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

Input:
Title: A COMPREHENSIVE MOLECULAR PHYLOGENY OF GEOMETRIDAE (LEPIDOPTERA) WITH A FOCUS ON ENIGMATIC SMALL SUBFAMILIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comments. See my full review.
Experimental design: See full review.
Validity of the findings: See full review
Additional comments: I read the manuscript 1.5x: my attention flagged in the taxonomically arcane Discussion on the second read. The science is sound. It represents an enormous amount of work and is abundantly worthy of publication. Foremost, the data set is among the largest and most comprehensive molecular study of a larger lepidopteran group, only data for butterflies might be richer at this point. Over a thousand taxa were sequenced, representing all but one world subfamily (Orthostixinae) and 93 tribes. The geographic coverage, while still rather incomplete for parts of Africa, Southeast Asia, and Australia, is outstanding. The eleven genes represent an enormous character set, of proven utility for reconstructing lepidopteran phylogenies. The phylogenetic analysis is state of the art, especially for such a large number of OTUs. The figures are handsome and a great asset to the manuscript.

One aspect of the study that gives the work extra merit is that many of the authors are among the most active and widely renown geometrid workers. Six of the authors have previously published on the systematics of the Geometridae.

Other commendable aspects of the work include the image album for (Archiearinae, Desmobathrinae and Oenochrominae), the numerous supplemental documents, and the comprehensive list of taxonomic changes organized into a single table.  The ICZN rules (and recommendations) and other nomenclatural details appear to have been adhered to.

The authors did a nice job of “folding in” morphological characters (~synapomorphies) into the Discussion. The morphological supplement is a nice addition.

The manuscript is ultradense in taxonomy and as a consequence will not attract readership outside of a small sphere of lepidopterists. But broad appeal has little to do with the importance or value of science—this manuscript will have a very long “shelf life” and can be used as a road map to guide future fieldwork, phylogenetic studies, geometrid classification, and revisionary work.

My only criticism is that the manuscript is still in need of editing and or minor re-tweaking of sentences that are awkward. Someone with strong grammatical skills and or keen eyes could help the effort. A couple different sets of eyes wouldn’t hurt. My Ph.D. student (Kevin Keegan) and I highlighted or annotated issues in the Abstract, Introduction, Methods, Results, and Conclusion, and then only sparsely elsewhere. Annotations have been made in two pdfs, which I will return.  Simple yellow-highlighting connotes a place where the team may want to revise. I did not attempt to edit the entire manuscript…that would and could be done much more efficiently with a Word file. (Totally pain with pdfs.)

Be forewarned that the Abstract embedded in the manuscript (beginning on line 33) is in better grammatical shape than the one that appears first in the pdf– that one should be jettisoned.

Minor matters:
Line 35:  use familial and tribal as your adjectives, throughout text (do global searches): family/familial and tribe/tribal are mixed in present text

I prefer use of the Oxford comma in scientific writing, especially when the elements of a series are themselves compound.  Not sure of the PeerJ policy.  Oxford commas absent here.

Line 94-104:  I am not a fan of claim staking which is largely what is being done here. Besides these claims and mention of manuscripts in prep reappear in the Discussion and some mentions again in the Conclusions. Geesh.

Line 177 needs redrafting. Not sure what is being said.  Wasn’t C01 used in some identifications? If so, say so.

1008.  Concluding sentence: “For this taxon and many tribes – old and new – we encourage morphological studies that attempt to find more apomorphies and that include a broader range of taxa.”
Not clear what this taxon refers to as several genera were mentioned in previous sentence. Also while the sentiment in the last sentence is good, the sentence is awkward or uninspiring.

I could suggest as a seed for something a little different.
“We hope the phylogenetic hypotheses shared here will open new paths of inquiry across the Geometridae. Morphological synapomorphies have not yet been identified for many of the re- and newly defined higher taxa circumscribed by our 11-gene data set. Likewise, there is great need, across the family, to begin the work of mapping behavioral and life history attributes to the clades identified in this work.”


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

Input:
Title: A COMPREHENSIVE MOLECULAR PHYLOGENY OF GEOMETRIDAE (LEPIDOPTERA) WITH A FOCUS ON ENIGMATIC SMALL SUBFAMILIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is written in clear, professional English, but would benefit from careful proof-reading by a native speaker (I've included numerous edits in the attached PDF). Literature is well cited and the context of this paper and taxonomic history of the groups are clearly spelled out. The format of the article conforms to the general structure of a scientific paper, figures, tables and supplementary files are well done and the sequence alignment ("raw data") is shared. A table with GenBank numbers is included, although it still has to be populated for the newly generated sequences. Overall, the amount of detail provided is actually exemplary! The entire publication is self-contained, with the hypotheses postulated in the introduction addressed and answered in the discussion and conclusion; more detailed analysis and taxonomic work has been shifted into separate / pending publications, which is to the benefit of this paper.
Experimental design: The manuscript is well within the aims and scope of this journal, addressing the higher evolutionary relationships between genera, tribes and subfamilies of the mega-diverse moth family Geometridae through molecular phylogenetics. It also implements taxonomic changes based on the results of this study, which is laudable. The need for this kind of study is discussed in the introduction and self-evident from the strong results. The taxon sampling is much broader than typical in the field and was carried out with preference for type species, which is an important aspect often ignored by other studies. Overall, this study is a milestone that greatly advances our understanding and the taxonomy of geometrid moths.

Taxonomic sampling, analyses of molecular data and taxonomic acts are all carried out to a high technical standard. At the same time, some additional data analyses would have been desirable as described below.

The methods and material / sampling is described with great detail and sufficient for replication of the experiment.
Validity of the findings: This study is the by far largest phylogenetic analysis of geometrid moth relationships and greatly advances our understanding of evolutionary relationships due to the unusually large taxon sampling. The methods and data seem robust, with data analyses being statistically sound and well-founded. All conclusions and taxonomic changes are well explained and always linked to and supported by the results. With the manuscript being of a taxonomic nature, it doesn't include speculative elements.
Additional comments: I was very pleased to see that taxonomic and molecular phylogenetic experts teamed up to deliver a very well-rounded research that will be a milestone for the systematics of Geometridae for many years to come. I am particularly impressed by the taxon sampling, the amount of detail provided and the great care taken regarding taxonomic questions. Very well done!

As always, there are some points that could be improved on. While I spell them out, this doesn't mean that the manuscript couldn't be published with no more than some editing by a native speaker - it is good and important as is, with conclusions substantiated by the results! My three points of concern are:

A) Apomorphies in support of the newly defined higher taxa. In all but one case you resort to listing molecular sequence data as diagnostic characters, which might be technically valid, but leaves a taxonomist longing for more. A diagnose should help others to identify unknown taxa as to belong to a taxon like a genus / tribe / subfamily or not. The molecular sequence data listed don't really achieve this, because the data from the other samples are needed, too, and the phylogenetic analyses are as much part of it as the actual sequence data - it's the result of the data analysis that "diagnoses" the species as belonging to a taxon. Furthermore, a diagnosis based on molecular data alone doesn't enable the inclusion / exclusion of other taxa based on morphology, which is the main source of information we have or can readily obtain for many of the taxa not included in your study. For many taxa no molecular-grade specimens are available, which is presumably the reason why you weren't able to include them in the first place. While I'm lamenting the lack of diagnostic morphological characters, I know from own experience how difficult it can be to find such characters and that this might have exceeded the scope of the project. At the same time, looking at the expertise shared between the authors, I wonder who if not you would be in a better position to do so? Hopefully you'll be able to follow up with this one day!

B) The data analyses were carried out carefully and according to current best practice. At the same time, I was surprised to see that only nucleotide data were analysed, mitochondrial and nuclear data combined. Especially for older relationships, it would have been highly advisable to also analyse the data with synonymous changes excluded (i.e., as Degen1 encoded nuclear or amino acid data) and with mitochondrial data excluded. Both synonymous changes and mitochondrial data (20% of this data set) are known to be particularly plagued by compositional heterogeneity due to their rapid evolution, which can mislead phylogenetic analyses. The conflicting result for the placement of Larentiinae (i.e., not as sister of Sterrhinae, but as sister to all remaining Geometridae) seems likely to be a classic case for this problem - more details on this in the comment in the attached text. It's easy to test for (e.g., Euclidean compositional distance tree; SymTest by Lars Jermiin) and easy to counter (e.g., amino acid analysis, recoding with Degen1). Maybe something worth still carrying out to avoid postulating a strongly supported yet possibly / probably incorrect hypothesis - happy to help if need be.

C) Major questions addressed in this paper concern taxa with an exclusive or largely Australian distribution, and places like the Australian National Insect Collection (ANIC) are mentioned in the supplementary text. Yet, as curator of the ANIC Lepidoptera collection, I'm not aware of any of the authors having made an attempt to check our collection (we hold just about every named and unnamed Australian species) or include Australian geometrid experts like Catherine Byrne or Peter McQuillan. While obviously not a must and maybe not always possible, this could have contributed valuable additional information and samples.

In any case, I'm looking forward to seeing the manuscript published - it's a great and important paper as is!


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

Input:
Title: A COMPREHENSIVE MOLECULAR PHYLOGENY OF GEOMETRIDAE (LEPIDOPTERA) WITH A FOCUS ON ENIGMATIC SMALL SUBFAMILIES
Review round: 2
Reviewer: 1

Output:
Basic reporting: As stated in my initial review “the manuscript is still in need of editing and or minor re-tweaking of sentences that are awkward. Someone with strong grammatical skills and or keen eyes could help the effort.” Even though authors state that “the text has been changed accordingly” there is much that could be done. I made about 70 suggestions over the first 16 pages and stopped, Mistakes with punctuation, between versus among, that vs which, subject verb agreement. Some of the new text introduced new problems. Elements of a series that contain commas should be separated with semi-colons. Etc.
Experimental design: Excellent.
Validity of the findings: See below.
Additional comments: The data set is enormous and represents a Herculean effort by an expert team of lepidopterists. Despite the analytical limitations and challenges mentioned by reviewer #2 and the writing mechanics, I don’t see a compelling reason not to move forward with acceptance and publication.

Given reviewer Andreas Zwick’s issues, it might be prudent to add one more sentence to the “Searching strategies and model selection” section of the Results that states some of the general uncertainties/misgivings/limitations about the results, and that this study is a best collective guess with the data at hand/methods. In essence, something that serves as a disclaimer given the reservations of Zwick. And then maybe in the Discussion/Conclusion, near the end, mention (again) what needs to be done in terms of taxon sampling, number of genetic markers, and ?analytical methodology to get a stable, robust higher classification for the Geometridae. This would be another way of conveying that this present effort is simply a best effort with the data at hand.

As stated in my initial review “the manuscript is still in need of editing and or minor re-tweaking of sentences that are awkward. Someone with strong grammatical skills and or keen eyes could help the effort.” Even though authors state that “the text has been changed accordingly” there is much that could be done. I made about 70 suggestions over the first 16 pages and stopped, Mistakes with punctuation, between versus among, that vs which, subject verb agreement. Some of the new text introduced new problems. Elements of a series that contain commas should be separated with semi-colons. Etc.

My misgivings are minor. Given the effort and new data being made available, I am in favor of having the publication go forward with but very minor additions, or even none at


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: EFFECT OF ADJUVANT RADIOACTIVE IODINE THERAPY ON SURVIVAL IN RARE OXYPHILIC SUBTYPE OF THYROID CANCER (HÜRTHLE CELL CARCINOMA)
Review round: 1
Reviewer: 1

Output:
Basic reporting: - The paper states important findings, but needs lot of revision from grammatical standpoint to be considered for "pass" state
- Sentences throughout the paper need to be paraphrased because of grammatical errors
- In discussion section, would suggest further comprehensive research into what is being done to address this issue from future standpoint. Are there any clinical trials to address this?
- Also, figures for OS and CSS combined stage I-II and III-IV, though all stages have different course, as a result would suggest looking at each stage individually to confer exact OS and CSS difference.
Experimental design: - Original and primary research which is addressed within the Aims and Scope of the journal
- Research question well defined, followed by clear evidence in results supporting it.
- Rigorous investigation performed and results stated clearly with the help of table and figures, with valid statistical analysis.
- Methods described well, with sufficient detail and information to replicate.
Validity of the findings: - All underlying data has been provided along with valid tables and figures
- Conclusion needs to be paraphrased like other sections given grammatical errors
Additional comments: Dear Drs. Yang & Zhao,

It was a pleasure reviewing your article on HCC. You stated some new findings, which need to be further looked at in prospective trials to further validate your results. However, before the paper is considered for publication I believe it needs major revision from grammatical standpoint.

There are lot of grammatical errors, as a result sentences need to be paraphrased. In addition to that, I would advise looking at all the AJCC stages separately - stage I, II, III, IV to evaluate the OS and CSS. Also, in the discussion section, would recommend highlighting what is being done at this point from future perspective, that is stating any clinical trials looking at this question, or what is the best way moving forward with this issue.

Regards,
PeerJ Reviewer


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: EFFECT OF ADJUVANT RADIOACTIVE IODINE THERAPY ON SURVIVAL IN RARE OXYPHILIC SUBTYPE OF THYROID CANCER (HÜRTHLE CELL CARCINOMA)
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1-	Use of English is of poor quality throughout the text and needs to be comprehensively revised. If some of the problems are just simple grammar misuse, other sentences are not understandable. Here are a few examples: «receipt of radiotherapy» (line 98); sentence in lines 110-112 need to be rewritten because it is difficult to understand; «Extrathyroidal evasion» (line 114). «At the end of the last follow-up» (lines 130-131); «Patients without receipt of radioiodine», «Reception of RAI therapy» - legend of Table 2 , «the prognosis were better» line 182; sentence in lines 223-224 does not make sense.
2- Manuscript is well organized, with helpful figures and tables.
Experimental design: No comment.
Validity of the findings: 1-	Authors should explain the fact that 7% of patients in the no-RAI group were not submitted to surgery. Taking into consideration that HCC is only diagnosed in histologic analysis, I wonder how the diagnosis was made in those patients. If no explanation can be found, this issue should be discussed.
2-	In the discussion, authors claim that factors associated with increased frequency of RAI therapy may be used in the future to guide the selection of the best candidates for RAI therapy. However, this is a retrospective study aiming at evaluating the survival benefits of RAI in patients with HCC, so the authors cannot claim that those factors should guide our future decisions to perform RAI treatment in patients to HCC. To add to this, performing a total thyroidectomy is a prerequisite to perform RAI treatment in most cases.
Additional comments: In this manuscript, authors evaluated the survival benefit of radioiodine (RAI) treatment in a large dataset of patients with Hurthle Cell Carcinoma of the thyroid gland.
This is a controversial issue, with recent data raising questions on whether HCC patients have poorer prognosis and worse response to RAI when compared with their non-HCC counterparts. Other strengths of the manuscript are: the use of a large well-characterized dataset of patients and the construction of a nomogram for prognosis stratification of patients with HCC, using widely available clinic-pathologic features.
However, authors need to address several points to make the manuscript acceptable for publication. Adding to the points previously mentioned:
1-	Authors systematically consider the married status of the patients in the analysis. The relevance of this should be explained in the discussion section.
2-	Minor issue: The phrase «vital status» is repeated in the same sentence in lines 86-87.


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: EFFECT OF ADJUVANT RADIOACTIVE IODINE THERAPY ON SURVIVAL IN RARE OXYPHILIC SUBTYPE OF THYROID CANCER (HÜRTHLE CELL CARCINOMA)
Review round: 1
Reviewer: 3

Output:
Basic reporting: The text should be improved because in some parts it is not clear and English is not perfect. Please use the list of recommended remarks.
Experimental design: Research question well defined, relevant & meaningful.Rigorous investigation performed to a high technical & ethical standard. Methods described with sufficient detail & information to replicate
Validity of the findings: Improtant findings.
Conclusions should be rewieved.
Additional comments: Quion Yang et al. aim was to find out in large population based research evaluating whether patinents with Hurthle cell carcinoma (HCC) can benefit from postoperative RAI therapy. Their study is interesting. However, there are some issues that have to be clarified before publication in the Peer J.
Major comments:
1.	Primary reference should be cited for the sentence of the Introduction (Paolo et al. is not appropriately cited) - lines 44-46
2.	Lines 100-115: text is not clear. Can you be more married? Please rewrite.
3.	Lines 145-148: State only those that are statistically significant. These are the only one that are important! Omit non-significant factors.
4.	Line 191 Refernce Allia at al. is not appropriate (it describes cell cultures and not HCC in human body)
5.	Line 202 Add reference Petric R et al. (BMC Cancer 2014, 14:777)
6.	Line 223-224: not clear: “It seems that RAI therapy acts absence of survival benefit in the treatment of HCCs patients.”
7.	Conclusions in the abstract section and at the end of manuscript should be as follows:
“In summary, this study suggests that RAI therapy was significantly associated with improved OS in patients with Hürthle Cell Carcinoma. However, there were no association between treatment with radioiodine and CSS, possibly due to small number of deaths that were related to HCC.”

Minor remarks
Lines 58-59: Sentence: “Radioactive iodine (RAI) therapy is known widely to be effective in the treatment of postoperative thyroid cancer.” Should be rewritten as follows: “Radioactive iodine (RAI) therapy is known widely to be effective in the treatment of thyroid cancer after thyroidectomy.”
Line 60 : insert “number of ” as follows: “….there are increasing number of studies……….”
Line 64: insert “patients with” as follows: “…..evaluating whether patients with HCC can benefit from……”
Line 127: instead “choose” use “be treated”
Line 218: instead “confirm” use “evaluate”
Line 221: Cox’s
Line 226 and 227: instead of “limited samples” use “incidents”
Line 238: instead: “solid elucidate” use “evaluate”


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: EFFECT OF ADJUVANT RADIOACTIVE IODINE THERAPY ON SURVIVAL IN RARE OXYPHILIC SUBTYPE OF THYROID CANCER (HÜRTHLE CELL CARCINOMA)
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript has been revised and outcomes and stated clearly now.
Experimental design: Research question is well defined and addressed by providing relevant evidence. Grammatical errors have been corrected, and now the manuscript flows much better.
Validity of the findings: Valid findings which will add to the current science knowledge.
Additional comments: Thanks for updating the manuscript as it flows much better 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: EFFECT OF ADJUVANT RADIOACTIVE IODINE THERAPY ON SURVIVAL IN RARE OXYPHILIC SUBTYPE OF THYROID CANCER (HÜRTHLE CELL CARCINOMA)
Review round: 2
Reviewer: 2

Output:
Basic reporting: This is an improved version of the manuscript, with a better use of English. However, there are a few details that need revision. Here are a few examples:
-	Abstract, line 37: the verb is missing in the first sentence (is??)
-	Introduction, line 43: «.. is a rare…»
-	Resultsl, line 174: «.. seems to be no difference»
-	Discussion, line 215-217: sentence needs to be rephrased
Experimental design: No further comments
Validity of the findings: No further comments
Additional comments: This is an improved version of the manuscript, with a better use of English. However, there are a few details that need revision. Here are a few examples:
-	Abstract, line 37: the verb is missing in the first sentence (is??)
-	Introduction, line 43: «.. is a rare…»
-	Resultsl, line 174: «.. seems to be no difference»
-	Discussion, line 215-217: sentence needs to be rephrased


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: EFFECT OF ADJUVANT RADIOACTIVE IODINE THERAPY ON SURVIVAL IN RARE OXYPHILIC SUBTYPE OF THYROID CANCER (HÜRTHLE CELL CARCINOMA)
Review round: 2
Reviewer: 3

Output:
Basic reporting: Clear and unambiguous, professional English used throughout.
Literature references, sufficient field background/context provided.
Professional article structure, figures, tables are O.K.
Paper is self-contained with relevant results to hypotheses.
Experimental design: Original primary research is within Aims and Scope of the journal.
Research question is well defined, relevant & meaningful. It is stated how research fills an identified knowledge gap.
Iinvestigation performed to a high technical & ethical standard.
Methods dare escribed with sufficient detail & information to replicate.
Validity of the findings: It is the first SEER based study on impact of RAI therapy in more than 2000 patients with Hurthle cell carcinoma. Rationale & benefit to literature is clearly stated. All underlying data have been provided; they are robust, statistically sound, & controlled. Conclusions are well stated, linked to original research question & limited to supporting results.
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: MARKED VARIATIONS IN GUT MICROBIOTA AND SOME INNATE IMMUNE RESPONSES OF FRESH WATER CRAYFISH, MARRON (CHERAX CAINII, AUSTIN 2002) FED DIETARY SUPPLEMENTATION OF CLOSTRIDIUM BUTYRICUM
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Dear Author,

The cumulative body of points indicate that a major revision is needed. There are lots of things that the authors should revised in the manuscript. Also, there are some questions and doubts which authors should address. find my comments and questions in details in the annotated pdf file.

Some comments:

First of all, after checking your gene expression results, I can say that your results would become more important in terms of scientific reality as it would be accepted as statistically significant if the expression rates of probiotic application exceeded 3 fold the rate of the control group. As far as I can see in the results, Probiotic has never increased or decreased the gene expressions 3 fold (at least) comparing to the control. Generally, statistical analyses is not performed if gene expression is not increased by at least 3-fold in scientific manuscript.

There is no clarity on the number of biological replicates used for different analysis, everywhere
it is mentioned “triplicate tanks”, which is not appropriate for parameters other than growth and FCR. Mention specifically the number of individual fishes (N=12) or pooled fish samples (N=4) used for immune parameters, gene expressions.... State this clearly in figure legends.
Only 4 fish samples were used for the immune parameters, gene expression analyses....of the fish. is it enough ?.

Why only intestine tissues were tested for immune genes expression?  hepatopancreas was the most important immune organ for crayfish immunology, and therefore, author needs to validate related gene expression in this organ.

Growth, biochemical compositions, lysozyme, haemolymph osmolality, THC..   statistical informations should be added.

The lysozyme plates were incubated for 20 minutes, but that time can be adequate or not depending on the stopped of the enzyme to perform the lysis. Authors have to justify why they choose this time

The methods and reference of the method used to determine the LD50 in line 174 is not described.
Based on which you select this dosage for injection? LD50 ? Did the diseased crayfish have the signs of vibriosis?. Should be detailed.

Line 104. Culture conditions should be added.
Line 106. Moreover, there is no validation of the actual dietary levels of C. butyricum after dietary incorporation. In fact, the methodology used for dietary
incorporation is not convincing. There are questions related to the preparation of the C. butyricum in fish diet, top dressing of feed pellets without vacuum and final proximate composition of the experimental diets.

Table 1. references and Product size (bp) of the sequences should be added.


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: MARKED VARIATIONS IN GUT MICROBIOTA AND SOME INNATE IMMUNE RESPONSES OF FRESH WATER CRAYFISH, MARRON (CHERAX CAINII, AUSTIN 2002) FED DIETARY SUPPLEMENTATION OF CLOSTRIDIUM BUTYRICUM
Review round: 1
Reviewer: 2

Output:
Basic reporting: Yes, reporting is very thorough.  Figures can be improved.
Experimental design: Yes; see below for more detailed comments.
Validity of the findings: Yes; see below for more detailed comments.
Additional comments: Marked variations in health status, gut microbiota and innate immune responses of marron (Cherax
cainii, Austin 2002) fed dietary supplementation of Clostridium butyricum (#34753)
by Foysal et al, PeerJ submission 34753.

The manuscript by Foysal et al describes the use of Clostridium butyricum as a probiotic supplement for marron, Cherax cainii, an increasingly important farmed crawfish in Australia.

This probiotic is a naturally found, acid producing bacteria, that resides in the gut of diverse animals and has been increasingly used as a probiotic in farmed organisms. Previously, the use of this probiotic has been shown to help reduce colonization by species of Vibrio and Aeromonas in fish, both of which are also known pathogens among crustaceans. The authors do admit, however, that in marron Vibrio are not yet known to cause disease (line 71).

Major points:

1.The authors demonstrate a correlation between molting and weight gain. Does age of the marrons factor in as well? Do the authors have some controls to take the age in consideration in these experiments.
2.In certain studies, the probiotic benefits of C. butyricum has been attributed to its ability to synthesize butyric acid (Duann et al 2017). Does this study explore the role of butyric acid in contributing to the probiotic effects of C. butyricum. Does modulation of fat synthesis by the probiotic lead to weight gain?
3.Authors demonstrate up-regulation of immune responsiveness induced by pre-exposure to probiotic followed by challenge with a potential pathogen.  Can the results of these studies be supported further by survival assays?

line-by-line points:

line 58: define the type of acid produced.
line 71: remove ",however,"
line 76: change "with recently developed high-tech stuffs" to something else like, "with modern technologies such as"
line 93: this statement is not clear -- "in three randomly replicated tanks for each feeding treatment," which makes it unclear to the reader how samples were "pooled" as in line 151
line 112 (Dissolved Oxygen) - spelling mistake
line 223, Provide reference for growth techniques - It isn't mentioned in the said reference - Ambas, Fotedar 2016. Ramirez et al 2017 defines the same growth charachteristics with a different equation. Which one would be justified to use?
line 127: cacpodylate is misspelled; should read "cacodylate"
line 127: this is unclear to me.  If the anticoagulant possesses fixative qualities, how do any of the cells or enzymes remain active for the assays?
line 135: please explain this assay a bit better; I believe that this solution of Micrococcus is a suspension of cell membrane components.  Maybe explain to the reader how a turbidimetric assay works in a short statement.
line 155: NanoDrop does not really tell you much about quality (as a bioanalyzer would)
line 173: how were the 8 marron selected?  Randomly?
line 173-74: a technical question, but why was the challenge bacteria not fed the way the probiotic was and instead injected?  Under crowded conditions of farming, wouldn't the more natural exposure be an oral one, although certainly exposure can come via injury as well.
line 180: change "grinded" to "ground"
line 193 (onwards) for the Bioinformatics, when it is said that "pipelines" are used, were they used with default values? If so, please state it explicitly in the beginning of the section and then it will not be necessary to repeat it.
line 252-from here and discussion section:  the authors should speculate on how it is possible that the use of a single probiotic species can somehow increase diversity in the gut? For example, enrichment of 3 specific genera as stated in line 270
line 318: it is suggested that probiotic supplementation increased abundance of the Clostridium genus.  But I'm assuming it should only increase abundance of the specific probiotic?  Unless the probiotic preparation is contaminated with other species?  Otherwise, how then (as elsewhere in the paper), how does the presence of the probiotic help to increase overall microbial diversity in the gut?
line 336 (and section, line 268): please make it clear that Piphillin is a program that makes predictions on the presence of functional pathways based on the composition of the microbial communities present.  And it does better of course with metagenomic data. For example, in line 337 is should be said "the analysis predicted significantly higher protein and energy metabolism" rather than saying that the analysis revealed...

Figures: overall the figures need to be improved.  The font is barely legible. Overall, the legends can be improved.
Figure 1 (A) is unclear.
Figure 5: in A and B, it seems easier if the colors were consistent, for example, if probiotic is RED in A, then it should be RED in B.
Figure 6: it is unclear why the dendrogram is needed in this figure.  What does the grouping tell us?


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: MARKED VARIATIONS IN GUT MICROBIOTA AND SOME INNATE IMMUNE RESPONSES OF FRESH WATER CRAYFISH, MARRON (CHERAX CAINII, AUSTIN 2002) FED DIETARY SUPPLEMENTATION OF CLOSTRIDIUM BUTYRICUM
Review round: 1
Reviewer: 3

Output:
Basic reporting: The language used is largely acceptable with clear technically correct text.
Introduction and discussion has been written nicely with relevant references and sound understanding of the subject.
The paper has been made more authentic by providing raw data set. The table, figures are appropriately described and depicted.
Overall the paper concludes with clear significant results.
Experimental design: The experiment has been designed with proper hypotheses to generate data to fill the knowledge gap. The experiments have been done conforming to the highest standards.
Methodology has described in detail for easy replication.
Validity of the findings: Clear results are obtained through robust data with excellent statistical analysis using latest bioinformatics tools available. Thus conclusions are well supported by results.
Additional comments: This is an excellent piece of work with lot of novelty in experimental design and execution. A very significant contribution has been made to support sustainable marron culture in Australia.


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: MARKED VARIATIONS IN GUT MICROBIOTA AND SOME INNATE IMMUNE RESPONSES OF FRESH WATER CRAYFISH, MARRON (CHERAX CAINII, AUSTIN 2002) FED DIETARY SUPPLEMENTATION OF CLOSTRIDIUM BUTYRICUM
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
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: MARKED VARIATIONS IN GUT MICROBIOTA AND SOME INNATE IMMUNE RESPONSES OF FRESH WATER CRAYFISH, MARRON (CHERAX CAINII, AUSTIN 2002) FED DIETARY SUPPLEMENTATION OF CLOSTRIDIUM BUTYRICUM
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have adequately addressed my concerns.
Experimental design: The experimental design is much clearer now.
Validity of the findings: The findings are robust.
Additional comments: Thank you for addressing these comments and concerns.


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

Input:
Title: PREVALENCE OF NEAR-DEATH EXPERIENCES IN PEOPLE WITH AND WITHOUT REM SLEEP INTRUSION
Review round: 1
Reviewer: 1

Output:
Basic reporting: Well constructed throughout, logical, good standard of English with appropriate supportive evidence.

Figures and tables relevant to the work. Figures highlighting some statements made by participants is fascinating and clearly upholds the claims made within the paper.

Line 68 and line 240 have unneeded hyphens between age- and sex-matched. These should be removed.
Experimental design: An interesting design using crowdsourcing, although limitations of this noted. Well described in terms of detail.

One question would be why you opened the survey for only 3.5 hours, as I read it (line294)? It would be beneficial to advise the reader why this decision was made as it appears to be a very short time frame to capture relevant data/allow participants to complete the survey especially given the global context and different time zones.

Unsure if using a survey dismisses incentive bias to please the investigator as with all surveys, the majority of participants have some engagement with the subject already but you have evidenced your comment.
Validity of the findings: Appropriate use of analysis tools bringing to the fore an obviously notable outcome in terms of statistics. Appears statistically sound.

Conclusion well drawn and consideration given to other outcomes or influences on the work.
Additional comments: A very interesting paper and was a pleasure to read.

I agree with regard to the positive nature of the GNDES and it would be good to be able to develop a new tool that offers the less positive experiences that some have demonstrated in your work.


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: PREVALENCE OF NEAR-DEATH EXPERIENCES IN PEOPLE WITH AND WITHOUT REM SLEEP INTRUSION
Review round: 1
Reviewer: 2

Output:
Basic reporting: Clear and correct english.
Some references can be added to strengthen the work
Tables and figures are acceptable
Experimental design: Ethical standards are duly fulfilled, however, technical standards are poor, since the information is obtained through an on-line survey.
Validity of the findings: Results must be handled as speculation and this must be identified as such
Additional comments: General Aspects
The present study depicts relevant information regarding the propensity of REM sleep intrusion in association to near-death experiences. However, the omission of arousal disorders weakens the strength of the work and of the data presented. Another limitation is that frequency and temporality of near-death experiences and REM intrusion is unknown, it is not known which of them occurred for the first time, so the results describe both phenomena barely. Authors should clarify if REM sleep intrusion is properly classified by the AASM. It is unclear if the intrusion of REM is different from sleep paralysis and if this phenomenon occurs only accompanied by narcolepsy, sleep paralysis may occur in non-narcoleptic subjects. Statistical analyzes have been well performed and are adequate. It would be interesting to know if subjects after the near-death experience present neurological damage. Sociodemographic data is the most interesting part of the work. Authors should have included in their instrument / scale / questionnaire more sleep parameters items, such as: diagnosed narcolepsy, epilepsy, sleep debt, insomnia, voluntary sleep deprivation, usual amounts of sleep, napping or not, apnea , etc. This would have strengthened its methodology.Authors should add information that supports or strengthens their work.
There are some aspects of the study which need to be revised.
Introduction
Lines 60 to 62, REM sleep definition must have a reference
Line 62, REM as a whole state cannot intrude into wakefulness, they are two different states, however, some characteristics of REM sleep can be present during awakening/wakefulness.
Results
Lines 161 to 164, Why were the truly life threatening and the not truly life-threatening not analyzed in independent groups?
Line 168, authors can consider that substance abuse (n = 33) could be removed from the sample. In these subjects the near-death experience must have been accompanied by altered states of consciousness. What was the rationale to include them?
Line 189, What is the evidence for REM intrusion? Is the scale score?
Discussion
Lines 255 to 260, consider to revise and improve this lines. References are correct but authors do not present the information correctly.
Line 256, consider to change “release”
It would have been important to know and describe the actual use of stimulants, alcohol and drugs of the survey participants.
Line 296 to 297, I ask the authors what are these different results?
Line 297 to 298, “sleep studies…….would have been impractical”. Authors should be more careful to make that statement, since the data that you present is obtained from subjective reports. Polysomnography is currently the gold standard for assessing sleep and it would have been desirable for the present or future studies,and a statement regarding this subject should be addressed in the limitations section.
I would recommend that authors can review the following references and if possible that some of them could be added to the manuscript:
Serotonin neurons in the dorsal raphe mediate the anticataplectic action of orexin neurons by reducing amygdala activity. Hasegawa E, Maejima T, Yoshida T, Masseck OA, Herlitze S, Yoshioka M, Sakurai T, Mieda M. Proc Natl Acad Sci U S A. 2017 Apr 25;114(17):E3526-E3535. doi: 10.1073/pnas.1614552114.
A clinician's guide to recurrent isolated sleep paralysis. Sharpless BA. Neuropsychiatr Dis Treat. 2016, 19;12:1761-7. Review
Sleep Paralysis, "The Ghostly Bedroom Intruder" and Out-of-Body Experiences: The Role of Mirror Neurons. Jalal B, Ramachandran VS. Front Hum Neurosci. 2017 Feb 28;11:92. doi: 10.3389/fnhum.2017.00092.
Sleep paralysis and "the bedroom intruder": the role of the right superior parietal, phantom pain and body image projection. Jalal B, Ramachandran VS. Med Hypotheses. 2014 Dec;83(6):755-7. doi: 10.1016/j.mehy.2014.10.002.
Sleep-related epileptic behaviors and non-REM-related parasomnias: Insights from stereo-EEG. Gibbs SA, Proserpio P, Terzaghi M, Pigorini A, Sarasso S, Lo Russo G, Tassi L, Nobili L. Sleep Med Rev. 2016 Feb;25:4-20. doi: 10.1016/j.smrv.2015.05.002.
Frequent lucid dreaming associated with increased functional connectivity between frontopolar cortex and temporoparietal association areas. Baird B, Castelnovo A, Gosseries O, Tononi G. Sci Rep. 2018 Dec 12;8(1):17798. doi: 10.1038/s41598-018-36190-w.
Neurobiological mechanisms for the regulation of mammalian sleep-wake behavior: reinterpretation of historical evidence and inclusion of contemporary cellular and molecular evidence. Datta S, Maclean RR. Neurosci Biobehav Rev. 2007;31(5):775-824. Epub 2007 Mar 12. Review.


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

Input:
Title: PREVALENCE OF NEAR-DEATH EXPERIENCES IN PEOPLE WITH AND WITHOUT REM SLEEP INTRUSION
Review round: 1
Reviewer: 3

Output:
Basic reporting: The authors in this study investigated: (i) the frequency of near-death experiences and REM sleep intrusions collected from an online crowdsourcing service; (ii) the correlation between people who report a near-death experience with the frequency of REM sleep intrusions. They found that from a large sample of 35 countries the prevalence of near-death experiences was 10%. Additionally, they confirmed the association of near-death experiences with REM sleep intrusion.

Results obtained support the main hypothesis of the study.

The occurrence of REM sleep intrusion is very low in the general population, although the vast majority of cases go unrecognized and its prevalence change in relation to age, with the use of antidepressant medications or in the setting of narcolepsy.
However, the authors discussed that " REM intrusion during wakefulness occurs frequently in healthy people" (see discussion section line 250). Please, make a citation on this sentence and also indicated in the discussion the percentage of REM sleep intrusion in the general population compared to your population.

I would ask whether it is possible to define in your population among people who had REM sleep intrusion the kind of medications (i.e antidepressive) used. Additionally, may you determine among people who had REM sleep intrusion people who may suffer from narcolepsy? Please indicate if it is possible this data in the results or makes comments on this.

Additionally, you addressed your hypothesis at the beginning of the section Materials and methods. Please, I would add the hypothesis and research question in the introduction at the of the paragraph.
Experimental design: Research question well defined
However, I do not totally agree how the REM sleep intrusion has been determined.
Validity of the findings: The study confirms the data already published from the previous study (Nelson at al 2006), in which the authors correlated the NDE with REM sleep intrusion.
 However, this study was criticized for several regions including the control group used.
I believe that in this study the association between the NDE and REM sleep intrusion is still speculative since the way how REM sleep intrusion has been determined is limited. To this, I would suggest to the authors to add the word "we speculate..." throughout the manuscript
Additional comments: No additional comments


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

Input:
Title: PREVALENCE OF NEAR-DEATH EXPERIENCES IN PEOPLE WITH AND WITHOUT REM SLEEP INTRUSION
Review round: 1
Reviewer: 4

Output:
Basic reporting: The paper is of interest and adds a relevant piece to the near-death experience (NDE) literature. It assesses the prevalence of NDEs in people presenting or not REM sleep intrusions. I am not a native English speaker, therefore, I cannot judge adequately about the quality of the writing. I will however note that the manuscript seems rather well written, clear and easy to read. Moreover, authors have made all the appropriate raw data available.

The introduction is brief, giving some context for both the study of NDEs and REM intrusion. However, it would benefit the reader to hear more about studies that have examined the incidence and prevalence of such experiences, although some of them were subject to methodological biases. Some of these studies have been mentioned by the authors in the Methods and Discussion, but more might be said about this in the Introduction. In this respect, see the following articles:
-Knoblauch H., Schmied I., Schnettler B. 2001. Different Kinds of Near-Death Experience: A Report on a Survey of Near-Death Experiences in Germany. Journal of Near-Death Studies 20:15–29.
-van Lommel P., van Wees R., Meyers V., Elfferich I. 2001. Near-death experience in survivors of cardiac arrest: a prospective study in the Netherlands. The Lancet 358:2039–2045. DOI:10.1016/S0140-6736(01)07100-8.
-Perera M, Padmasekara G BJ. 2005. Prevalence of Near-Death Experiences in Australia. Journal of Near-Death Studies 24:109–116.
Experimental design: Overall, I believe that this original and meaningful research fits the aims and scope of the journal. Nonetheless, some points need clarification:

>Authors write that they also inquired about unpleasant experiences. Could they detail/specify the question(s) they asked in the methods section?

>Authors claim that they have been inspired by the study by Nelson et al. (2006) which suffered a selection bias, and that the anonymous nature of their online survey enabled the avoidance of a selection bias. However, it seems to me that the title of the survey, which places emphasis on NDEs, could have introduced a selection bias by overestimating the number of participants having experienced a NDE. I would suggest that authors comment on this point.
Validity of the findings: Overall, data are relevant and well analyzed. Nevertheless, t is not clear to me why the alpha level was set at 0.01 in the case of a multivariate logistic regression. Therefore, I wonder whether the authors could comment on that point?
Additional comments: Overall, authors need to check the references in the text which are not always written in a coherent manner.

ASTRACT:
There is an extra space at the end of the Methods part.

MATERIALS AND METHODS:
>Line 110: authors state that the survey ended whenever participants declared not having experienced a NDE. Besides, in Table 1, NDEs are defined as events occurring in life-threatening situations or in situations that just felt so. Therefore, it is not clear to me how they ended up with a sample of experiencers that also included individuals that have experienced a NDE during meditation or prayer. Could authors comment on this point?

RESULTS:
>Line 184: some participants stated having had two or three NDEs or NDEs-like, but can authors ensure that participants only referred to one experience when completing the GNDES? That they have not indicated all the characteristics they have experienced during their various NDEs (which could, in this case, lead to false positives on the GNDES)?
>Line 190: I recommend using the acronym GNDES, as it was previously done in the manuscript.

DISCUSSION:
>Line 218:
-I would suggest removing the p-value from this part of the manuscript;
-On the same line, authors mention that almost half of all experiencers labeled their experience as “stressful”: was this determined on the basis of the written narratives or by using the additional close-ended question about the valence of the experience?
>It could be interesting to cite the following recent article in which a proportion of 14% of negative NDEs was identified. Interestingly, and in accordance with authors’ assumption, this study also included NDEs of different etiologies (and not only cardiac arrests) and showed a higher proportion of suicidal attempts among negative NDEs:
Cassol, H., Martial, C., Annen, J., Martens, G., Charland-Verville, V., Majerus, S., & Laureys, S. (2019). A systematic analysis of distressing near-death experience accounts. Memory, 1–8. https://doi.org/10.1080/09658211.2019.1626438
>Line 222: authors state that NDEs occurred equally in life-threatening as well as non-life-threatening situations, supposedly unlike other studies, nevertheless, the studies that they cite are not adequate to illustrate this point. Indeed, Thonnard et al., 2013, Charland-Verville et al., 2015 and Cassol et al., 2018 voluntarily chose to screen participants who had experienced NDEs in life-threatening situations. These participants come from the database of the Coma Science Group which also comprises NDEs experienced after non-life-threatening situations (see Charland-Verville et al., 2014). Regarding Martial et al., 2017, authors do not specify the context in which their participants experienced their NDE.

TABLES:
>In Table 3, I recommend to use a dash instead of a semicolon when reported the IQR.
>In the legends of Table 4 and Table 5, authors should remove “n – number of participants”.


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: PREVALENCE OF NEAR-DEATH EXPERIENCES IN PEOPLE WITH AND WITHOUT REM SLEEP INTRUSION
Review round: 2
Reviewer: 1

Output:
Basic reporting: No  further comment
Experimental design: No further comment
Validity of the findings: No further comment
Additional comments: Thank you for the amendments made. This reads well and is a very interesting subject.


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: PREVALENCE OF NEAR-DEATH EXPERIENCES IN PEOPLE WITH AND WITHOUT REM SLEEP INTRUSION
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Authors really improved the manuscript. It should be noted that changes strengthen the work. The work is now clearer and contains more information about near-death experiences and iits relation with REM sleep intrusion. Details clarified in the online survey strengthen the work. Improvements at the same time strengthened the 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: PREVALENCE OF NEAR-DEATH EXPERIENCES IN PEOPLE WITH AND WITHOUT REM SLEEP INTRUSION
Review round: 2
Reviewer: 3

Output:
Basic reporting: Thanks the authors for fully and clearly replay to the answers.
Experimental design: Ethical standards are duly fulfilled. However, there are limitations given by an on-line survey.
Validity of the findings: The erects support the main hypothesis
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: PREVALENCE OF NEAR-DEATH EXPERIENCES IN PEOPLE WITH AND WITHOUT REM SLEEP INTRUSION
Review round: 2
Reviewer: 4

Output:
Basic reporting: All comments have been addressed.
Experimental design: All comments have been addressed.
Validity of the findings: All comments have been addressed
Additional comments: Most issues have been addressed; however, it seems to me that the authors may not have quite seized one of my concerns. Yes, authors have stated several times in the manuscript that the near-death experience phenomenology may also occur during non-life threatening situations. However, in their survey, participants were asked the following question: "(…)near-death experiences are exceptional experiences that you may have when you are dying or feel as if you were dying. Have you ever had such a near-death experience – either during a true lifethreatening event or an event that just felt so?". Therefore, even though participants may not have been in serious danger, they should have at least felt like it in order to pursue the survey (in case of negative answer, the survey ended there). Knowing that, it is surprising to note that the final sample also includes participants who have experienced a NDE phenomenology during meditation or prayer, which in principal are not life-threatening contexts, whether objectively or subjectively. Could authors comment on this point? Did they gather information that could inform on such a result?


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: EFFECTIVENESS OF SMALL ROAD TUNNELS AND FENCES IN REDUCING AMPHIBIAN ROADKILL AND BARRIER EFFECTS AT RETROFITTED ROADS IN SWEDEN
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Helldin and Petrovan Review: Effectiveness of small road tunnels and fences in reducing amphibian roadkill and barrier effects; case studies of retrofitted roads in Sweden

The work done by Helldin and Petrovan presents an important contribution to our understanding of the use of tunnels and drift fencing on amphibian roadkill mitigation. I think the study is effective and the conclusions are justly drawn. However, there are several comments that I think would make the paper even more effective and increase its citability. I would be happy to review a revision of this paper in the future.

Brandon Hedrick

Major comments:

1)	Please include the supplemental information on each site in the main manuscript. Considering the PeerJ has a digital format, I wouldn’t want this information to be lost in the supplement. When I read the MS, I wanted a more thorough explanation of the site differences earlier on and I think this would remedy the problem.
2)	I would strongly consider combining figures 2–4 into a multiple panel figure with the images of the tunnels in the supplement. While table 1 lists the differences between tunnels, a visual would really help show the differences.
3)	I was a little confused until I looked at Table 2 what you meant by ‘before mitigation’ and ‘after mitigation’. There isn’t any mention of the years in the actual text and it reads like the ‘before mitigation’ and ‘after mitigation’ were several weeks apart.
4)	There are a few instances where methods are not adequately described. I am not sure how the data from lines 300–307 was generated. Please add this to the methods. More information on how the Hels and Buchwald equation was applied at each site (what the variables were) could be presented more clearly, perhaps in a table. I was also a little confused as to how the data were standardized. Please add in more details to the methods to remedy this.

Line by Line:

Line 25: Please spell out versus for the abstract

Line 26: consider ‘In mitigated road sections, the estimated number..’. I had a little trouble following as written.

Line 37: change to ‘by road mortality, barrier effects of roads, and traffic’

Line 42: ‘in attempt’

Line 40-42: Is there any evidence of road avoidance in amphibians? I have seen many salamanders and frogs walk across roads blissfully unaware of traffic. Can you add a citation?

Line 47: Please add the recent Hedrick et al. (2019) pub here: https://www.biorxiv.org/content/10.1101/569426v1

Line 65: Change ‘where’ to ‘were’

Line 105: Since there is no standard for monitoring tunnels, I think the supplement would be better moved back into the main paper. I would hate for this important information to be lost in the supplement. The supplement will need to be edited some in order to pull it into the main manuscript.

Line 114: I’m not sure why site 2 was so different having read the supplement. Is it just that there were temporary fences before mitigation?

Line 130: ‘..information presented by Hels and Buchwald..”

Line 142: For site 3, did you use the same speeds for the two newt species and for the two frog species in the Hels and Buchwald equation? I think more explanation is needed here.

Line 162: What do you mean when you say ‘largely following the number that was killed before mitigation’?

Line 179: Roadkill should not be a verb throughout
Line 192: The fence end effects can be curbed by putting a loop at the end of the fences to reorient the amphibians. See page 9 here: https://files.ontario.ca/environment-and-energy/species-at-risk/mnr_sar_tx_rptl_amp_fnc_en.pdf
Line 205–216: Add in the recent Hedrick et al. (2019) paper here as well. That study focused on this issue in particular.
Line 221: Gibbs and Shriver 2005 can also be cited here.  https://link.springer.com/article/10.1007/s11273-004-7522-9
Line 241: ‘…have a significant, but complex..’

Line 244: Longer than at the other sites right?

Line 262: Change ‘not unaffected’ to ‘affected’

Line 283: Change to ‘In contrast’

Line 286: I’m not sure what the sentence starting with ‘neither’ means. Reword.

Line 300–307: I’m not sure how this data was generated. Can you add this to the methods or delete?

Line 308: Gibbs and Shriver (2005) above used the Hels and Buchwald equation for a different species and confirmed its use.

Line 314: I think another very important point here is to identify the hotspot. It is not uncommon for tunnels to be placed near a hotspot, but not on it. In this case, the tunnels are not used at all.

Line 320: See the comment about the fence loop above.

Line 333: I completely agree! Too often tunnels are placed based on scant data and their impact is not assessed.

Line 350: Very good point about the juveniles. I wish they were easier to monitor. I have also always ignored this important factor in tunnel studies.

Table 2 makes it look like some sites were not measured before or after mitigation. Can you add in a row that says something like ‘manual monitoring’ to show how they were monitored?


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: EFFECTIVENESS OF SMALL ROAD TUNNELS AND FENCES IN REDUCING AMPHIBIAN ROADKILL AND BARRIER EFFECTS AT RETROFITTED ROADS IN SWEDEN
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is very good. The whole paper is very well written, explains the problem that is to be addressed clearly, and places the research firmly within a body of existing literature. The structure conforms to PeerJ standards and the figures and tables are sensibly structured and clear. I enjoyed reading it and found the results interesting, even though the different methods used at the different sites took some digesting.
Experimental design: The question posed is a sound and relevant one – how effective are amphibian tunnels in reducing road mortalities? The rationale for tackling this is also very sound, with two levels of controls – before and after the intervention was implemented (repeated measures) and mitigated vs unmitigated sites. The three sites provide some degree of replication which is also good. What is problematical, however, is that the study is collating information from three projects that were designed in rather different ways in terms of logistics, species, timings, tunnel/fencing design etc.  This is very common in ‘real world’ conservation practice where the original goal was to perform an intervention rather than perform a rigorous experiment. I am therefore very sympathetic to the challenges faced in delivering a rigorous experimental outcome given these constraints. The fact remains, however, that the inherent inconsistencies in how these three projects were carried out present some significant issues in terms of rigorous analysis. To overcome this, the authors have valiantly attempted to standardize the data from the three studies as far as they can so comparisons can be made. Nevertheless, this means that the analysis is entirely descriptive without any statistical testing. Personally, I can live with this given the design, but it does mean that there are numerous imponderables that make direct comparisons between before and after, and between mitigated and unmitigated sites, rather difficult. The authors acknowledge these issues and discuss them in a very articulate way in the paper. However, it does leave the reader with a feeling that there may be some doubt about the veracity of the findings. I am not sure how to address this, unless some sort of test (probably non-parametric) of association can be performed between (for example) ‘Change in number of deaths on road’ and ‘before and after mitigation’ and ‘mitigation vs non-mitigation’. Whether or not the paper warrants publication without some formal hypothesis test may therefore be an editorial matter.
Validity of the findings: There is compelling descriptive analysis (based on the standardization procedure) that the mitigation interventions reduced road mortality of amphibians. However, as described above, these descriptive analyses do make a range of assumptions about the data (e.g. percentage mortality can be modelled from an estimate of traffic intensity based from a different study), and there are range of possible confounding factors that might have influenced the results (e.g. seasonal changes in behaviour, natural population fluctuations between years, differences in survey effort between sites etc). The Discussion is sensibly cautious in that it flags such issues, and I suspect the authors are correct in their interpretation of the data. But it does raise the question of whether the study is technically and analytically sound enough to meet PeerJ  editorial requirements. Again, working in practical conservation myself I understand the constraints and can live with this, but ultimately I think this issue is an editorial call.
Additional comments: Line 63: I suggest ‘three independent sites’ rather than ‘hot-spots’ is more informative here.
Line 65: ‘were’ rather than ‘where’.
Lines 109-113: Some information is given in the supplementary information about the number of personnel involved in the surveys. How did the standardization procedure control for variation in personnel and expertise?
Lines 113-115: I wonder if site 2 is too anomalous to include in the study. The actual number of amphibians that appeared to be killed actually increased after the mitigation (Table 3). The reasons for this are explained, but it does rather throw the story.
Lines 126-127: Sorry, I can’t follow the rationale about how the number killed on the road could represent a certain number that survived.
Lines 255-257: This is an honest and pragmatic admission, but it does raise some doubt about the reliability of the interpretation of the increases/decreases.
Lines 261-265: Ditto last comment.
Lines 267-270: I suspect the authors are correct in this assertion, but a purist might argue otherwise.
Lines 281-282: I think the assertion that decrease in breeding adult deaths = increase in adult survival = benefit to conservation makes some assumptions. Amphibians have complex life cycles and density dependence may operate at different stages. It is therefore plausible that reduced survival in the adult stage could relax competition in tadpole/juvenile stage and thereby compensate for the mortality. As the paper deals with ‘deaths on roads’ rather than ‘survival’ (sensu stricto), it may be safer just to remove this sentence unless there is supporting literature.
Lines 290-291: A possible reason to remove site 2 from the analysis?
Lines 302-306: I quite like the idea of calculating thresholds like this. Any idea how these theoretical estimates related to traffic at these sites in reality?
Lines 306-307: Another reason to dump site 2 from the analysis?!
Lines 326-333: Without wanting to divert the paper down an economic line, this interesting point does raise the logical question of when does it become more cost-effective to build new habitat on one side of the road rather than build a tunnel?
Lines 333-338: Following on from the last point (and with the same qualifier), all these points are very good ones with which I entirely agree. However, the stark reality is who pays for this research to be done? I would envisage road building companies, government, research funders and conservation agencies all passing the buck to each other. This is a very common problem with no easy way forward.
References: Check these carefully. A few have capitalized first letters of words in article title.
Table 1: ‘Height’ not ‘Hight’
Figure 5: I assume that y-axis plots ‘number of evenings/nights’? May be wise to explicitly label it.


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: EFFECTIVENESS OF SMALL ROAD TUNNELS AND FENCES IN REDUCING AMPHIBIAN ROADKILL AND BARRIER EFFECTS AT RETROFITTED ROADS IN SWEDEN
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Helldin and Petrovan Review #2

The authors have responded to all of my previous comments adequately and I feel that the paper is quite strong now. I have suggested several additional minor changes to correct spelling and grammatical errors that should be incorporated before publication. However, I do not think that it is necessary for me to review the manuscript again before publication. Great work! I am excited to see that this is getting out.

Brandon Hedrick

Minor Comments:

Line 58: I originally read road tunnels as tunnels for cars. Can you change to ‘road culverts’ or ‘under-road tunnels’ to clarify?

Line 105: Change ‘Swedens’ to ‘Sweden’s’

Line 113: Change ‘trapping in tunnels was conducted in during’

Line 133: Change ‘significant’ to ‘large’ or something like that throughout. Otherwise, define what constitutes a ‘significant migration night’ statistically.

Line 139: ‘therefore’ spelling

Line 123, 149, 179: Please delete the last sentence at all sites and move it to the end of the section before site descriptions. It is repetitive as is.

Line 202: Change to ‘should also represent’

Line 208: This sentence is a little awkward. Please change

Line 311: Change to: ‘…may thus have a significant, but…’

Line 321, 341, 348: I would prefer a word other than significant. Significant is associated with a p-value. Maybe substantial would fit better.

Line 371: Add (2001) to Hels and Buchwald here

Line 372: ‘..while that study was well conducted, the results were based..’

Line 382: I can’t find the citation, but there have been suggestions in mitigation plans of having a loop at the end of fences to funnel amphibians back into the tunnel system. You might discuss that here. It looks like some of the fences you used incorporated this to varying degrees (Fig 3D, F)

Line 483: Need new paragraph

Table 4: I find it really interesting that you had such variable tunnel success at sites 2 and 3. Less than half of the critters entering the tunnels at site 2 actually exited. However, 80% used the tunnels successfully at site 3. I wonder why.


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: DETECTING THE IMPACT OF LAND COVER CHANGE ON OBSERVED RAINFALL
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment - very well presented.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: I think the authors should continue to investigate this topic as one of the case studies - the Queensland one - proved probably inconclusive.  They should also try and see if they can find instances of increase of land cover and increased rainfall.

Minor points:
Line 50 – problem with El Nin ̃o
Lone 97 – problem with Koppen
Queensland study area – did not have much tree cover initially.
Line 216 – I would have thought Spearman would be more appropriate
Line 306 – below??
Lines 360-361 – something wrong with figure reference


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

Input:
Title: DETECTING THE IMPACT OF LAND COVER CHANGE ON OBSERVED RAINFALL
Review round: 1
Reviewer: 2

Output:
Basic reporting: Line 34, maximum temperature at what height? surface temperature?

Line 39, "in both space and time", omit "in"

Line 41, better to add more details/examples about "those complex set of interactions"

Line 48, "On the longer time scale", compare to daily which is not mentioned in previous sentence. This paragraph is comparing local vs. larger scale, not on the time scale

Line 54, should this topic sentence and this paragraph not restricted to "predict Australian rainfall"? but rainfall at any locations?

Line 90, either use "the largest" or "the worst", they mean the same thing

Figure 3, no (a) and (b) were labeled in the graph, or use (left and right).

Table 1, extra space between row "2002: and row "2005"
Experimental design: Line 92, "..due to the drought and cold condition", the reference for that scientific conclusion is from News, a reference from a scientific journal would be more appropriate

Line 113, please include scientific name (Latin) for those tree species.

Line 115, previous sentence authors mentioned about great height, so here better to add details of height. "...20 years to mature (~? m)"

Line 115-116, changing subjects. "Land clearing" is the subject, but not for the rest of the sentence.

Line 125, add more details, t test for the averaged precipitation before (1979-2003) and after (2004-2015) the disturbance? Or move this sentence to the section of "statistical analysis" in below

Line 208, ", this variable was included" what variable? subject changes within a sentence, rewrite

Line 225-231, those paragraphs are results, do authors need them to be here for explaining a further step analysis? or should be moved down to Result section

Line 236-246, those two paragraphs sound like a discussion? should it be moved down to Discussion section?

Line 287-301, should these two paragraphs be combined with each individual analysis below? I don't see any reason to give a summary first and then introducing each analysis
Validity of the findings: Line 363-367, those are not key findings and should be moved to Method section.

Line 377-382, except the first sentence, the rest should be moved down to the discussion section

Line 386, "..compared to the number in QLD region"

Line 394, put the key findings as the subheading text instead of "step trend test" would be better

Line 395-398, those sentences are illustrating how to read the figure, should be put in the figure caption, not here in the Result section

Line 400, what is a "reasonable relationship"? authors should use more statistically sound words to describe the results

Line 404, a difference of what?

Line 411, if authors knew it was difficult to see, why not regraph the data to make it easy to see?

Line 421-429, All those sentences read like Method to me

Line 439 and 440, "strongest evidence" "weak evidence"? could it be more statistically described? like p value or with how much variance explained?

Line 476, the discussion point of "recovery" is worth to expand. Not only the evolution model from Figure 3 which only illustrates the mass/area, the species composition might be changed too. And ties into the evapotranspiration rate.

Line 526, "Possible future work could focus on a non-drought period.." I think research on lands with disturbance(e.g. fire, drought, etc) is also important even though it is hard to study as reported from this study.
Additional comments: This paper uses sophisticated statistical approaches to identify impacts of changes in land cover on rainfall from 1979 to 2015 in two regions in Australia. Results show lands under historical bushfire had significant changes in precipitation, but not for lands with tree clearing. I am sure this paper adds an important value to the community. However, this paper has a very detailed Method section which includes some results and discussion. Some part of the Method section also includes a summary test first and then introducing each analysis. This would cause repetitive sentences. For example, line 303 "As indicated, the step trend test is a modified version of the Mann-Whitney U statistic (Hirsch and Gilroy 1985).", this sentence is repeating line 289 in the summary. I think the summary is not necessary and should be merged to each analysis.
I suggested authors to reorganize the whole paper and keep the sections simple and clear (not overwritten with details not necessary for readers to understand the analysis/paper). Or if authors think those sentences in the Method section are necessary, put them in the appendix, so the main body of this paper can be simplified. I understand authors trying to describe all their trails, thoughts and steps in analyzing those data in this paper. This is my personal opinion. I will let the editor decide the format of this paper.


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

Input:
Title: DETECTING THE IMPACT OF LAND COVER CHANGE ON OBSERVED RAINFALL
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comments
Experimental design: No comments
Validity of the findings: No comments
Additional comments: In this paper, Vervoort and his colleagues reported their finding about the impact of land cover change on observed rainfall. The analysis is very comprehensive. And I learned a lot from this manuscript. The authors used statistic approach to analyze observed data, to detect the change of precipitation after forest cleaning and bushfires. They found different influences after two changes. The methods are quite reliable, and conclusions can be trusted. I think this paper has great potential to be a high-cited paper. But the paper still needs to be further improved before publication.

1.	Not only the statistically analysis, but also the physically meaning should be further discussed. Theoretically, the change of vegetation will impact on ET, especially in dry seasons, because land cover change will impact on the root zone storage capacity, which provides an important buffer to increase resilience to drought (Gao et al., 2014). This should impact on downwind precipitation.
2.	Several important papers, on the impact of land cover on precipitation, are still missed in the reference list (e.g. Keys et al., 20142).

Gao, H. , Hrachowitz, M. , Schymanski, S. J. , Fenicia, F. , Sriwongsitanon, N. , & Savenije, H. H. G. . (2014). Climate controls how ecosystems size the root zone storage capacity at catchment scale. Geophysical Research Letters, 41(22), 7916-7923.
Keys, P. W. , Van, d. E. R. J. , Gordon, L. J. , Hoff, H. , Nikoli, R. , & Savenije, H. H. G. . (2012). Analyzing precipitationsheds to understand the vulnerability of rainfall dependent regions. Biogeosciences, 9(2), 733-746.


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: DETECTING THE IMPACT OF LAND COVER CHANGE ON OBSERVED RAINFALL
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Well done with the attention to detail in the revision.


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

Input:
Title: DETECTING THE IMPACT OF LAND COVER CHANGE ON OBSERVED RAINFALL
Review round: 2
Reviewer: 2

Output:
Basic reporting: None
Experimental design: None
Validity of the findings: Line 387-406, I think these sentences can be merged with different sections below and the Conclusion section. No need for a separate section at the beginning of the Discussion. But it is fine if authors want to keep it that way.
Additional comments: I really appreciated authors hard working on revising this manuscript. I find this paper is acceptable as it is.


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

Input:
Title: CELL-FREE SUPERNATANTS FROM CULTURES OF LACTIC ACID BACTERIA ISOLATED FROM FERMENTED GRAPE AS BIOCONTROL AGAINST SALMONELLA TYPHI AND SALMONELLA TYPHIMURIUM VIRULENCE VIA AUTOINDUCER-2 AND BIOFILM INTERFERENCE
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article #35559 shows two lactic acid bacteria identified as Weissella viridescens and Weisella confusa, which were isolated from fermented grapes with antagonistic activity against Salmonella Typhi and Salmonella Typhimurium. In addition, it was demonstrated that the cell-free supernatant of these lactic acid bacteria exerts the antagonistic activity and inhibit the formation of biofilms by blocking the signal of the AI-2, which is one of the three known types of autoinducers related to Quorum Sensing in Salmonella.
The manuscript is written correctly, clearly and unambiguously.
My first recommendation is about the title, when I read “Metabolites”, I think it is based on the identification of metabolites, so I consider authors could either perform new experiments for identifying the active metabolites or at least their chemical nature, or amend the title accordingly.
In the introduction section more details are required. In the second paragraph, it would be appropriate including information about the other types of autoinducers involved in Quorum Sensing in Salmonella (AI-1, AI-2 and AI-3) and, the reason for choosing AI-2. On the other hand, hypothesis is missing in this section, so it is suggested to include it based to the conclusion.
More than 70% of the references are from 2015 to 2019, which may be indicative of the current status of the addressed topic.
Experimental design: This research addresses a current topic within an adequate subject for publication in this journal.
I think the problem and research question are adequate.
The methods used are correct and in a general way are described correctly. In some cases the information may be enhanced:
	1.- In Isolation of LAB from fermented foods method a reference is missing (lines 137-139) or mention it if you have validated that method previously.
	2.- In Agar well diffusion test is important to mention the height the agar reaches in the plate or the volume of agar that was placed in each plate.
        3.- In the MIC and MBC method (line 170), please specify if 105 CFU/mL is the final concentration or the concentration added in the 10 μl for each well and, also the final volume in each well must be indicated.
The developed experiments were designed correctly; however an experiment that helps to make a more specific identification would enrich the work.
The statistical analysis is accepted but the statistical design is missing. In the statistical analysis section, it is written “Turkey´s HSD test”, which could change for “Tukey´s HSD test”.
Validity of the findings: I wonder why authors mention the lactic acid bacteria isolated from fermented grapes are “potent” What do they mean with potent? It should be indicated based on which scale or respect to what?
Results show a good anti-salmonella and anti-biofilm activities of LAB-CFCS tested. Data presented seems robust, clear and sound, and conclusion is adequate.
In the discussion, in lines 409-410, it is mentioned that CFCS contain other metabolites than organic acids, which is not clear from the methodological approach or results obtained, so that these lines are speculative.
Additional comments: Abstract:
A period is missing after each subtitle, i.e., “Background. Salmonella Typhi…”

Introduction:
Citation in line 71, replace “Alum et al., 2016” by “Alum, Chukwu & Ahudie, 2016”.
Citation in line 72, is not correct, replace “Bhutia & Narain, 2010;…” by “Bhatia & Narain, 2010;…”

Materials & Methods:
In line 122, the word “this” could be changed for “the” or be omitted.
In line 214, the word “Salmonella” must be in italics.
In line 229, change the parenthesis position. “… as described by (Liu et al., 2014)…” “… as described by Liu et al. (2014)…”

Results:
In line 277, delete the letter “s” in “…Tables 2…”
In line 277, you mentioned “…19 selected LAB-CFCS…” but in the table 2, that number does not coincide.
In lines 323-324, the sentence “…Therefore, they were preliminarily identified as members of the genus Lactobacillus or Lactobacillus-like microorganisms (Liu et al.,2014).” must be change to Discussion and remove italics from “Liu”.

Discussion:
Please include in the manuscript discussion an explanation to the following questions:
Why does a bacterial strain have more antagonistic activity against Salmonella Typhi compared to Salmonella Typhimurium?
Why do two strain of the same bacteria exert different antagonistic activity against a bacterial pathogen?

References:
The reference in lines 529-531 is badly located; it should be in alphabetical order after “Rutherford ST, Bassler BL. 2012 ...”


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: CELL-FREE SUPERNATANTS FROM CULTURES OF LACTIC ACID BACTERIA ISOLATED FROM FERMENTED GRAPE AS BIOCONTROL AGAINST SALMONELLA TYPHI AND SALMONELLA TYPHIMURIUM VIRULENCE VIA AUTOINDUCER-2 AND BIOFILM INTERFERENCE
Review round: 1
Reviewer: 2

Output:
Basic reporting: Line 80 – reform the sentence of action driven salmonella having goals into more neutral
Line 81 – maybe to highlight the most common symptoms of food poisonings?
Line 85 – better wording for “bacteria sense them” or reforming wording?
Line 94 – Several serovars… move sentence before the previous sentence at Line 91 “Biofilm…” for more comprehensive and logical explanation order
Line 100 – A range … maybe to add also the food hygienic practices to emphasise the initial need to produce in hygienic and controlled conditions to AVOID the contamination possibilities as much as possible, especially in food production facilities.

Table 2 – Why are all the SDs 0.00 and all the values 10-folds only? Are these the correct values or table needs to be updated?

Tables and figures – clear, understandable and good amount of information

Introduction – clearly written in a good narrative order considering the relevant aspects of the subject in good detail and covering the wider aspects of the subject. Some minor polishing.
Experimental design: Line 134 – reduce word fermented to only the first for easier reading to avoid repetition

Materials and methods – well and clearly structured and written containing good amount of detailed information with appropriate references
Validity of the findings: Line 261-268 – replace this source of isolations with materials and methods line 134 description
Line 269 – Start new paragraph
Line 277 – Change Tables -> Table

Results – section is very well written, constructed and formatted to present the results in structured, clear and understandable way. The chain of validation of the functionality and effectiveness are well structured in the analysis to ensure best possible results with good outcomes.

Line 336 – This might be too straightforward conclusion of the usability and to compare with the more difficult options. Maybe this would be more justified if you can name some commonly used, or prominent control methods, which then are more difficult to apply in practice?

Line 367 – This due -> this is due
Line 412 – Remove -> It would not be surprising, -> These Weissella…  OR Prior better understanding, these Weissella…
Additional comments: Very well and clearly written article with detailed information to support the experiments and findings. The study was well structured, and the presentation of the study and findings further highlight the authors’ abilities to present complex issues in clear format. The study is an interesting and topical, and the findings can be applied to wide range of applications and is very useful for further studies to be utilized.

I would want to encourage the authors to continue with this line of work and explore the possibilities to implement these findings in practice to ensure better food safety procedures in the future. This was a good study and hopefully the work will continue so that the findings will not remain only at the laboratory and academic level but are created into easy-to-use real-life options for industry.


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: BRIDGING THE TB DATA GAP: IN SILICO EXTRACTION OF RIFAMPICIN-RESISTANT TUBERCULOSIS DIAGNOSTIC TEST RESULTS FROM WHOLE GENOME SEQUENCE DATA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Text is written in an understandable way with only minor mistakes, such as word doublings (line 172), missing punctuation marks (line 144 - 147), etc.
Figure legends names goes a little confused and i would have chosen other figures, which fit better to the results. Tables are well labeled and described, however, I would cite the paper in legend for table 1.
Introduction and background quite long compared to results part, however, I miss a part how RDT's work in general.
Experimental design: The study is well designed and the program is first trained with in-silico data and afterwards checked with "real" data.
The method part is written in detail and easy to understand so that the analyses can be reproduced. In my eyes, however, the results do not reflect the aim of the study. In my opinion the results support the fact that the high resolution of the NGS data should not be broken down to RDT. For comparisons of different years it would be enough to compare resistant status R/S, so that even different GeneXpert results could be compared. Line 86 says "comparison within and between countries can be difficult due to use of differing technologies", but this is still the case when one country uses Xpert classic and the other uses Xpert ultra, isn't it?
Validity of the findings: For me the validity of the study is not clear. It is stated that this tudy should close a knwoledge gap, which I don't see. Of course it makes sense to make different technologies comparable, but I see no reason to break down such a high-resolution technology as NGS to such a primitive technology as RDT. The author stated that this will make surveillance easier (line 231) or allowing for longitudinal analysis of RR-TB prevalence, but in my mind for surveillance and prevalence estimation a status such as R for resistant or S for susceptible would be enough. The knowledge of the specific probe is not a knowledge gain but loss of information compared to a specific SNP you received from the sequencing. Especially the last sentence of the clonclusion is missleading. Noone would take the RR-TB results as marker for transmission. Even the results here show, that there are a few SNP prominently circulating but to get an idea of transmission one would genotype the strains and when WGS data is available it would be possible to do a sufisticated contact tracing by SNP distances.
Additional comments: How you decided on the name of the tool? When I searched for TBGT in github there were 5 hits and two other software were named exactly the same with small letters. Google had several hits but on the first pages your TBGT was not found at all. Additionally I don't get the connection of transforming NGS data to RDT data and test a TB Genome. That is why I would think of a new name for the tool.
I am not so sure, who needs such a tool. I liked the previous publication about the correlation of RDTs and SNPs, but in my mind a cool story would be the transformation the other way round, although I know, that this is not possible with Xpert Classic results for example.

In the supplementary tables I would always put Rif susceptible on top or on the bottom of the table to make them comparable.


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: BRIDGING THE TB DATA GAP: IN SILICO EXTRACTION OF RIFAMPICIN-RESISTANT TUBERCULOSIS DIAGNOSTIC TEST RESULTS FROM WHOLE GENOME SEQUENCE DATA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The author have developed a useful tool that transforms SNP results to RDT format. This will be helpful to public health systems (National Health Control Programmes) in several countries that utilize the relevant technologies in their programmes. The study is aimed at converging all data(past and present) n the public domain to offer offer the most effective therapies to their patients.
The language used in the paper is professional and has clarity.However the repetitive word "all" in line 172 needs to be removed. Authors should explain the significance of the results in relation to public health initiatives so that it is easier to understand the function of this tool.
Contemporary references have been quoted to illustrate the background
however the  figures 2a, 2b,3a, 3b do not describe the units of the y-axis
Experimental design: The article meets the aims of the Journal and identifies a critical gap in the utility of genomic data generated at different levels. The TBGT tool is an attempt to converge this data in a more powerful format for betterment of TB treatment and control. This utility needs to be described better to the reader for easier comprehension.
Please mention in Methods (line 131) whether vcf file is araw file or a filtered vcf file. If the latter, then the parameters used to filter the file shouold be mentioned.

In the TGBT, the output is in the form of a text format. It would be helpful if the result format  was in excel as well to ease comparison of data
Validity of the findings: The utility of this approach to capture data from multiple data repository sites would be useful to mention. The approach to undertake this could be mentioned in the discussion especially for countries like India where consolidation of data is likely to be fragmented.
Additional comments: No additional comments