methods and reminding the reader of that reasoning would go a long way in telling your story. For example, on line 204, you could add in something like, “We assessed alpha and beta diversity metrics to detect differences between the species, sites, and sexes.” The end of line 221 could read, “We used PICRUSt to gain a better understanding of the possible functions of the bacteria in our skin and gut samples.” Similar context and reasoning, by adding sentences or introductory phrases, would also be useful in the results to make the manuscript easier to follow.

The supplemental figures need an associated file of captions. It’s not clear what all the figure parts represent otherwise.

Figures: I think the manuscript would look cleaner if you only kept figures 1,2,5,and 6 in the main paper, and put the rest into the supplemental. For Figure 4, you could easily report the averages and standard deviations in the paper text.
Figures 2c, 5c, and 6c – instead of only looking at the top 10 taxa as 100%, it’s important to be able to visualize what proportion of the total the top 10 taxa comprised. Consider adding an “other” portion of the bar charts such that the entire community is 100% and the top 10 comprise just a portion of that total. In the text (Lines 268-274, 315-320, 339-347), you discuss the relative abundances of different taxa, but because the figure seems to re-scale the total community to only include the top 10 taxa, it’s not possible to know how the relative abundances differ between the species/sites, since the “other” category has been discarded. Also in these figures, I see that there’s a portion of phyla that are unidentified/unclassified. This is very rare if only the bacterial OTUs are kept. Make sure you’re only keeping “Bacteria” in your OTU filtering, and add that step to the methods.

Discussion: You did a good job of comparing your results with others published, and the discussion has a lot of detail. For the reader, it would be helpful to condense, or even remove, some of this information to make the discussion more focused. While explaining every result has its merits, focusing on your hypothesis is important. There are a few places where you could simplify without losing much content while gaining better flow. For instance, on line 400, you could elaborate on why you’d expect the toads to differ between the sites, and remove the sentence that goes from 400-402. The rest of this paragraph could also be simplified and condensed.
The question I kept asking myself while reading this ms was whether the fact that they’re new to the environment means they’re less likely to be selecting for any specific microbes on their skin, allowing them to harbor/keep a wider variety of the microbes they come across. On lines 423-424, you write that a richer microbiome could aid in host spread to new environments, but I wonder if the opposite could also be true.

Regarding the comparison with the Woodhams et al. 2015 anti-fungal dataset: In the methods, you should note what % similarity you used as a cut-off for your comparison against the Woodhams dataset. Did you re-run closed-reference OTU picking? For your results and discussion of these results, you can identify which of the taxa you detected were initially isolated from Madagascar, or toads elsewhere (since no toads from Madagascar are represented in that data set). In the discussion (~line 454), it’s interesting that the high prevalence of chytrid in India could be associated with the lower proportion of Bd-inhibiting bacteria. If they’re not selecting for inhibitory bacteria in the face of chytrid, do they have other forms of resistance/tolerance (more of a curiosity, not necessarily needed to add to your manuscript)?

Minor suggestions/comments:
Title – Including your results into a more descriptive title would go a long way in bringing in readers
Lines 55, 205 - semicolon should be a comma
Line 62 – ecosystem’ missing the final “s”
Line 88 – would be helpful to indicate that the insect in question is invasive
Lines 132-133 and 322-323 – There’s a discrepancy between the sample sizes (8 male and 8 female per site, but then no females from site 3). Please identify the number of samples analyzed somewhere other than the supplemental tables (at least a range of sample sizes per sex/site)
Line 139 and Fig1 caption – fix “Moore et al. (Moore et al. 2015)”
Lines 150+ – the first time you use “gut,” please explain what part of the gastro-intestinal tract you mean. Some authors use gut to mean the entire GI tract, others the entire intestine, and others just a portion of the intestines. Also, were there any further steps for the gut samples, such as opening them and rinsing out the gut contents? It would be important to note whether the gut microbes in your study are also representing whatever the animal has eaten.
Line 169 – when you pooled the samples “according to band size,” did you quantify the band brightness to do it?
Lines 198, 200 and 201 – either here or in the results, state what # of reads you rarefied to for datasets B and C
Lines 207-209 – Alpha diversity metrics often don’t fulfil the assumptions of parametric tests, especially with your lower samples sizes of dataset C. Consider replacing these with non-parametric tests.
Line 211 – cite the phyloseq package
Line 212 – what predictor variables did you include in your PERMANOVAs? From the results, it seems like you analyzed the interaction between species and site, but that’s not explicitly stated.
Lines 253-256, elsewhere – for non-significant p-values, report fewer decimal places to make it look less messy.
Line 251 – give the p-value for Shannon diversity ANOVA, not just <0.05, since you elsewhere write out significant p-values
Line 260, Figure 2b – it’s surprising that the weighted unifrac permanova detected differences between the species, because in the figure it looks like the two species greatly overlap considerably. Consider double checking this result
Line 281 – fix “Thia”
Line 284 – Figure 2b does not show this.
Lines 285-288 – Figure S2 doesn’t have a “b”. Was the wrong figure s2 uploaded?
Line 291 – You write that the frog only had OTUs from Proteobacteria, but Figure 2C clearly says otherwise. I think you mean that the differentially abundant OTUs were all in Proteobacteria. Clarify the wording in this paragraph.
Lines 311+ – Instead of writing that there were “no significant differences…except,” you could write that Shannon was the only alpha diversity metric that was significantly different between the sexes. With these sex analyses, it’s unclear why you would repeat the analyses that determine if site were significant, since you already analyzed that variable with dataset A.
Line 315 – change from “factors was” to “factors were”
Lines 365, 367, 369 – fix the citations
Line 374 – change “impoverish” to “impoverished”
Line 378 – this comma seems misplaced
Lines 399-404 – check your grammar in these sentences
Line 404 – add “in” after “investigated”
Line 406 – change “comparing” to “compared”
Line 420 – change “as” to “such as”
Line 460 – change “Similarly, to” to “Similar to”
Figure 2 – For people less familiar with frogs, consider adding species names somewhere on the figure itself.


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: CHARACTERIZATION OF THE MICROBIOME OF THE INVASIVE ASIAN TOAD IN MADAGASCAR ACROSS THE EXPANSION RANGE AND COMPARISON WITH A NATIVE CO-OCCURRING SPECIES
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment, the authors have successfully revised their manuscript.
Experimental design: No comment.
Validity of the findings: No comment
Additional comments: No additional comments


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

Input:
Title: CHARACTERIZATION OF THE MICROBIOME OF THE INVASIVE ASIAN TOAD IN MADAGASCAR ACROSS THE EXPANSION RANGE AND COMPARISON WITH A NATIVE CO-OCCURRING SPECIES
Review round: 2
Reviewer: 2

Output:
Basic reporting: This manuscript describes microbiomes in and on an invading toad species in Madagascar, further comparing the communities between the toads and a native frog species. The manuscript is well written, with sufficient literature cited, and I suggest some minor edits below.
Experimental design: The research question here was adequately explained, and besides the low sample size for gut microbiome comparisons, the research goals were generally well assessed.

Methods notes:
Line 145-146 – What is meant by “swabs were kept dry”? Were they air dried, or just placed in tubes without preservative? If not "dried", state how long they were stored at 4C. I have concerns that not freezing them sooner could have affected the communities. I am aware of a couple studies assessing effects of short-term storage at 4C but not longer term storage (e.g., Kim et al. 2017 Microbiome, Lauber et al. 2010 FEMS Micro Letters)

I’m not convinced that LEfSe is very meaningful with the small sample sizes of the gut samples. Because of the small sample size and pooled samples, it is important that not too much weight is put on the quantitative results from the gut microbiome data.
Validity of the findings: No comment
Additional comments: Minor comments:
It would be helpful to stick to either using the Latin or common names for your study species throughout the manuscript (preferably common name). There is a lot of jumping back and forth between the two, within and between sentences and paragraphs.

In the discussion, you should acknowledge that similarity to bacteria that can inhibit Bd does not necessarily mean that the bacteria on your toads actually have, or could have, that function. Your 97% similarity cut-off for identifying potentially Bd-inhibitory/enhancing bacteria could include a wide amount of diversity in those species.

Lines 32-33 – It’s not clear what “greater differences at lower taxonomic levels” means

Line 52 – Be careful about invasive species versus alien or non-native species. By definition, invasive species have negative effects on some aspect(s) of the ecosystem they’re invading.

Line 55 – ecosystem’ to ecosystem

Line 140 – If you want to re-introduce the main study species in the methods, input the full Latin name earlier (line 133-134), not here.

Line 148 – euthanized to euthanize

Line 150 – input sample sizes, or a range, for P. mascareniensis (per pooled sample)

Line 162 – we amplify to we amplified

Line 189 – OTUs to OTU

Line 201-202 – the note of “…, for a total of 12 analysed individuals of D. mel. And 12 individuals of P. mas)” is confusing, since you didn’t analyze 12 individuals, you analyzed pooled samples. Suggest removing this information.

Line 209 – effect of the sex  effect of sex

Line 219-220 – instead of “lack of replication”, maybe clarify with “low sample size and pooled samples do not allow for thorough statistical analysis…”

Line 252 – OUT picking to OTU picking

Line 256 – by proportions, do you mean proportions of OTUs or proportions of the total reads? This isn’t clear here and in the results. Also, clarify that these bacteria are “potentially” Bd-inhibitory/enhancing

Often in the results you refer to many panels of a figure, when you could refer to fewer at a time or in some cases, refer to those figures fewer times. For example, if line 269 refers to Fig 2a-d, don’t need to refer to them again at the end of the sentence. Another example, line 280 could refer just to Fig 2E, with line 281 referring to Fig 2F, or refer to them both once.

Line 286-287 – Which distance matrix are you referring to for the analysis of dispersion? Since both weighted and unweighted PERMANOVA were significant, should run this analysis with both distance matrices.

In lines 290-298, 340-344, and 371-380, you eye-ball differences between groups in relative abundance of different taxa, but then later provide the LEfSe analysis results. This is especially notable in the males vs females comparison, where you list differences, but then state that there were no differentially abundant taxa in either sex (line 346). I suggest you move the lefse results up to where you describe the communities, and you can specify, if need be, where taxon abundances appear different even though not significantly so.

Line 317 – taxa to taxon

Line 336 – missing a parenthesis

Line 337, 338 – consider replacing “using” (or line 338’s “using the”) with “assessing” or “comparing” or something similar

Line 354 – exhibiting to exhibited

Line 374 – double check the “10 abundant families”. Looks like 9, since “unclassified” isn’t a family, and may represent many families

Line 375 – Based on what you consider to be high abundance, it looks like P. mascareniensis has 7 families with fairly high abundance. Are you saying there are two families with higher relative abundance than in the toads, or that there were only two families with high abundance?

Line 385 – was to were

Line 405 – “on the gut microbiome the invasive species” to “on gut microbiomes of invasive species”

Line 407 – influence to influenced

Line 412, 540 – you didn’t really look at how the microbiomes “changed” across the toad’s expansion range, just looking at how they differ.

Line 413 – what makes a site “impoverished”? Not clear what you mean here.

Line 414 – host “identity” to host “species”. Identity could be read to mean individual frogs/toads

Line 415 – the “and at a lesser extent with sex” reads very out of place here. Missing a word maybe?

Lines 430-432 – Why do you think urban/town environments would impoverish the bacterial pool? Are there references to show this? The toads, which were found in more diverse, human-use areas, had greater bacterial richness than the frogs, found in the grass.

Lines 439-440 – It’s not clear what the second axis is in the “trend” of increase in alpha diversity across toad sites. The sites don’t seem to represent a gradient. Maybe better to say that the local populations differ?

Line 460 – assessed to assess

Line 498 – amphibian’ to amphibian

Lines 508-510 – Specify that you’ve switched topics to the gut microbiomes

Line 542 – change “more diverse than the one of the” to “more diverse than a”

Line 543 – toad to toad’s

In the taxa bar charts, consider having higher % total abundance cut-offs for inclusion. There are some colors in the legends that aren’t visible in the figures.

Including asterisks in figures to denote significant differences, or providing the statistical results in the figure captions, would be helpful.


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: HELICOBACTER PYLORI PREVALENCE IN HEALTHY MEXICAN CHILDREN: COMPARISON BETWEEN TWO NON-INVASIVE METHODS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The study objectives methodology is clear.
Experimental design: The experimental design also is good.
Validity of the findings: The authors found that the detection of H Pylori in the stool sample was more reliable compared to that in Saliva and dental plaque.
Additional comments: Well organised 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: HELICOBACTER PYLORI PREVALENCE IN HEALTHY MEXICAN CHILDREN: COMPARISON BETWEEN TWO NON-INVASIVE METHODS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Abstract:
Although some of the children declared some dyspeptic symptoms, these were no (should be: not) related to H. pylori.

Main text:
Results:
Most of the children (91.8%, 157/171) live  (should be: lived / were living)   in a rural community.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: No comment.


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

Input:
Title: HELICOBACTER PYLORI PREVALENCE IN HEALTHY MEXICAN CHILDREN: COMPARISON BETWEEN TWO NON-INVASIVE METHODS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The paper entitled Helicobacter pylori prevalence in healthy Mexican children: comparison between two non-invasive methods conforms to professional quality standards in terms of its verbal expression.
This paper has sufficient bibliographic background although I would suggest another text on CagA and Vac Apara to make the bibliographic background information in Mexico more consistent, since there are few research groups in Mexico and this will help even more to strengthen the research groups.
The experiments show their methodology and results in a clear, consistent and easy to understand way for the reader, which coincide with the hypothesis test applied through statistical analysis.
Experimental design: The work is Original, and also meets the expectations and scope.
Besides considered as novel since in some way it complements much of the literature reported in the world and in addition to strengthening lines of research that support the knowledge here in Mexico and thus contributing to the strengthening of many other groups in the world.
In addition, it was observed that ethical care is taken since it shows the work record evaluated by an Ethics committee which, having been authorized by means of a support of CONACYT( Ministry of Investigation in Mexico) which should have also reviewed the berry nature of the document, especially for the reason of working with minors in Mexico which also shows the authorization of their parents or guardians
Validity of the findings: The statistical methods are adequate, clear and cosiso, also the conclusions are adequate since it mentions the limitations of the SAT method on the genotification of Cag A and VacA which is important to mention
Additional comments: Include an article on CagA and VacA also carried out with the patient population in Chiapas in Mexico since the future comparison of similar communities in the same country is interesting, which would provide future information of scientific interest.

Molecular detection of Helicobacter pylori based on the presence of cagA and vacA virulence genes in dental plaque from patients with periodontitis.
Flores-Treviño CE, Urrutia-Baca VH, Gómez-Flores R, De La Garza-Ramos MA, Sánchez-Chaparro MM, Garza-Elizondo MA. J Dent Sci. 2019 Jun;14(2):163-170. doi: 10.1016/j.jds.2019.01.010. Epub 2019 Mar 27.


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: HELICOBACTER PYLORI PREVALENCE IN HEALTHY MEXICAN CHILDREN: COMPARISON BETWEEN TWO NON-INVASIVE METHODS
Review round: 1
Reviewer: 4

Output:
Basic reporting: The manuscript meets the basic criteria of publication, but there are some control experiments missing in its current version.
Experimental design: The design of the experiments employed in this work is sound, but negative control/loading control/normalization control need to be included.
Validity of the findings: The findings are relevant and might help to get new insights into the H pylori detection methods.
Additional comments: The manuscript entitled “Helicobacter pylori prevalence in healthy Mexican children: comparison between two non-invasive methods” presents a comparative study on three detection methods for H. pylori. Authors analyzed saliva and dental plaque using PCR method and stool samples by immunochromatography. Authors report that there is a high prevalence of H. pylori in asymptomatic children and amongst the multiple methods, detection by stool antigen test is most feasible method. The manuscript presents useful data here, but has some limitations. As the paper compares three different methods, my main concerns are pertaining to the technical details of the methods.  Here are my comments:
1) Authors need to include information on the negative controls in the various methods tested. It is not clear to me if they were included. Authors need to specify this. If they were not included in the study, it would be important to include these. Also, did they have any normalization control/loading control? How did they rule out that the difference in the signal is not because of the difference in the input (bacterial load)? Please address these issues with respect to all the three methods described in the paper.
2) The methods are qualitative and results may be interpreted empirically. It would be impractical to represent these data in categorical (yes/no) format.
3) Authors may need to include a representative image (may be as supplementary data) for the PCR based detection method (both negative and positive samples).
4) Authors need to include additional data on the sensitivity and specificity of all the methods used. Did authors ensure the assays did not give any false positives and false negatives?
5) Author may consider presenting the data by Venn diagrams with overlaps representing the samples detected positive by multiple methods.
6) Did authors have technical replicates (all assays) included for each sample tested? It would be relevant to include a statement on this.


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

Input:
Title: HELICOBACTER PYLORI PREVALENCE IN HEALTHY MEXICAN CHILDREN: COMPARISON BETWEEN TWO NON-INVASIVE METHODS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The English must use Clar, good standards and expression
Experimental design: It is very clear, simple and specific
It is stated how research fills an identified knowledge gap.
Validity of the findings: The information shown in this work is important and must be published since its scientific value will have a reflection or impact on the epidemiology and control of transmission mechanisms.
Additional comments: It is very important that this work be published since the information provided by this work is of great scientific value


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: HELICOBACTER PYLORI PREVALENCE IN HEALTHY MEXICAN CHILDREN: COMPARISON BETWEEN TWO NON-INVASIVE METHODS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The revised manuscript meets the basic criteria of publication. The work is well executed and technically sound. The data support their conclusions.
Experimental design: Experimental design is sound.
Validity of the findings: All the conclusions that are justified and based on clear results.
Additional comments: The revised manuscript addresses my concerns satisfactorily. I recommend the paper for acceptance 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: IRON DEFICIENCY ANEMIA AMONG CHILDREN AGED 2–5 YEARS IN SOUTHERN ETHIOPIA: A COMMUNITY-BASED CROSS-SECTIONAL STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The research question, relevance , methodology, results/interpretation as well as conclusions derived are good.
There are lots of editorial issues. Authors should read through carefully to correct them. Some will be highlighted alongside some other comments.
1.	Line 31 including replace with involving
2.	Line 42 ‘last week’ replace preceding week
3.	Line 89 focusing on the assessment of iron rich food
4.	Line 106-107 to be rephrased for clarity
5.	Line 131, ferritin was omitted.
6.	Line 136-137. Rephrase sentence.   .... transported in cold box to Ethiopia.
7.	Line 138-139. Mention the principle of reaction of Cobas e601 and 501.
8.	Line 143 ‘sea level’
9.	Line 173 Anthropometry measurements
10.	Line 311, comparable with prevalence
11.	Line 312-13. Revise sentence. The prevalence of IDA......
12.	Line 325-327. Revise this sentence; the Information being passed is not clear
13.	Line 338. Rwanda and Brazil have also
14.	Line 321-322. ...treatment of IDA with iron supplementation
15.	Line 334-335. Are the associations between demographics /socioeconomic characteristics and iron rich food consumption/IDA? The sentence does not read as such. Rephrase for clarity.
16.	Line 355. Furthermore not further.
17.	Line 357. ......public health problems among our study participants
18.	Line 461-463, repeated in line 464-466
19.	According to the table 1, 10 children included in the study were on iron supplements.  Were any of them in the IDA group?  This information should be in the write up for readers’ information.


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: IRON DEFICIENCY ANEMIA AMONG CHILDREN AGED 2–5 YEARS IN SOUTHERN ETHIOPIA: A COMMUNITY-BASED CROSS-SECTIONAL STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: Generally OK
Abstract:
Reword sentence beginning with:  “Three-5 mL of venous blood” to clarify this was only one tube….
Experimental design: Generally OK
Methods:
Was a traceable reference material used to calibrate the ferritin assays?
Validity of the findings: Results:
L. 247.  Data cited for underweight do not seem to match numbers on Table 1.
L. 275-276 – Data cited for moderate and mild anemia do not seem to match Table 4.
Additional comments: Timely and useful paper given the 2020 WHO Guideline on Use of Ferritin.  Presentation is straight forward and will be particularly valuable to those working in research and policy.

L. 164  and references– Gibson, RS is the correct citation (Rosalind is the given name)
Discussion:
Specifically, what was the reason for selection of the "Higher ferritin-cut-off adjustment" approach for reporting your IDA data?
More in-depth comparisons among the current results and additional papers from the BRINDA project might be useful.  Consider [1-3]
Editorial Comments:
Line 54 and references – Central
Line 143 – sea
Review throughout for noun and verb plural/singular agreement.

1.	Namaste, S.M., et al., Methodologic approach for the Biomarkers Reflecting Inflammation and Nutritional Determinants of Anemia (BRINDA) project. The American Journal Of Clinical Nutrition, 2017. 106(Suppl 1): p. 333S-347S.
2.	Engle-Stone, R., et al., Predictors of anemia in preschool children: Biomarkers Reflecting Inflammation and Nutritional Determinants of Anemia (BRINDA) project. The American Journal Of Clinical Nutrition, 2017. 106(Suppl 1): p. 402S-415S.
3.	Merrill, R.D., et al., Factors associated with inflammation in preschool children and women of reproductive age: Biomarkers Reflecting Inflammation and Nutritional Determinants of Anemia (BRINDA) project. The American Journal Of Clinical Nutrition, 2017. 106(Suppl 1): p. 348S-358S.


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: IRON DEFICIENCY ANEMIA AMONG CHILDREN AGED 2–5 YEARS IN SOUTHERN ETHIOPIA: A COMMUNITY-BASED CROSS-SECTIONAL STUDY
Review round: 1
Reviewer: 3

Output:
Basic reporting: The opening sentence in line 53-54 is not indicated the most recent figure. please check the most recent figure from global nutrition report 2020.

You assumption (line 56) about the causes of anemia is not the same any more. Please read

This review

file:///C:/Users/bekel005/Downloads/nutrients-08-00693.pdf

Global experts opinion on harvest plus website

https://www.harvestplus.org/viewpoints/new-findings-iron-deficiency-anemia-experts-weigh

I recommend you to revise your manuscript based on the new findings from this review and experts view in the field.

There is repetition of sentence like line 56 and 62.

you second paragraph don't have all the figures required. you mentioned poor, low, most … It's better to indicate the actual figures to the readers.
Experimental design: You have been using and justifying the prevalence in the background from demographic and health survey 2016. Why you calculate your sample size using central statistics agency 2012 figure ?

when exactly you collect the data ?

your previous publication referred says from February 15, to March 30, 2017 ? but the sample size was 100. Can you tell us the duration of this study for 340 children?

Who did the blood sample collection

why the two machine for laboratory analysis ?

did you use internal quality control?

How do you know if the machine is reading the right quantity?

What method of laboratory analysis used?

We don’t know how you used the 24 hr recall data? What you did before data analysis?

Was the 24 hr dietary recall qualitative or quantitative or semi quantitative?

in line 198, we don't know which questionnaire you are referring. is it 24 hr dietary recall or 7 day FFQ?

For which dietary data you applied FAO nine food categories ? is it for 7 day recall or 24 hr dietary recall ?

line 202 - Now you added another dietary assessment method. Please descript each dietary method separate: how you collect the data? What you did before data analysis? what analysis you did at the end.

What you described in the dietary assessment section is mix of all methods and it’s not detail enough.
Validity of the findings: The conclusion is not clear to which community, and geographic area.

Any reason why all variables P-value< 0.3 included in the multiple logistics regression?

Your finding about child immunization card is not clear. What does this result mean? What if the mother lost immunization card which is most common?

current breast feeding total n is 330 where as your percentage is 100%. You mentioned in the title your N is 331?

Something is wrong with this figure and variable names. please check and report wasting and underweight separate with the right figure.

please check the Weight-for-height (wasting) score naming of sub categories (which says Under weight?)
Additional comments: Make sure you comparison your findings with other similar findings are not with different age categories and geographic coverage. If you did that please make it clear to your reader so that they will be aware of it. Your interpretation of findings has to be inline with the current information bout causes iron deficiency anemia.


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: IRON DEFICIENCY ANEMIA AMONG CHILDREN AGED 2–5 YEARS IN SOUTHERN ETHIOPIA: A COMMUNITY-BASED CROSS-SECTIONAL STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: Fairly ok. Authors to pay attention to grammar use. In view of this make corrections to line 129-130 and 272.
Experimental design: Well defined
Validity of the findings: Ok
Additional comments: Generally, the revised article reads better as most queries has been revised.


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: IRON DEFICIENCY ANEMIA AMONG CHILDREN AGED 2–5 YEARS IN SOUTHERN ETHIOPIA: A COMMUNITY-BASED CROSS-SECTIONAL STUDY
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: Lines 134-140.  “serum” is mentioned several times on these lines, but L 130 says blood was collected by using a lithium heparin plasma separator test tube.  Clarify if ferritin and CRP were analyzed in serum or in plasma
Validity of the findings: No comment.
Additional comments: Line	Comment
37	..child had both..
48	..women’s
54	..anemia are due..
95	Medicine and Health Sciences
115	33%
137	..kept in..
202	≤3
207	..and the volume..
208	..intake were not measured.
215	Responses scored ‘never’..
219	..guidelines.
243	..variables was checked..
257	pairs
266	..one was..
272	..mothers participated..
273-274	..home, 75%..
362	..results have to..
374	..women’s education


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: GENOME-WIDE ASSOCIATION STUDY UNCOVERS NEW GENETIC LOCI AND CANDIDATE GENES UNDERLYING SEED CHILLING-GERMINATION IN MAIZE
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In this work,the authors used 43,943 SNPs and FarmCPU method to perform GWAS of five germination-related traits during seed chilling-generation in maize. They detected 15 significant signals under a Bonferroni multiple-test threshold, among which, they further analyzed 3 loci and 10 candidate genes associated with multiple traits. Overall, this manuscript was well organized, and I favor its acceptance after a few of minor editions.
1. The manuscript would be much improved after professional-English editing. The results section was particularly verbose.
2.Please clearly define the differences among chilling, cold and freezing stress and confirm the correct use of these three words in MS.
3.What were the criteria for selecting seeds (eg. size, weight...)? Also, germination experiments required a normal temperature control. It would be more convincing if some germinating pictures of different types of maize lines were provided.
4.Please provide the evidence that the chilling condition was chosen at 10℃.
5.Most figures weren’t at sufficient resolution as the request of Peer J.
6.Lines 136-137: I can’t find the LD decay regions with 220K in Conneely & Boehnke (2010). It should be described in Method or cited the relevant published article.
7.Lines 130-133: FarmCPU is a widely used GWAS method with high power and low false positive and false negative. That is well known and not an important point in MS. I suggest to delete this paragraph.
8.Fig. 3: The threshold was nearly 1.3, which was different from GWAS threshold (P<2.03*10-6). How to get the threshold?
9.Lines 207-208: “Statistical Analysis System v 9.3” should be abbreviated to “SAS v 9.3”. Alternatively, remove it here and describe it in Method instead.
10.Line 211: The name of the candidate genes “abh4” should be “ABH4”.
11.Section 4.2: This part compared the overlaps between present genetic loci of chilling tolerance and previous published data. Readers would be put off by searching for so much genetic information in so many articles. So I suggest making a list of previous studies.
12.Fig. 5: Heat maps shouldn't just have two colors. This title needs to be reconsidered.


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: GENOME-WIDE ASSOCIATION STUDY UNCOVERS NEW GENETIC LOCI AND CANDIDATE GENES UNDERLYING SEED CHILLING-GERMINATION IN MAIZE
Review round: 1
Reviewer: 2

Output:
Basic reporting: I think that there are no problems with the authors' writing and format in the manuscript.
Experimental design: In this manuscript, the authors investigated five seed germination-related phenotypes such as the germination rate or growth under the cold stress among 300 inbred lines. By using these data, they performed the GWAS analysis and identified several SNPs that were associated with the cold-tolerant phenotypes. These SNPs gave positive effects to the expression of four genes in response to the cold stress. Their experimental design was clear.
Validity of the findings: I agreed that the ten genes that were associated with multiple traits were good candidates of the important genes for germination under the cold stress.
Additional comments: I listed several concerns below.

・The authors should transfer the description about Fig.5 to the Result section. It shows how important the SNPs that they identified are for germination under the cold stress. I think they should show how different the germination phenotypes under the cold stress are between the five elite lines and the others.

・In L170, the authors wrote that Hap2 and Hap3 showed significantly larger SL values than that of Hap1. Is it correct? I look like that there is rather a significant difference between Hap2 and Hap3 in their figure.

・It is difficult to see where three groups are divided in Table 2.

・I think that some “Figs.” are typo of “Fig. S”.


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: FARMING PRACTICES AFFECT THE AMINO ACID PROFILES OF THE AQUACULTURE CHINESE MITTEN CRAB
Review round: 1
Reviewer: 1

Output:
Basic reporting: ok
Experimental design: ok
Validity of the findings: ok
Additional comments: this paper has good structure and high quality.


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: FARMING PRACTICES AFFECT THE AMINO ACID PROFILES OF THE AQUACULTURE CHINESE MITTEN CRAB
Review round: 1
Reviewer: 2

Output:
Basic reporting: General structure of the paper is pretty basic and nothing to complain about, but the flow between the sections can be improved. My main probe with the basic reporting is a poor readability of the text. English grammar is not that problematic, so I would not recommend to show paper to the native speaker (thought its never hurts per se), but what I will recommend is to drastically improve the text. Now text is stuffed with wordy, complex unreadable sentences, which are confusing and not always conveying what authors seems to want to say. I marked numerous examples of that in the annotated pdf, but in short, even experienced authors can always improve their writing. I think that Hotaling (2020) gave excellent tips to do just that. I will strongly recommend to re-write the main text with Hotaling’s tips in mind (https://aslopubs.onlinelibrary.wiley.com/doi/full/10.1002/lol2.10165). I think that paper’s readability, enjoyment of the readers and, eventually, number of citations will gain immensely from trimming overly wordy text into the more concise manuscript.
Experimental design: Experimental design raised my main issue with a manuscript. Authors have only sampled 5 crabs of each sex from each of the sampled ponds. I was surprised that a) it was always 5 (10) crabs per pond without correction of the sampling effort for the pond’s size, even though surface area of the sampled ponds varied from >4ha to over 20ha; b) I was confused by the following statement (lines 94-96) “Since the hepatopancreas and
gonads from a single crab had relatively low total weight, the tissue from 5 crabs of each sex per
pond were pooled to form and at least two replicates were then analysis in each culture area. “ Does authors pooled the tissues from 5 crabs into two batches at least? Than those are not replications, those are pseudoreplications, as you are sampling your tissues from the same pool of 5 animals. Therefore I am a bit concerned about small sample size. I would like authors to address the possible pseudoreplications, small sample size and why was not the amount of the specimens sampled corrected for the surface area of the sampled pond
Validity of the findings: I looked through the analysis and the raw data and the conclusions of the authors seems to be well supported by data. Regrettably, authors did not used open-source software, so checking the code is not an option. I think that description of the statistical outcomes and analysis are fuzzy in some places and language that authors use to talk about stats should be elucidated. Please see pdf annotations for the details. In general, while I find the finding valid and supported by data, my issues with sample size and adjacent parts of the experimental design (which I described above) should be addressed first for the proper evaluation of the results.
Additional comments: I commend this paper for looking into the important issues of the development of sustainable acculturate in one of the largest world economies- China. I think paper asks interesting questions and supports its answers with appropriate data, but considerable efforts should be taken to improve the text’s readability and to better explaining experimental design.
I think paper can be accepted after the major review is implemented.


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: FARMING PRACTICES AFFECT THE AMINO ACID PROFILES OF THE AQUACULTURE CHINESE MITTEN CRAB
Review round: 1
Reviewer: 3

Output:
Basic reporting: In this manuscript, literature references and background information have been provided.
Experimental design: no comment
Validity of the findings: This manuscript has shown the impactful and novel results where the rationale and benefit is clearly stated.
Additional comments: In this paper, the author investigated and observed differences in farming practices and amino acids contents of three edible tissues among the pond-reared mitten crabs across Jiangsu Province. The results showed that the snail supply, macrophytes coverage, total commercial feed and the ratio of trash fish to total feed input have influence to the average individual size and commercial yield of mitten crab. The author also discussed the nutrient value and taste of mitten crabs from different locations (i.e., north and south area of Jiangsu). All the information would be very important and indicative to the farming practices.

Questions:
- [line 119 and 120] the eqn 5 and eqn 6 are the same at the right side.
- About the Table 1, it would be helpful to reformat the Table 1. Part of the information is out of the margin right now.


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

Input:
Title: FARMING PRACTICES AFFECT THE AMINO ACID PROFILES OF THE AQUACULTURE CHINESE MITTEN CRAB
Review round: 3
Reviewer: 1

Output:
Basic reporting: After authors' revision, the manuscript has become clear and unambiguous, professional english used throughput.
Experimental design: The original research aims and scope has been well demonstrated in this manuscript. Research questions are well defined and illustrated.
Validity of the findings: Meaningful data and results have been provided and statistically analyzed. All of them are robust, statistically sound and controlled.
Additional comments: The authors have answered my questions and comments in the revised manuscript. I would like to support this manuscript publish in the PeerJ journal. Thanks.


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: COMPREHENSIVE TRANSCRIPTOME ANALYSIS OF PERIPHERAL BLOOD UNRAVELS KEY LNCRNAS IMPLICATED IN ABPA AND ASTHMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Huang et al. performed a comprehensive transcriptome analysis of peripheral blood in order to unravel key lncRNAs implicated in ABPA and asthma.The authors identified high-confidence lncRNAs from the RNA-seq data from three types of human peripheral blood.Then they integrated a few transcriptome analysis methods to identify crucial lncRNAs. Differential expression analysis and differential network analysis were performed to conform and filter the important candidate lncRNA. The study is well organized.However, there are some specifice comments：

There are some obvious typos, even in the Abstract.For example, “Our analysis indicated that these lncRNAs exhibits in the loss-of-function
networks”and “a high-confifidence dataset of lncRNAs were identifified using a stringent fifiltering pipeline”. So I suggest the authors to go over the text carefully.
Experimental design: Before the analysis, the authors should provide some indexes to show the quality of sequencing data, such as mapping rate.
The threshold values of the correlation coefficients are totally different in three types of networks. The analysis process is somewhat subjective. Suggest the authors to show some evidence to support your selection.
Validity of the findings: According to my understanding, the analysis of this study is based on the RNA-seq data.It is a little confused that some variants (~26,。2% )via GATK analysis pipeline located in intergenic region.
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: COMPREHENSIVE TRANSCRIPTOME ANALYSIS OF PERIPHERAL BLOOD UNRAVELS KEY LNCRNAS IMPLICATED IN ABPA AND ASTHMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: This paper was overall well written, with sufficient data analysis and proper statistics.
Experimental design: The experimental design is OK, with 2 disease groups, and 1 control group.
Validity of the findings: The methods part includes clear information. Literature reviews also validate some of their findings. However, more details should be added to their validation parts.
Additional comments: Review of  Manuscript 56161, " Comprehensive transcriptome analysis of peripheral blood unravels key lncRNAs implicated in ABPA and asthma"

This paper analyzed the RNAseq data of PBMC from a total of 27 unique individuals, including 7 asthma patients, 12 ABPA patients and 8 healthy individuals considered as control group. The raw data was mapped to human genome to get lncRNAs, including unknown lncRNAs through a series of careful designed procedures.  In addition, the sequence variances were also identified from the RNAseq sequences using GATK tools. Then, both the genetic variances and expressions among these 3 groups were systematically analyzed. Immune related modules were enriched from the DETs among these 3 groups. After that, the authors built a network between the DETs and other transcripts, network alterations among these 3 groups were identified to find sub-networks for each disease, namely loss-of-function network, gain-of-function network, and anti-function network. Finally, network hubs were analyzed and 4 of these nodes were chosen for validation.

Main comments:
1.	Line 151, please add a few details of how GlueGO work. Is the function enrichment based on differentially expressed genes based on a certain p value? Or it was ranking based/GSEA-style enrichment?
2.	Line 154-155, please also explain why the correlations between DETs were not calculated and compared between groups.
3.	Line 188, how did the authors perform the “across samples by Fisher Exact probability test”? From the supplementary file, they were using Fisher Exact test. In this case, it should be “across groups” rather than “samples”.
4.	Line 207, how did the authors perform “cluster analysis”. Cluster analysis based on what? They should write it clearly to make the readings more smoothly.
5.	In the validation part, the authors should explain why they chose those 4 transcripts for validation purposes. Also, they validated the results from 4 different GEO datasets, please explain why those 4 datasets were chosen. Also, did they validate the 4 transcripts in all 4 datasets? Or they validate them as one dataset for one transcript? Why not using one convincing dataset to validate all transcripts? They may provide a Venn diagram of the DETs of the other dataset and their hub genes.

Minor points:
1.	Line 119, should be “Figure 1”, rather than “Figure S1”. Please check.
2.	There are some other typos, such as in Line24, “It complicates and aggravate” should be “It complicates and aggravates”… etc. please read carefully and check.
3.	In figure 5, please add a title to each network.
4.	In table1, sex information should also be included to exclude the co-variance effects.


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: COMPREHENSIVE TRANSCRIPTOME ANALYSIS OF PERIPHERAL BLOOD UNRAVELS KEY LNCRNAS IMPLICATED IN ABPA AND ASTHMA
Review round: 1
Reviewer: 3

Output:
Basic reporting: The introduction are very clear and written well.
Experimental design: Number of samples are insufficient for this analysis.As per data, less than 1%of asthma patient were affected by ABPA.So to predict the analysis ,the sample number should be high.
Also , Author didn't mention the type or stage of the asthmatic patient.The experiment is not  well designed.
Validity of the findings: The findings are valid.But to prove the conclusion the sample number of asthama,ABPA and control should be high.Also the stages of the disease has to be noted clearly.
Additional comments: The author work is done very informatively and clearly.I really appreciate the author research.I suggest author may also include cystic fibrosis patients.


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: COMPREHENSIVE TRANSCRIPTOME ANALYSIS OF PERIPHERAL BLOOD UNRAVELS KEY LNCRNAS IMPLICATED IN ABPA AND ASTHMA
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: COMPREHENSIVE TRANSCRIPTOME ANALYSIS OF PERIPHERAL BLOOD UNRAVELS KEY LNCRNAS IMPLICATED IN ABPA AND ASTHMA
Review round: 2
Reviewer: 2

Output:
Basic reporting: In the revised manuscript, the authors addressed all my concerns.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In the revised manuscript, the authors addressed all my concerns.
minor concerns:
1. In line 228, still "across samples" though the author claimed that was changed in the rebuttal letter.
2. In table S2, could the authors also add one column that showed some basic information of the GEO datasets? Should include the basic experimental design, types of sequencing(RNAseq vs microarray), the tissue where RNA was extracted, etc.


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: ALPHA- AND BETA- ADRENERGIC RECEPTORS REGULATE INFLAMMATORY RESPONSES TO ACUTE AND CHRONIC SLEEP FRAGMENTATION IN MICE
Review round: 1
Reviewer: 1

Output:
Basic reporting: I feel as though the authors mention health problems and inflammation, but don't draw a strong and explicit enough link between increased inflammation and health problems. The reader might find a few citations linking prolonged inflammation to health.
Experimental design: While I do understand and appreciate your use of female mice, it could use a little more explanation, perhaps with some citations. There is some evidence of variation of the inflammatory response across the estrous cycle in mice. Since your mice were P21, were they not yet sexually mature? Do you not expect this to be an issue for your study? Please elaborate.
Validity of the findings: I suggest removing all mention of trending results from the results section to make the text more clear. Statistical purists will always say that your finding is either significant or not with nothing in between.
Additional comments: A well written and interesting study. I have very little to add.


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: ALPHA- AND BETA- ADRENERGIC RECEPTORS REGULATE INFLAMMATORY RESPONSES TO ACUTE AND CHRONIC SLEEP FRAGMENTATION IN MICE
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The present study determined the inflammatory response to acute and chronic sleep fragmentation and assessed the modulating effects of  α- and β- adrenergic receptors antagonists on these responses. Findings of this study added meaningful data about the sympathetic nervous system mechanism of the stress responses resulted from sleep disorders.  Advice for the author:
A.Give evidences or reasons for the choice of sampling sites in the central parts and peripheral organs/tissues in the introduction or discussion section.
B. What suggestions for clinical practices could be drawn from this study findings?


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

Input:
Title: ALPHA- AND BETA- ADRENERGIC RECEPTORS REGULATE INFLAMMATORY RESPONSES TO ACUTE AND CHRONIC SLEEP FRAGMENTATION IN MICE
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I have no further comment on the revised 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: USING AN INTEGRATIVE TAXONOMIC APPROACH TO DELIMIT A SIBLING SPECIES, MYCETOMOELLERIUS MIKROMELANOS SP. NOV. (FORMICIDAE: ATTINI: ATTINA)
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript is describing a new species of the fungus-growing ant genus Mycetomoellerius using a wide variety of techniques: phylogenetics, behaviour, chemistry and morphology. This piece should be used as an example of how modern taxonomy should be done!

Kind regards
Pepijn Kooij

Basic reporting
The manuscript is well written and follows the journals structure with only few spelling corrections are needed. Introduction and background are mostly sufficient, however, I would like to suggest the authors to introduce the junior synonym M. balboai already in the introduction. The figures are relevant and of sufficient quality, however, I did detect one issue. It seems that the figure legends for Fig 3 and Fig 4 have been mixed up. All raw data has been made available, however, for the sequence data there is only the final edited and concatenated alignment available. It is unclear to me from the methods whether there were any ambiguous regions the authors had to deal with, and if yes, how they did this.
Experimental design: Experimental design
The research is well within the scope of the journal and has well defined, relevant and meaningful research questions. I fully agree with the authors that it is important to identify and describe species. I would like to congratulate the authors on the amount of work they put into this manuscript. Their rigorous investigation with a variety of techniques and thorough analyses of the data are, in my opinion, a perfect example of what modern taxonomy should look like.
Validity of the findings: Validity of the findings
All underlying data has been provided and are robust and statistically sound. Conclusions are well stated linked to original research question & limited to supporting results.
Additional comments: General comments
L48-56 “Diverse studies … 1995 to 2019”
I suggest the authors to split this sentence in two. Finishing after “taxonomists” and continuing with “In fact, an average …”

Also, here and further throughout, I suggest the authors to consider the recently published paper on the new leaf-cutting ant genus Amoimyrmex (Cristiano et al., Austral Entomology, 2020) as one of their examples as well as in their counts of new species.

L58-62 “The lower attines … Leucoagaricus gongylophorus”
The recurring assumption among scientists in this field is indeed that the lower attines
cultivate “undomesticated” fungal cultivars, however, this wording is in my opinion incorrect. From the data so far published (e.g. the by the authors cited Mueller et al 2018), it is clear that the clades of fungi grown by lower attine ants are predominately domesticated with only a few exceptions of “free-living” taxa/specimens. Due to the low frequency of these free-living specimens, this phenomenon could easily be explained by rare occasions of fungi escaping the mutualism. Furthermore and in light of this, I would still consider these fungi as obligately mutualistic as they normally not able to live without the ants, i.e. the free-living taxa/specimens seem to be different species based on the published phylogenies.

L79-82 “It is this … (Appendix Table 8).”
This is the first table referred to in the text, I suggest the authors to number the tables, also those in the appendix, by order of appearance.

L104-105 “Based on … Mycetomoellerius mikromelanos sp. nov.”
Because this is the first appearance of the name Mycetomoellerius mikromelanos in the main text I suggest the authors to add the citation to the name.

L112-113 “Colonies of … Republic of Panama”
Please, add the total number of colonies collected/used here.

L162 “We set k = 2 … iterations.”
I am wondering whether the authors chose this number arbitrarily or performed a test to see which value of k to use. I suggest the authors to perform such a test to exclude any ambiguity.

L182-183 “We used … same colony”
I have to admit that this is not entirely clear to me. Does GLMM make sure that these samples, which are technical replicates, are seen as that? If not, these values should be averaged first before being used for statistical analysis.

L299 Section “Phylogenetic analysis”
I suggest the authors to mention here as well the total number of positions of the alignment and the number of informative characters.

L371
Chance “Lichtl” into “Licht”.

L376
Chance “underround” into “underground”.

L386-388 “A name … unavailable”
The authors mention here that a previously used name for this was incorrect and unavailable. Related to this, but not in this line, I noticed that in various of the supplementary files (especially the R scripts) the name “fovouros” was used. I suggest the authors to correct this throughout the documents.

L415-416 “Pilosity and color … ferrugineous-orange”
I was wondering if there is a colour referencing method for insects. For fungi, I know we use the following publication to identify the colour and give it a number code “Kornerup, A., Wanscher, J.H., 1978. Methuen Handbook of Colour. Eyre Methuen, London.”

L574-576 “Escovopsis, … M. mikromelanos.”
While it is commonly assumed that Escovopsis is a parasite, the published evidence in the cited references is far from strong, mostly because this was only tested on young colonies only. Among some of my colleagues we are starting to believe this is an opportunistic commensal fungus that can switch mode to parasitism (following the mutualism-commensal-parasitism continuum) when conditions are right. It is quite striking almost never adult colonies are found severely infected. I suggest the authors chance this to “an assumed micro-filamentous fungal parasite” or similar.

L834-842 “For example, … higher attines”
I have several concerns regarding the cited publication. I helped review the paper in question and feel that our concerns were not taken seriously. The genetic data in the cited paper has been manipulated (completely removing ambiguous regions) in such a way that it lost a lot of species signals in there. Ambiguous regions should have been recoded using one of various available software packages, but comments regarding that were ignored. As a result the naming of clade A (which is a single species) and clade B are possibly misinterpreted, including the placement of some of the taxa. rDNA in these taxa behaves quite strangely, especially in the polyploid Leucoagaricus gongylophorus grown by leaf-cutting ants. The possibility of L gongylophorus having adopted some higher attine fungal nuclei is not discussed or considered. In light of this, I suggest the authors to use this citation and it’s conclusions with caution.

Legends of Fig 2 and Fig 4 (currently Fig 3)
In the legend it says “Scp = Scape” but in the figures there is “Sc”.

Appendix table of literature research
Please, correct the spelling of my name in the corresponding author column: “Pepijn Kooij”


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: USING AN INTEGRATIVE TAXONOMIC APPROACH TO DELIMIT A SIBLING SPECIES, MYCETOMOELLERIUS MIKROMELANOS SP. NOV. (FORMICIDAE: ATTINI: ATTINA)
Review round: 1
Reviewer: 2

Output:
Basic reporting: Thank you for the opportunity to review this manuscript. This paper deals with the description of a new species of the ant genus Mycetomoellerius based on integrative taxonomy tools. The approach employed lend firm support for the species described here. The work is competent, very well written, and pleasingly illustrated. I'm glad to recommend publication under minor revision.
Experimental design: Sadly, I'm not familiar with most of the analyses and techniques employed in this manuscript, but I'm familiar with ant taxonomy and morphology. This is a great contribution to ant taxonomy, especially regarding the classification of fungus-farmings ants.
Validity of the findings: The paper is certainly original. All underlying data have been provided; they are robust, statistically sound & controlled.
Additional comments: I am happy to recommend publication, subject to the author's consideration of the minor points raised in the revised version of the manuscript.


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

Input:
Title: USING AN INTEGRATIVE TAXONOMIC APPROACH TO DELIMIT A SIBLING SPECIES, MYCETOMOELLERIUS MIKROMELANOS SP. NOV. (FORMICIDAE: ATTINI: ATTINA)
Review round: 1
Reviewer: 3

Output:
Basic reporting: The English language is clear. Sufficient literature references are provided. The article structure, text, figures, tables, and all supplementary files comply with the journal standard.
I suggest just minor revisions that don't need to be re-reviewed.
- re-evaluate the use of the terms gyne and queen (text and figures)
- improve the introduction presenting the genus species number, and more details of the history of M. zeteki taxonomy and its occurence
- provide dorsal imagens of all castes
- change the epithet fovouros for mikromelanos (supplementary files)
Experimental design: The entire research project is sufficiently detailed and directed to answer the research question.
Validity of the findings: The findings have scientific validity and suitability, and contribute to the construction of knowledge about the genus Mycetomoellerius and to the use of a multiple approach for the investigation of species potentially involved in groups of cryptic species.
Additional comments: I would like to thank the authors for reviewing this impressive manuscript, which use an integrative approach to overcome the challenges that cryptic species imposes to morphological taxonomy. It should be noted that all the manuscripts of the original descriptions cited taxa are listed in the references. This is not common, but extremely important for the recognition of basic sciences such as taxonomy, especially due to the funding metrics. This recognition of taxonomy is also highlighted in the manuscript when the deposition of vouchers in well-curated and accessible natural history museum collection and the importance of species identification are mentioned.


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: USING AN INTEGRATIVE TAXONOMIC APPROACH TO DELIMIT A SIBLING SPECIES, MYCETOMOELLERIUS MIKROMELANOS SP. NOV. (FORMICIDAE: ATTINI: ATTINA)
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I gladly recommend the paper for acceptance now. The authors have worked well on addressing the comments from myself as well as those of the other two reviewers.

I still have a personal concern with the following statement at L868-870: "For example, higher attine ants
grow two clades of fungi according to several studies (Solomon et al., 2019; Schultz et al., 2015; Jesovnik et al., 2017; Shik et al., 2021; Mueller et al., 2018)."

I disagree with the notion that higher attines grow two clades of fungi. The main single clade includes L. gongylophorus and has recently been divided into two clades without strong support (see cited references) and only a few cases of higher attines growing "lower attine fungi" have been found so far. However, this is all part of the current debate, and I accept that the authors might have different opinions regarding the interpretation of the current phylogenies. I hope I will be able to convince them otherwise in the near future with proof I am currently working on.

I believe I had mentioned this in the previous review, but I would congratulate the authors on the variety of techniques used to identify these species, because I believe that this is the way modern taxonomy should be done.

Best regards
Pepijn Kooij


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: USING AN INTEGRATIVE TAXONOMIC APPROACH TO DELIMIT A SIBLING SPECIES, MYCETOMOELLERIUS MIKROMELANOS SP. NOV. (FORMICIDAE: ATTINI: ATTINA)
Review round: 2
Reviewer: 2

Output:
Basic reporting: After carefully comparing the previous version with the present one and following the rebuttal letter provided step-by-step, I am convinced that authors have fully addressed all the issues pointed out in my revision. Also, I now understand that several of the problems I raised regarding the figures were artifacts of the software I was using to open them, and I apologize for that. The paper can certainly be published in this new format.
Experimental design: Ok.
Validity of the findings: Ok.
Additional comments: You have done a wonderful job joining different and informative tools to delimit this new species. Thanks for being such an inspiration for different generations of myrmecologists, old and new!


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: EXPRESSION OF THERMOPHILIC TWO-DOMAIN LACCASE FROM CATENULOPLANES JAPONICUS IN ESCHERICHIA COLI AND ITS ACTIVITY AGAINST TRIARYLMETHANE AND AZO DYES
Review round: 1
Reviewer: 1

Output:
Basic reporting: This paper describes a newly characterized two-domain laccase from аctinobacteria Catenuloplanes japonicas. This laccase is able to decolorize triarylmethane and azo dyes and  could be considered as a promising enzyme for biotechnological applications because of its high thermostability, рН-stability, ability to oxidize different phenolic substrates and resistance to inorganic ions at alkaline conditions.

In general, the information enclosed in this work is original and valuable.
The introduction and the background are all right and show context. The literature is well referenced and relevant. The manuscript structure follows to PeerJ standards, and the raw data supplied is satisfactory.

Nevertheless the English language should be improved. Some examples where the language could be improved include lines 57-59 and 86-90, the current phrasing makes comprehension difficult.
Figures are relevant, high quality, and generally well labeled.
Experimental design: The primary research is within the Scope of the journal.
The Research question is well defined and relevant.

The methods are described with sufficient detail and information to replicate.
Validity of the findings: The impact of the findings is assessed.

All underlying data have been provided and they are robust. However statistical analysis of data is not mentioned in material and methods, neither in results. The number of assay replicates should be mentioned and standard deviation added.

Conclusions are well stated and linked to original research question.
Additional comments: Line 27:  “inhibitors of classical fungal laccases (azides, fluorides), Low redox potential was found…
Please replace the comma before “Low” with a period.

Line 38:    “ A novel two-domain recombinant laccase CjSL turned out…”
The term “turned out” in this phrase is inadequate, please write it in another way.

Line 43-44:  “The pH optima of CjSL for oxidation of inorganic and organic substrates were 3.6 and 9.2 respectively”
Please replace with: “The pH optima of CjSL for oxidation of ABTS and 2,6-DMP substrates were 3.6 and 9.2 respectively”

Lines 56-60
“It contains four copper ions in the active site. Four copper atoms arrange into three types of metal centers (T1, T2 and T3) take part in catalytic act (Solomon et al., 1996;  Bento et al., 2005). Meanwhile, four electrons which are necessary for complete reduction of oxygen to water laccase are subsequently taken away from the substrates (a wide range of organic and inorganic compounds) (Morozova et al., 2007).”
This sentence is not clear, please rephrased it.

Line 69      Only bacterial laccases “can” contain two or three structural and functional domains.
Please delete “can”.

Line 78          It was used (in complex with mediator) in detoxification of dyes.
Please replace with: They are used (in complex with mediator) in detoxification of dyes.

Line 83         members of this group, searching, obtaining and investigation of new two-domain
                     proteins…
Please replace with:  members of this group, searching, obtaining and “investigating” of new two-domain proteins…

Line 94  “Microorganism, cloning of cjsl gene”
Modify to: Microorganism, “and” cloning of cjsl gene

Line 112    “plasmid pQE/cjsl”
Please replace plasmid pQE/cjsl with  pQE::cjsl

Line 120
Why do you use such long induction times?

Line 162  “putative substrates by detecting the changes in the absorption spectrum after a 24-h incubation”.
Please replace 24-h with 24 h

Line 163   Compounds were assayed at pH 3.6 or pH 9.2 for non-phenolic and phenolic compounds…
Please replace with: “The substrate specificity was” assayed at pH 3.6 or pH 9.2 for non-phenolic and phenolic compounds…

In Parragraph 162-173 (and all over the ms)
Please replace period with comma in substrate names, for example: 3.4.5-trimethoxyphenol with 3,4,5-trimethoxyphenol

Line 173 The substrate concentration of 1 mM was used.
Please replace with: The substrate concentration used was 1 mM.

Line 203     An experimentally shown structure of another trimeric…
Please replace with: An experimentally determined structure of another trimeric …

Line 257 At pH 9.2, “NaN3 decreased” the laccase

Line 275 The purified laccase could efficiently decolorize only azo dye Congo Red at pH 4.0 when
taken alone.
Please replace with: The purified laccase “alone” could efficiently decolorize only azo dye Congo Red at pH 4.0.

Line 293     In this study, we cloned “a” new laccase gene from Catenuloplanes japonicas…

Line 296    … copper center only in the second domain. In “the” structural aspect,

Line 301        … elevation of aerobic induction temperature up to 22°C, resulted in increase...
Induction conditions are not coincident between this line and lines 120-121.  Was the induction temperature 22°C or 18°C and 25°C? Please clarify the situation.

Lines 351-355   The grammar of this sentence should be improved.

Line 379    Fungal “laccases are” known to

Table 2:
1 unite quals = 1 unit equals??

Table 4:
Where no data available, replace  – by c.

Table 5:
Replace: Positive amino acids (with histidine) with Positive amino acids (K,R with/plus H)

Replace: Negative amino acids (with tyrosine) with Negative amino acids (D, E with/plus Y)

Replace Pka with pKa

Table 1-4  The reported values are the mean of how many replicates? Add this info to mat and methods, and in table legend. Please add standard deviations.

Figure 1:
Replace 2.6-DMP with 2,6-DMP

Figure 2:
Replace comma with dots

Figure 3A  Lines labels are missing. Please explain which line corresponds to each substrate.

Figure 4 and 5: How many replicates did you do? Please add error bars.

Figure 5: Please explain what K means. Control?

I believe that with this corrections, and the improvement of the English language and grammar the manuscript will increase its quality and would be suitable 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: EXPRESSION OF THERMOPHILIC TWO-DOMAIN LACCASE FROM CATENULOPLANES JAPONICUS IN ESCHERICHIA COLI AND ITS ACTIVITY AGAINST TRIARYLMETHANE AND AZO DYES
Review round: 1
Reviewer: 2

Output:
Basic reporting: without comments
Experimental design: without comments
Validity of the findings: without comments
Additional comments: The work is a very good contribution regarding the type of laccase obtained for various biotechnological applications. The authors cloned the laccase from Catenuloplanes japonicus in Coli.
The laccase obtained has high thermostability, рН-stability and the ability to oxidize a wide range of different phenolic substrates and resistance to inorganic ions at alkaline conditions. Also, is able to decolorize some dyes. The manuscript is well organized and structured. The bibliography is current. The methodology is very well described and complete.
The results are thoroughly analyzed and integrated. I have no comments about the manuscript.


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

Input:
Title: GENOME-WIDE ANALYSIS, IDENTIFICATION, EVOLUTION AND GENOMIC ORGANIZATION OF DEHYDRATION RESPONSIVE ELEMENT-BINDING (DREB) GENE FAMILY IN SOLANUM TUBEROSUM
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1. English language and manuscript write-up is of sufficient quality.
2. The authors provided up to date literature according to the background of the study
3. The MS is written and organized well. All the figures and tables are of good quality and understandable. The raw is shared and provided in supplementary files
4. No comment
Experimental design: 1. The paper fall in aim and scope of journal
2. The research question is clear and authors identified the knowledge gaps.
4. No comment
5. Methods are described very well.
Validity of the findings: 1. The research have novelty as very few findings published in this area.
2. All the data is analyzed statistically very well
3. The conclusion can be improved more
4. no comment
Additional comments: The work of Mushtaq and colleagues presents interesting results on Genome-wide analysis, identification, evolution and genomic organization of Dehydration Responsive Element Binding (DREB) gene family in Solanum tuberosum, and something important to highlight, is the importance of DREB gene family in abiotic stresses.
I couldn´t find any limitations. Perhaps the authors limit the role of DREB in abiotic stresses. The strengths of the work are the novelty of results, bioinformatics, good introduction, and in general, the ms. is well written

Comments
1.	In abstract line 35 ‘S. tuberosum’ should be Solanum tuberosum’ and line 46’ S. lycopersicum’ should be ‘Solanum lycopersicum’
2.	In abstract section line 40 word ‘strengthened’ to be replaced by ‘Supported’
3.	The concluding sentence, line 50, can be written like this, this study provides a comprehensive and systematic understanding of precise molecular mechanism and functional characterization of StDREB genes in abiotic stress responses and will lead to improvement in S. tuberosum
4.	The introduction can be written more better like include some sentences on gene duplication and evolution.
5.	In result section in sentences structure ‘the’ is use too many times. Remove unnecessary ‘the’ from sentences.
6.	In discussion section few more (2-3) relevant references aligned with the subject study can be added.
7.	In Table 1, the different colors represent what ? Indicate it to make it more meaningful
8.	English language and manuscript write-up is of sufficient quality. The quality of figures is appropriate.
9.	The data underlying the study is significantly provided in article and supplementary files.
10.	The manuscript has a good correlation between the objectives, methodology and results.


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: GENOME-WIDE ANALYSIS, IDENTIFICATION, EVOLUTION AND GENOMIC ORGANIZATION OF DEHYDRATION RESPONSIVE ELEMENT-BINDING (DREB) GENE FAMILY IN SOLANUM TUBEROSUM
Review round: 1
Reviewer: 2

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

Intro need to be improved&.
Literature well referenced & relevant.

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

Figures are relevant, high quality, well
labelled & described.

Raw data supplied
Experimental design: Original primary research within Scope of
the journal.

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

Rigorous investigation performed to a
high technical & ethical standard.

Methods described with sufficient detail &
information to replicate.
Validity of the findings: There are literature present on DREB but rare in S. tuberosume

All underlying data have been provided;
they are robust, statistically sound, &
controlled.

Conclusions are well stated and can be improved more, linked to
original research question & limited to
supporting results
Additional comments: The study has worked out the Genome-wide analysis, identification, evolution and genomic organization of Dehydration Responsive Element Binding (DREB) gene family in Solanum tuberosum. The work was taken up in StDREB gene family was comprehensively analyzed using bioinformatics approaches. The study is of general interest and not providing any novel information which can be exploited in crop improvement.
Comment 1: I have concern that did the studied was taken under any abiotic stress ?
Comment 2: A lot of literature is present on DREB family so please update literature throughout.
Comment 3: Do you perform identification of cis-acting regulatory elements in StDREB promoters?
Comment 4: Did you include the transcriptional level of the StDREB Genes? If yes then provide if not then why?
Comment 5: Did you compare the DREB family composition between S. tuberosum and other species? If not then why?
Comment 6: The discussion can be improved more.
Comment 7: Check English spellings and sentences throughout

Overall conclusion: The paper is well drafted and I will accept it after incorporation of these modification


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: GENOME-WIDE ANALYSIS, IDENTIFICATION, EVOLUTION AND GENOMIC ORGANIZATION OF DEHYDRATION RESPONSIVE ELEMENT-BINDING (DREB) GENE FAMILY IN SOLANUM TUBEROSUM
Review round: 1
Reviewer: 3

Output:
Basic reporting: The article is well drafted, clear and professional English used throughout

The references need to be updated

The article is organized well and raw data is also provided

No comment
Experimental design: The article falls in aim and scope of the journal
The research question is well defin3ed, relevant and meaningful. The knowledge gaps are already identified
The research is performed under high technical and ethical standard

The methods are described well self explanatory
Validity of the findings: The literature is already present on DREB family in different crops but scarce in potato
All data has been provided and is robust, statistically sound and controlled
Conclusion need improvement
No comment
Additional comments: In this article the authors comprehensively analyzed StDREB gene family  using  bioinformatics approaches which is quite scare in potato crop. The article is well drafted and organized, however I have few comments to improve the article more understandable for readers. Abstract is well written. In introduction the last paragraph need drastic improvement (Line 123-130). The same sentences were repeated many times. A lot of information is present on DREB family, so update your references accordingly.  Materials and methods are well drafted. Results are also written well. However discussion need improvement. Do not repeated the sentences again in the discussion. Conclusion also need improvement and only report your main findings.


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

Input:
Title: GENOME-WIDE ANALYSIS, IDENTIFICATION, EVOLUTION AND GENOMIC ORGANIZATION OF DEHYDRATION RESPONSIVE ELEMENT-BINDING (DREB) GENE FAMILY IN SOLANUM TUBEROSUM
Review round: 2
Reviewer: 1

Output:
Basic reporting: Article is okay now
Experimental design: Article is okay now
Validity of the findings: Article is okay now
Additional comments: All the comments were incorporated by the authors and I have no other 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: GENOME-WIDE ANALYSIS, IDENTIFICATION, EVOLUTION AND GENOMIC ORGANIZATION OF DEHYDRATION RESPONSIVE ELEMENT-BINDING (DREB) GENE FAMILY IN SOLANUM TUBEROSUM
Review round: 2
Reviewer: 2

Output:
Basic reporting: Improved by the authors
Experimental design: No comment
Validity of the findings: Improved by the authors
Additional comments: I read the manuscript after the addition of comments and I have no comments. The article is now good


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: GENOME-WIDE ANALYSIS, IDENTIFICATION, EVOLUTION AND GENOMIC ORGANIZATION OF DEHYDRATION RESPONSIVE ELEMENT-BINDING (DREB) GENE FAMILY IN SOLANUM TUBEROSUM
Review round: 2
Reviewer: 3

Output:
Basic reporting: No more comments. The article is substantially improved
Experimental design: No more comments. The article is substantially improved
Validity of the findings: No more comments. The article is substantially improved
Additional comments: The article is substantially improved and I have no more comments. Now it is 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: INTRA-SUBJECT SAMPLE SIZE EFFECTS IN PLANTAR PRESSURE ANALYSES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is fairly clear, well organised, and self-contained. The plantar pressure data is publicly-available and easy to access.

Regarding the cited literature, it is generally good. However, I would also add the following paper to the references:

* Keijsers, N. L. W., Stolwijk, N. M., Nienhuis, B., & Duysens, J. (2009). A new method to normalize plantar pressure measurements for foot size and foot progression angle. Journal of Biomechanics, 42(1), 87–90. doi:10.1016/j.jbiomech.2008.09.038

Note Fig. 4 in that paper and the discussion thereof. It uses intra-class correlation to estimate sample sizes for plantar pressure studies and is therefore highly relevant.

Regarding manuscript clarity, there are some clarity issues in the data analysis section. Most notably, it is not clear when the authors intend to use what they call simple metrics, and when they use vector analysis. In particular, it is unclear when pSPM is used. Also, equation 1 has some formatting problems (I assume that the pixel number, k, should be in brackets and noted on the sum).
Experimental design: The research question is clear and meaningful. The design of the experiments is generally good. I am happy to see the authors using statistical bootstrapping techniques to evaluate different sample sizes. I believe that that is the right way go do. I am also happy to see the authors evaluating sample sizes at different walking speeds.

My one criticism is that the authors aggregate their results across the entire plantar surface in equation 1. In quantitative plantar pressure analyses, it is very rare to aggregate results over the whole plantar surface. Usually, results are presented per pixel or aggregated over regions of interest. By reporting a single result over the whole plantar surface, as is done in this manuscript, it becomes difficult to interpret what impact the sample sizes have on traditional plantar pressure analyses. For example, I would expect that a region-of-interest plantar pressure study might require less samples than a pixel-by-pixel study because the within-region variability gets discarded before the statistical analysis is performed. Also, the paper I mentioned earlier by Keijsers et al. suggests that different foot regions require different numbers of samples in order to get reliable estimates of the mean peak pressures. If my study were to focus only on, say, the hallux, would I still need 400+ samples? Given the way the experiments were setup in this paper, I cannot get a clear answer to this question.

I feel that the paper would be much stronger if the authors were to report their sample size results on a per region or per pixel level. This way, I can easily determine how many plantar pressure samples I should collect based on the foot region I’m interested in and the type of statistical analysis I intend to perform. The current results do not make that easy to figure out.
Validity of the findings: The results presented agree with what I would expect and their interpretation is performed properly. I am happy to see that the authors have listed the limitations of their study, particularly the use of the treadmill for measurement and the choice of pressure metric.

The one criticism I have is on the discussion of the sample sizes recommended in this study. The authors note in their discussion section that multiple studies have recommended between 8-30 samples being necessary for reliable mean plantar pressure estimates. The same can be said for the work of Keijsers et al. that I mentioned earlier. However, the authors propose that around 400 samples are required. This is a difference of an order of magnitude. It would be nice if the authors could comment on why there is such a large discrepancy between their results and those from previous studies. Is it because of the choice of pressure metric, the choice of analysis technique (e.g. region-of-interest, per pixel), the use of the treadmill, the reliability of the sensors, or something else? Some extra discussion here would be very helpful.
Additional comments: Overall, I am very enthusiastic about the paper and would like to see it get published. That being said, I feel that the paper can be made much stronger by (a) reporting results at either the per region or per pixel level, and (b) further discussing the sample size discrepancy between their results and those in previous studies.