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: ANATOMICAL NOTES AND DISCUSSION OF THE FIRST DESCRIBED AETOSAUR STAGONOLEPIS ROBERTSONI (ARCHOSAURIA: SUCHIA) FROM THE UPPER TRIASSIC OF EUROPE, AND THE USE OF PLESIOMORPHIES IN AETOSAUR BIOCHRONOLOGY
Review round: 1
Reviewer: 3

Output:
Basic reporting: The MS by Parker and Martz is a competent analysis of  the traditional key aetosaur taxon Stagolepis robertsoni that deserves to be published in the “Peerj” after several modifications suggested below.

 It is also important to stress that I am not an English native speaker, so I did not preformed an in depth review on the use of the English language, which seems mostly adequate. Yet, I would be me more comfortable if an English native speaker (the other reviewer or the editor) could perform such a revision.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In the introduction should be mention the new material from the USA assigned to Stagonolepis based on the osteoderm morphology (e.g. patron and type of ornamentation, proportions L/W in Long & Murry 1985, Lucas&Heckert 2001)  and the new species S. olenkae described by Sulej 2010 to the same genus from Poland.

In the Material section should be mention a table with all the information from the specimen analyzed in this contribution were referred to Stagonolepis robersoni. (e.g. NHMUK R4787 a cast- left side of the skull and mandibule)

The diagnosis should be check , because some autopomorphies mention by the authors are share with other aetosaur taxa, such as:

There are some autopomorphy characters from the diagnosis and combination that also distinguished to be check:

-The randomly arranged (reticular patron) of ornamentation described in Stagonolepis robertoni by the authors is also present in Chilenosuchus forttae (Casamiquela 1980, Desojo 2003). Although the validity of Chilenosuchus as an aetosaur is debate (see Desojo et al. 2013), it should be include in the comparisons and discussion section of the present manuscript.

-The autapomorphy: trunk paramedian osteoderms lack ornament along the posterior edge in the region of the dorsal eminence should be described in more detail and check in more aetosaur taxa, because it vary among the differ regions of the dorsal armor of one specimen (e.g. cervical, dorsal, sacral, caudal regions). see Taborda et al. 2015, Schoch & Desojo 2016 Fig.2 A-D.

- Character from the osteoderm onramentation should be compared with Chilenosuchus and Aetosauroides scagliai (see attach file).

The osteoderm description section (line 328) should be improve with more comparison with Chilenosuchus (see Desojo 2003) for the ornamentation pattron and ventral osteoderm comparison, as well as with Aetosauroides scagliai (see Taborda et al. 2015) for the anastomosing ornamentation definition (previously used by Taborda et al) and the dorsal eminence variation among paramedian osteoderms of a single carapace. This variation of ornamentation of the paramedian dorsal eminence affects the diagnosis provided by the authors here.

line 492: This statement is very interesting, but no forget that the ontogenetic variation could be work here,
Minor comments:
The first mention in the text of Stagonolepis olenkae is between quotation marks, could you justify it?

line 227: no really, it is possible to observe in S. olenkae and Aetosauroides scagliai new material (UFSM 11050) on the left side

line 230: also present in S. olenkae

line 248: it is difficult to see in the figure 3. Could you indicate it? This dorsal protuberance is also present in other aetosaurs (e.g. Paratypothorax, Neoaetosauroides), please check it.

line 271: also in Aetosauorides scagliai (see Taborda et al. 2015 PVL 2059)

line 287: a well development ventral keel is present in Neoaetosauorides (Desojo&Baez 2005) and absent in Aetobarbakinoides (Desojo et.al. 2012). so, I am not sure about your statement (291) of its variability. More information about intraspecific variation should be done to confirm or not it.

line 295: this lamina was described by Desojo & Ezcurra 2011 (pag. 599) as present in S. robersoni and A. scagliai , among other aetosaurs, named posterior infradiapophyseal lamina; please check.
There are several references in the text but not in the references section (e.g. Reese 1915, Heckert et al. 2010, etc.), please, check. Also, few citations are no in the text but in the references section, and the correct year of publication for Benton & Walker is 2011.

Fig. 1A: There is a bone on the right side of the right surangular indet, could be a right angular? please, check.
Could you indicate the 5th premaxillary tooth crown that you mentioned?

Fig.2a: Could you indicate de foramina? at least 4 foramina are present on the maxilla body.
Should be good if you can indicate the six alveoli in Fig.2B
2B: could you indicate the interdental plates and the alveolus?

Fig 3: Could you indicate the specimen number and the Premaxilla/maxilla contact? Also, should be indicate the premaxillary 4 teeth.


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: ANATOMICAL NOTES AND DISCUSSION OF THE FIRST DESCRIBED AETOSAUR STAGONOLEPIS ROBERTSONI (ARCHOSAURIA: SUCHIA) FROM THE UPPER TRIASSIC OF EUROPE, AND THE USE OF PLESIOMORPHIES IN AETOSAUR BIOCHRONOLOGY
Review round: 2
Reviewer: 1

Output:
Basic reporting: Nothing more to add.
Experimental design: Nothing more to add.
Validity of the findings: Nothing more to add.
Additional comments: None.


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: ANATOMICAL NOTES AND DISCUSSION OF THE FIRST DESCRIBED AETOSAUR STAGONOLEPIS ROBERTSONI (ARCHOSAURIA: SUCHIA) FROM THE UPPER TRIASSIC OF EUROPE, AND THE USE OF PLESIOMORPHIES IN AETOSAUR BIOCHRONOLOGY
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors did a great job with the suggestions  and modifications. Congratulations.
Only two comments:

1-my original comment: line 227: no really, it is possible to observe in S. olenkae and Aetosauroides scagliai new material (UFSM 11050) on the left side
-authors replied: This material has not been formally published and is being worked on by someone else so I have not included it.

 I understand and respect the position, Thank you. On the other hand, actually the manuscript is in press:
Osteology of the first skull of Aetosauroides scagliai Casamiquela 1960 (Archosauria: Aetosauria) from the Upper Triassic of southern Brazil (Hyperodapedon Assemblage Zone) and its phylogenetic importance
PONE-D-17-29053R2
Dear Ana Carolina Biacchi Brust,
We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.
Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.
Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.
If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.
With kind regards,
Thierry Smith, Ph.D.
Academic Editor
PLOS ONE

2-In the introduction should be mention the new material from the USA assigned to Stagonolepis based on the osteoderm morphology (e.g. patron and type of ornamentation, proportions L/W in Long & Murry 1985, Lucas&Heckert 2001) and the new species S. olenkae described by Sulej 2010 to the same genus from Poland.
-authors replied: This has been dealt with in many previously papers and I did not want to burden the reader with a complete rehash of the taxonomic history of all of the genera previously synonymized with S. robertsoni since it had already been covered several times in other recent papers. Instead I just dealt with the history of the Scottish specimens of interest.

-It is really a pity, because the topic of this paper is specifically on the taxonomy and anatomy of Stagonolepis robertsoni, so, a short taxonomy account of the  genus and species should be improve 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: DISTRIBUTION OF PORPHYROMONAS GINGIVALIS FIMA AND MFA1 FIMBRIAL GENOTYPES IN SUBGINGIVAL PLAQUES
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is a well described manuscript from a corresponding author who has been doing pioneering work in the area of P. gingivalis fimbrial protein biochemistry and genetics. The findings of the study challenge some of the previous reports indicating that certain types of fimA genotypes (such as type II) are strongly associated with severity of periodontitis. The study is significant and its scientific merit is high.
Experimental design: Specific comments:
1. You performed sequencing of 16S rDNA amplicons followed by BLAST search.  Are the results of this analysis included in the supplemental File 2? (please mention this in the text).
2. Thanks for providing the raw data and calculations in the Excel files. However, it is difficult to grasp what each column/row represents and what is being done to the data.  It would be nice if you could provide clear identifiers and some description in the figure legends as to what each File/sheet presents (currently there are no figure legends).
Validity of the findings: Overall, the study was performed with high rigor, data are presented well and proper conclusions are drawn.
Additional comments: This is a well described manuscript from a corresponding author who has been doing pioneering work in the area of P. gingivalis fimbrial protein biochemistry and genetics. The findings of the study challenge some of the previous reports indicating that certain types of fimA genotypes (such as type II) are strongly associated with severity of periodontitis. The study is significant and its scientific merit is high.


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: DISTRIBUTION OF PORPHYROMONAS GINGIVALIS FIMA AND MFA1 FIMBRIAL GENOTYPES IN SUBGINGIVAL PLAQUES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript by Nagano et al examined association between P. gingivalis fimbrial genotypes and periodontitis severity. Experiments indicated that there was no association between fimbrial genotypes and periodontitis severity.  Major concern is the novelty of this study, since several similar studies have been reported.
Experimental design: 1.  Ln 151-152, please indicate why dental plaque samples were resuspended in either RTF or water.
2.  Ln 161, need details on DNA purification.
3.  Ln 172-173, “Plaque samples in RTF or water were directly used as PCR templates”.  How did you collect DNA?
4. Ln204-206, “Among the 100 samples, both fimbrial types were detected in 63 and a single type in 15 samples, whereas 22 had no fimbrial genes.  Why not use Pg 16s to determine Pg level.  Is no fimbrial gene means no Pg?
Validity of the findings: Table 1 did not include statistical analysis.
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: NMR STRUCTURE OF THE C-TERMINAL DOMAIN OF TONB PROTEIN FROM PSEUDOMONAS AERUGINOSA
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this paper, the authors outline the structure determination of Pseudomonas aeruginosa TonB C-terminal domain, confirmed it to be monomeric, and explored its dynamics at the atomic level.

The paper is generally very well written. There is a good introduction, with relevant literature referred to. However, a schematic figure would be beneficial here, illustrating the general set-up of TonB proteins, in the context of the OM and CM, and their relationship to TBDTs. This would be very useful for readers unfamiliar with this system.

The plug domain is first mentioned (lines 67-68), along with the ‘mechanical model’,  but no context has been given for these terms. Could the authors introduce these ideas i.e. how the plug domain of the TBDT is proposed to mechanically unfold in the ‘mechanical model’?

The chemical shifts and structures have been deposited in public databases (lines 202-204), but have the restraints used in the calculations, NOESY spectrum peaks, and relaxation data been deposited also?

Line 248-250: ‘However, the changes were not observed in the 15N spin relaxation analysis, presumably because the time scale of the fluctuation is not sensitive to the 15N relaxation.’ – Should this be the other way around? i.e. the 15N relaxation is not sensitive to the time scale of the fluctuation?

Line 76 ‘intertwined dimer’ – does this situation qualify as a domain swap dimer? Would this be a better term to use?
Experimental design: The experimental work looks very sound and thorough, and all methods were well described.
Validity of the findings: The vast majority of their findings were robust and well stated.

I did identify a potential problem in the analysis of the predicted interaction with TBDTs, which influenced some of their conclusions. I would recommend they look at these conclusions again before publication. Specifically this relates to line 267 and Figure 6. Here the authors have shown the Paeruginosa TonB sequence (beta-5 strand) and how it might bind to the Ecoli BtuB sequence. They used the Ecoli sequence since there is no structure available for the Paeruginosa BtuB or other TBDT. However, there is a sequence (as opposed to structure) available for the Paeruginosa BtuB protein, and a sequence alignment could be carried out to look at the actual interacting residues in the correct species. I carried out my own such alignment, and I think some of the conclusions in this section may change when this sequence is used (for example, that in line 312, as well as others near line 267).

It would also be useful to state In the results (around line 188) what the similarity/identity of the PaeruginosaTonB sequence is to that of Ecoli.
Additional comments: There are a number of typos/grammatical errors that should be cleaned up prior to publication:

Line 32 ‘terminal terminus’ – either of these terms would be fine alone, but one needs to be removed.
Line 76 ‘composed’ – should be ‘were composed’.
Line 81 ‘as observed NMR’ should be ‘as observed in NMR’
Line 86 ‘the significant’ should be ‘a significant’
Line 88-89 ‘The major differences between the monomeric EcTonB without intertwining locate at residues 235-239 in the C-terminal end (Fig. 1b).’ – Can this sentence be clarified? E.g. ‘The major differences between the monomeric EcTonB and the intertwining dimer, pertain to residues 235-239 in the C-terminal end (Fig. 1b).’
Line 89-92 – ‘In the NMR structure of EcTonB-137, these residues fold to an additional beta-strand (beta6) and join in an anti-parallel fashion with the preceding beta-strand. On the other hand, the C-terminal end is either extended or largely invisible in the crystal structures (Fig. 1b). ‘ Would be better written as: In the NMR structure of EcTonB-137, these residues fold into an additional beta-strand (beta6) and join in an anti-parallel fashion with the preceding beta-strand, whereas in the crystal structures of monomeric EcTonB constructs, the C-terminal end is either extended or largely invisible  (Fig. 1b).
Line 97 ‘On the other hand’ – would be better as ‘In constrast,’
Line 222 ‘by additional’ should be ‘by the additional’
Line 252 ‘affect to the effective’ should be ‘affect the effective’
Line 254 ‘with the lower order’ should be ‘with lower order’
Line 271 ‘phenylalanine of’ should be ‘phenylalanine in’
Line 285 ‘interfere the interaction site’ should be ‘interfere with the interaction site’
Line 316 ‘unveil structural’ should be ‘unveil the structural’


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: NMR STRUCTURE OF THE C-TERMINAL DOMAIN OF TONB PROTEIN FROM PSEUDOMONAS AERUGINOSA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall the manuscript is well-written, but requires further proofreading to remove several minor errors.

Specific minor revisions needed:

The first paragraph of the Discussion introduces new modelling/docking information that would be more appropriate in the Results section.

The are number of citations to Ollila et al. 2018, which although submitted is still unpublished material. I'm unsure that this is acceptable according to PeerJ guidelines.

The HSQC depicted in Figure 2 requires darker contours.

L99 – A reference needed to specify exactly what crystal structures are referred to (dimers in crystal structures or complex of TonB Box and -terminus).

L179 – A reference for the Redfield equations would be useful.

L226/227 – Additional background information on the dynamics probed by parameters T1, T2, NOE etc. (i.e., timescales) would be helpful to guide non-NMR experts through interpretations.
Experimental design: No comments.
Validity of the findings: L108 to 111 – The authors state that the PaTonB-96 had an additional three residues because of cloning. Can they please state explicitly what these residues are and where they are located? For instance, residues located on the C-terminus have a dominating influence on the dynamic and structural findings which are of central importance to study.

L168 – Specific details are needed regarding the criteria used to assess the equilibration of MD trajectories.
Additional comments: No comments.


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

Input:
Title: NMR STRUCTURE OF THE C-TERMINAL DOMAIN OF TONB PROTEIN FROM PSEUDOMONAS AERUGINOSA
Review round: 1
Reviewer: 3

Output:
Basic reporting: The authors report an NMR structure of a pseudomonas TonB protein C-terminal domain fragment.

The TonB protein is part of a mechanism for active transport across the outer membrane of Gram-negative bacteria, using energy derived from interactions in the cytoplasmic membrane below. In particular, the C-terminal domain of TonB interacts with outer membrane transporter proteins, via a conserved TonB box motif, presumably enabling structural changes in the transporter protein. The interaction is believed to involve the plug domain, which serves as a gate for the transporter protein channel.

The authors report a monomer structure with an extended and flexible C-terminal tail, in contract to some previous X-ray and NMR studies on TonB constructs from E. coli.

--- Possible corrections:

Abstract: "outer membrane" for "outermembrane"

89: are located

136: citations for VnmrJ and NMRPipe.

154: "chemical shift values ... were used ... for the structure calculation".

How? If additional software was used, citations are needed.

172: Lennard-Jones

195: were assigned

249: presumably because 15N relaxation is not sensitive to fluctuations at that time scale.

299: is located
Experimental design: The authors perform an NMR structure determination according to accepted methods, using NOE distances. They also use an appropriate set of NMR experiments to characterize backbone dynamics, and supplement these with molecular dynamics simulations.
Validity of the findings: The reported structure has good verification statistics, so the authors have made a good case that their structure is valid.

The molecular dynamics simulations correspond well with the NMR relaxation parameters, supporting the authors' reasonable claim that the protein has a flexible C-terminal end.
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: NMR STRUCTURE OF THE C-TERMINAL DOMAIN OF TONB PROTEIN FROM PSEUDOMONAS AERUGINOSA
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: I am happy with the modifications made by the authors, and now recommend the paper for publication.


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

Input:
Title: NMR STRUCTURE OF THE C-TERMINAL DOMAIN OF TONB PROTEIN FROM PSEUDOMONAS AERUGINOSA
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have done a great job in addressing all of my previous suggestions. I have no further comments to add.
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: NMR STRUCTURE OF THE C-TERMINAL DOMAIN OF TONB PROTEIN FROM PSEUDOMONAS AERUGINOSA
Review round: 2
Reviewer: 3

Output:
Basic reporting: The authors report an NMR structure of a pseudomonas TonB protein C-terminal domain fragment. They have enhanced their report with a description of TonB-dependent energy transduction system.

The TonB protein is part of a mechanism for active transport across the outer membrane of Gram-negative bacteria, using energy derived from interactions in the cytoplasmic membrane below. In particular, the C-terminal domain of TonB interacts with outer membrane transporter proteins, via a conserved TonB box motif, presumably enabling structural changes in the transporter protein. The interaction is believed to involve the plug domain, which serves as a gate for the transporter protein channel.

The authors report a monomer structure with an extended and flexible C-terminal tail, in contract to some previous X-ray and NMR studies on TonB constructs from E. coli.
Experimental design: The authors perform an NMR structure determination according to accepted methods, using NOE distances. They also use an appropriate set of NMR experiments to characterize backbone dynamics, and supplement these with molecular dynamics simulations.
Validity of the findings: The reported structure has good verification statistics, so the authors have made a good case that their structure is valid.

The molecular dynamics simulations correspond well with the NMR relaxation parameters, supporting the authors' reasonable claim that the protein is monomeric and has has a flexible C-terminal end.
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: A COMPREHENSIVE REVIEW OF CIRCRNA: FROM PURIFICATION AND IDENTIFICATION TO DISEASE MARKER POTENTIAL
Review round: 1
Reviewer: 1

Output:
Basic reporting: The review gives a clear, well written account of the current understanding of CircRNA. While there are some minor errors, the authors give a very clear description of CircRNAs for people outside of the field, from their biogenesis, to their functions and future roles in molecular biology.
Experimental design: The review is structured into sections, first describing how CircRNAs are generated, followed by their function within the host system, followed by a list of methods of CircRNA detection including their costs and benefits depending on the method. Given what is known about this relatively novel RNA form, I feel the review gives appropriate time to each section, with appropriate citations. The sections are ordered appropriately for readers new to the field or with little knowledge of CircRNAs.
Validity of the findings: no comment.
Additional comments: The review is competently written and gives a good account of CircRNA for people new to the field. There are a few minor issues the authors could look to correct (I may have missed some).

47-62. The authors do not make clear how far reaching CircRNAs in their different forms are across the tree of life. Would it be possible to include a tree with presence/absence blocks for the differing forms of CircRNA (ecRNA/ciRNA/elciRNA) and their differing functions (miRNA sponge/RBP/pseudogene/regulation/splicing/translation), as I am unsure from the review is these forms/functions are ubiquitous or specific to some orders of life.

82-83. The sentence should be rewritten as 'Interestingly, not all exons form a circRNA...'

165: A small point, but it is a long time between RBPs being defined and first used, maybe restate here?

123-201: Another small point, but the order of sections here are confusingly ordered in relation to positions of illustrations in Figure 3. Possibly reorder the sections or labels within the figure.


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 REVIEW OF CIRCRNA: FROM PURIFICATION AND IDENTIFICATION TO DISEASE MARKER POTENTIAL
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript contains many typographic and drafting errors. English must be considerably improved. The manuscript includes sufficient background. It shows clearly the context.
The structure of the article is according to the required format by PeerJ. Figures are very illustrative, appropriately labelled and described.
Experimental design: The review is organized logically into coherent subsections. However, the “CircRNA in disease development and progression” section is poorly developed. The only paragraph in this section is difficult to understand and contains poor information.
Validity of the findings: No comment.
Additional comments: The review entitled “CircRNA: as a disease marker potential and research strategy” by Xu et al. addresses a very interesting and novel topic. In this review, authors explain relevant topics about CircRNAs including biogenesis, properties and functions. In general, these topics were correctly written and illustrated with appropriately described images. The “CircRNA in disease development and progression” section is not according to previous sections, this one is very poor. I consider that, since title is about CircRNAs and diseases, this section must be more extensive and complete. The “Methods of CircRNA detection and characterization” section is basically focused on methods of purification and identification of CircRNAs. This one is a small part that I consider to be a research strategy (as included in the title). Authors must discuss more about these topics and include Why CircRNA could be a disease marker potential?

I think that, if the authors address the previous comments and the following minor comments, the manuscript could be considered for publication.

Minor comment:

Section 7 is loss.

Line 48-49. Change “functional polypeptides to function” to “functional polypeptides”.

Line 88. Change “cricRNA biogenesis” to “circRNA biogenesis”.

Line 121-122. “CircRNAs play diverse biological roles due to the fact that average half-life of circRNA in most species is much longer than its linear counterpart.”  What is the relationship between biological roles and longer half-life?

Line 127 . Change “cricRNA” to “circRNA”.

Line 130. Change “The systematically validated circRNA such as circRNA such as ciRS-7” to “The systematically validated circRNA such as ciRS-7”.

Line 141. Change “In generally, there are only few circRNAs” to “In general, there are only few circRNAs”

Line 157. Change “small nucleonucleo proteins (snRNPs)”  to “small nuclear ribonucleo proteins (snRNPs)”

Line 180. Change “In eukaryotic system, the some circRNAs” to “In eukaryotic system, some circRNAs”
Line 218. Change “In particularly, their migration” to “Particularly, their migration”

Line 256. “cyclRNAs”. It is not the same nomenclature used through the manuscript.
Line 258. Change “The squence” to “sequence”
Line 268. Change “cricRNA” to “circRNA”.


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 REVIEW OF CIRCRNA: FROM PURIFICATION AND IDENTIFICATION TO DISEASE MARKER POTENTIAL
Review round: 2
Reviewer: 1

Output:
Basic reporting: In the presented manuscript, Xu et al present a review of what is currently known about circular RNA (circRNA), including their biogenesis, function and future directions for their use. While the review is of great interest, the authors have addressed many of the previous commends and the review provides a lot of information concerning circRNA, there is still room for improvement concerning the manuscripts writing.

Primarily, the authors make many claims throughout the manuscript not backed up by citations, maybe a further reread is required to insert correct or missing citations?

Additionally, as commented on below, the many of the sections are confusingly structured and the sentences within are poorly written. I feel the entire manuscript could benefit from a very careful reread and rewrite to clarify what is meant by each sentence, and to make sure the English is a lot clearer than it currently is.
Experimental design: no comment
Validity of the findings: As a review paper, the authors do not make any novel findings, however many of the claims of what circRNA can do are not backed up with citations, it would be useful if these citations could be provided.
Additional comments: I also have some comments on specific sections of the manuscript, I may not cover all grammatical errors and problems due to the sheer number of them.

Line 37: This sentence should be rewritten as: “Depending on their sequence, circRNAs can perform differing biological functions, including …. derivation of pseudogenes.”

Line 43: ‘Here we describe…’

Line 46: Is circRNA the correct pluralization? It reads strangely next to ncRNAs.

Line 55: this sentence should be rewritten as ‘Recent research shows that the lengths of mature circRNAs dictate…’

Line 70: Should this subheadings be here?

Line 72-109: The authors mention Alu elements several times during this section, they appear to be highly important for the generation of circRNAs but it is not clearly explained how important they are, maybe Alu elements could get a better description during this section?

Line 77-78: This sentence is very difficult to parse and should be rewritten entirely.

Line 86-88: This sentence is very difficult to parse and should be rewritten entirely.

Line 151-162: Maybe a better explanation of what species circRNAs are found in, and what species the examples given are in? As Ago2 is specifically siRNA in many organisms, and miRNA in only some.

Line 163-186: Should the ITCH system explained later also be mentioned in this section as a miRNA sponge?

Past this point there are many minor grammatical issues and confusing sentences, but no large problems, I recommend the authors still give the manuscript a thorough read through to fix the English as errors are present in every section throughout the manuscript.


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

Input:
Title: A COMPREHENSIVE REVIEW OF CIRCRNA: FROM PURIFICATION AND IDENTIFICATION TO DISEASE MARKER POTENTIAL
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors have adequately addressed most of the reviewers comments. However, the manuscript still contains many typographic and drafting errors (for example, line 256 “circ-Foxp3” instead of “circ-Foxo3” or line 308 “ripamycin” instead of “rapamycin”). English must be considerably improved.

Minor comments:

The sentences in line 286-288 and line 377-378 are not clear.

I don´t understand the sentence in line 334-335.


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 REVIEW OF CIRCRNA: FROM PURIFICATION AND IDENTIFICATION TO DISEASE MARKER POTENTIAL
Review round: 4
Reviewer: 1

Output:
Basic reporting: The article reports on the function and recent findings concerning circular RNAs. The authors also report on circRNAs involvement in different diseases and cancers in humans. The article is written sufficiently to explain these factors, in an interesting fashion.
Experimental design: NA, the manuscript is as previous.
Validity of the findings: NA, the findings are as reported previously.
Additional comments: The english has improved drastically from the previous iteration of the manuscript, there are a few minor errors not worth reporting, following a reread by the authors, the manuscript is sufficient.


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

Input:
Title: A COMPREHENSIVE REVIEW OF CIRCRNA: FROM PURIFICATION AND IDENTIFICATION TO DISEASE MARKER POTENTIAL
Review round: 4
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: English has been considerably improved. The authors have adequately addressed most of the reviewers 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: COMPARATIVE ANALYSIS OF WHOLE FLOWER TRANSCRIPTOMES IN THE ZINGIBERALES
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Almeida et al. assembled floral transcriptomes for seven linages in the Zingiberales, revealing unique gene conservation and duplication patterns which may be related to divergent and unique floral morphology within the family. These transcriptome assemblies will be useful for the comparative genomics and floral development research communities. I have a few issues that I feel should be addressed to strengthen this manuscript prior to publication.

Major issues:
1. Analyses related to orthology and conserved genes are potentially problematic.
Blastp or blastx  would return more hits than blastn, especially in divergent taxa. This explains why only 2-5% of transcripts from the Zingiberales BLAST to Arabidopsis orthologs; the nucleotide divergence between monocots and eudicots is too high. Hits to Elaeis are also probably lower than expected given it’s phylogenetic placement. Using blastp or blastx would help classify more proteins which could affect the interpretation of gene conservation  and lineage specific changes.
A more robust way to establish orthology would be to use orthoMCL or orthofinder. The resulting orthogroups can then be assigned functional categories based on pfam domains or Arabidopsis homology.

2. More analyses related to gene conservation/ lineage specific changes would be interesting. BLAST results are capturing only a rudimentary survey of gene-level changes. Analyses through a pylogenetic framework would be useful to identify gene duplications and ka/ks divergence related to adaptive evolution of floral morphology. Such analyses may me beyond the scope of this manuscript.

Minor:
Line 122 on. The build/version for each bioinformatic program should be provided.


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

Input:
Title: COMPARATIVE ANALYSIS OF WHOLE FLOWER TRANSCRIPTOMES IN THE ZINGIBERALES
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: Obviously replication of the experiments would be desirable but that is not provided. Nonetheless the resources described here would be beneficial to the community.
Validity of the findings: No comment
Additional comments: The authors have developed transcriptomics datasets to compare the gene expression profiles of members of the Zingiberales order. The goal was to shed light on the evolution and development of these flowers. The task is at hand is extremely difficult and while the data presented is limited, it provides a decent basis for further work. There are a few things that I would recommend changing and have given some suggestions regarding analysis or discussion that could be included.

1.	There are a lot of numbers thrown about in the manuscript, which reduces the readability of the manuscript. I would recommend reducing the prevalence of this in the text (although some are absolutely necessary). For example, “The number of filtered coding sequences observed in this study is similar to already described numbers of floral unigenes of other non-model plants…”, which goes on to list many numbers. To improve the readability I would keep the sentence in quotes above, remove the rest and make a table or simply give the upper lower limit examples while keeping the references.
2.	Ln 102: “whole developing flowers of…”
-	Could the authors be more specific here? It would be nice to know if flowers at several different stages were used. What morphological markers guided the collection strategy?
3.	Ln 92: “we present a comprehensive analysis…”
-	This wording is a bit strong.
4.	Ln 293: I don’t understand why Table 5 is referred to here when discussing chromatin remodeling proteins.
5.	I missed a discussion on the relevance of the floral homeotic protein activity during the manuscript, although the upstream activation of AG was discussed. Perhaps it would be good to include how many of the genes identified as commonly expressed and “differentially” expressed are putative or likely targets of the homeotic proteins as there is a considerable amount of work done on this in Arabidopsis (Kaufmann 2009; Kaufmann 2010; Pajoro 2014; O’Maoileidigh 2013; Wuest 2012). I’m not insisting on a new analysis but a discussion of how the activity of these proteins might be important (or not) during the development of Zingiberales flowers might improve the context of some of the findings.
6.	Similar to point 5, some work has been on the stage-specific expression of genes during Arabidopsis flower development. The authors could look into these data as a comparative resource (Wellmer 2006; O’Maoileidigh 2015; Jiao 2010). Again I’m not insisting on a new analysis but it is something the authors can consider for now or later.


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: PENTOXIFYLLINE DECREASES POST-OPERATIVE INTRA-ABDOMINAL ADHESION FORMATION IN AN ANIMAL MODEL
Review round: 1
Reviewer: 1

Output:
Basic reporting: The study conduct and reporting was excellent, fulfilling all the required criteria.
Experimental design: The experimental design was excellent, fulfilling all the required criteria.
Validity of the findings: The main issue with this study, is the lack of correlation with general woudn healing. While adhesion reduction was demonstrated with PTX, could the same impact on fibrosis affect healing of abdominal wounds and anastomoses? Before clinical trial can be recommended this question needs to be carefully answered in animal models. This warrants specific discussion in the manuscript.
Additional comments: This is a great study, but some caution regarding wound healing effects needs to be exhibited and discussed, prior to simply recommending clinical trials.


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: PENTOXIFYLLINE DECREASES POST-OPERATIVE INTRA-ABDOMINAL ADHESION FORMATION IN AN ANIMAL MODEL
Review round: 1
Reviewer: 2

Output:
Basic reporting: Literature searching of about of prevention of adhesion with PTX should be done again. Some studies related to the subject are given below. These references should be included in the study.
1. Durmus AS, Yıldız H, Yaman M and Simsek H. 2011. The effects of heparin and pentoxifylline on prevention of intra-abdominal adhesions in rat uterine horn models: histopathological and biochemical evaluations. Revue Med Vet 162(4): 198-203.
2. Jafari-Sabet M, Shishegar A, Saeedi AR and Ghahari S. 2015. Pentoxifylline increases antiadhesion effect of streptokinase on postoperative adhesion formation: involvement of fibrinolytic pathway. Indian J Surg 77(3): S837–S842.
Experimental design: Line 32 and 104: Seventy male BALB/c mice were randomized into one of three groups: How were 70 mice divided into 3 equal groups? Number of "n" must be specified in the text.
Validity of the findings: no comment
Additional comments: Some spelling mistakes are given below.
1. Line 38: tissue tPA: “tissue” should be removed.
2. Line 276: Peritoneum Injury: Peritoneum injury.
3. Line 298: such as (TNF-α, IL-1 and IL-6): such as TNF-α, IL-1 and IL-6.

References should be re-controlled (References, journal names etc).
Some references are same.
Line 345: diZerega GS. and Line 347: diZerega GS.
Line 348: diZerega GS, and Campeau JD. and Line 350: diZeregal GS, and Campeau JD.


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: PENTOXIFYLLINE DECREASES POST-OPERATIVE INTRA-ABDOMINAL ADHESION FORMATION IN AN ANIMAL MODEL
Review round: 1
Reviewer: 3

Output:
Basic reporting: Manuscript is well written and easily understandable. Literature is well referenced and relevant to the study. Regarding supplemental data, if the data is to be published, I would recommend it to be in a more user friendly format so that I could be understood by most readers even if they are not experts in interpreting statistical data. I would suggest data to be summarized in a table format.
Experimental design: Experimental design is well defined and seems to address the goal of the study appropriately. Scoring system and investigations to assess adhesion formation are detailed and extensive. However, I would suggest further clarification or revision in following areas of methodology:
a)	In the experimental design (lines 103-118), it is not clear what was done to the Sham group of Mice. My interpretation is that the Sham group is the one without peritoneal adhesion. It is not clear whether this group underwent any kind of surgical intervention or not. Whether there was no intervention at all or this group had open laparotomy and closure without abrading the cecum, needs to be clarified.
b)	Line 120 mentions that mice were euthanized on post operative days 3 and 7. I would suggest more detailed explanation on how many mice were euthanized on days 3 and 7 respectively, from each group. I assume out of the 3 groups created out of 70 mice, some would be euthanized on day 3 and others on day 7. I would suggest explanation of those numbers and whether they were randomized or not.
Validity of the findings: Study seems to be well structured and replicable. I would suggest  a comment whether any of the experimental mice incurred any side effects (e.g death leading to exclusion from study) from pentoxyfilline administration.
Additional comments: Since Lauder scoring system was used to classify the severity of adhesions, a table detailing the Lauder system would be very beneficial for readers


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: BODY SIZE INFORMATION IN LARGE-SCALE ACOUSTIC BAT DATABASES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Clear language:
I had edit and re-read the manuscript several times to understand the material as written. The language is often unsophisticated or casual. Moreover, preparation appears rushed (especially the Supplemental Materials), as formatting, names, and style are inconsistent and difficult to follow. I urge the authors to consult a native English speaker to increase the quality of the manuscript.

The intro and background:
The use of a citizen-science project seems central to the manuscript topic, but the authors fail to demonstrate that the quality of the data is appropriate for the study of bat body size measurements and acoustic call variation.
I'm unclear as to the most important broader implication of this research as written. What's the central idea? Is it about the applicability of citizen science for acoustic data collection? Or that both bat calls and body size show a consistent trend in relation to geography and climate, making calls an appropriate proxy for body size in future studies?
I expected more detailed background on P. pip or relevant conspecifics given that a single species, in particular, was featured in the research. Also, why was this species a model system for this research over any other species? Why is France appropriate for demonstrating variation in climate and geography?

The referenced literature:
Only two cited papers were published post-2015, suggesting that the authors have not updated the manuscript references to account for recent findings.
Citations are sometimes not provided where needed (e.g., lines 55, 81, 106, 173, etc.).

The structure:
Overall, I recommend restructuring the introduction to clarify the aims, questions, and reasoning for the study. In particular, the final paragraph could be more succinct, and the hypotheses made clearer.
The methods, especially, could have been easier to follow if consistent identifiers were used throughout. For instance, reference to the French citizen bat survey changes throughout when it could have been referred to as Vigie-chiro (which is named only in the abstract). Is it the "mist-net" dataset or "capture" dataset? Moreover, although I understand the difference between Fc and SlopeQCF, they are hard to keep track of because they are not consistently named. I believe that more attention should be given to the writing to ensure consistent language.
Since there are two PCA results reported, I suggest naming the axes with respect to the dataset for clarification when reading. I suggest looking at other papers describing results of PCA for tips on language usage.

Figures and tables:
Maps of the study locations should be provided in the main manuscript. This can be used to show the trends in important data. For instance, sampling points could use different sized icons to illustrate larger or smaller values.
Since PCA was a major analysis for the study, I suggest a table of each variable and its corresponding eigenvectors for the two important axes.

Raw data:
Supplied, though some columns are written in French.
Experimental design: The topic:
Is original and within the scope of PeerJ

The research questions:
I recommend using numbers to distinguish between each question/aim and hypothesis in the order of how they will inform one another.

Technical and ethical standard:
I did not see permitting information or ethical standards reported in the main manuscript.

Methods:
I do not believe the methods in the main manuscript is clear without referring to the Supplemental Materials.
Sample sizes and descriptions of each variable should be provided in a table within the main manuscript.
"Clutter" must be defined.
The authors did not report their settings for acoustic analysis they used to filter calls. They also did not report how they chose the quality of calls to retain for analyses, nor how many calls per recording they used. Did they average the 10 highest quality calls per recording? Per sampling segment? The sampling protocol, processing, and assumptions for acoustic recordings are crucial for the validity of the findings.
For the analyses of bat calls, I was not clear on the sample size after re-reading. If a single sampling circuit (n=78) was cut into segments (n= 1133) and used for acoustic analyses, thus increasing the sample size by non-independent data points, this would lead to pseudo-replicated.
I was unclear on how climatic data was acquired and whether temporal and spatial autocorrelation was addressed. For instance, climatic variables for more than just the season of capture would matter for body traits because climate has affected the population over time.
How do differences in bat calls by sex affect the acoustic results given that the authors state call frequency varies by sex?
Validity of the findings: Replication:
I question is the acoustic data was pseudo replicated if sampling circuits were split into multiple segments for analyses outside of averaging bat call parameters (which is unclear as written).

Data usage:
Given that body size is the typical variable used in these types of studies, I was surprised that the authors did not show evidence from their dataset that body size is consistently correlated with forearm length across the sampling area. If the data were available, why not make the analyses more robust by showing the correlations between multiple body trait measurements?
How representative are the measurements made for P. pip of variation across France? The spatial distribution of the data point were not similar between the two datasets that were compared. Moreover, within a dataset it is clear that the sampling was not random nor distributed geographically. Given that this is a study looking at geographic and climatic variation, I would have expected a more representative sampling distribution.

Conclusions:
What is the relationship between forearm length, geography, and climate for P. pip? (Rules have not been verified for this species.)
I am not comfortable with the conclusion that a single parameter of bat acoustic calls can be used as proxy for forearm length given the analyses performed here. The differences in the sampling distribution of the two compared datasets is massive and, as stated by the authors, are not distributed across the geographic or climatic ranges of France.

Speculation:
There should be more discussion about the validity of Bergmann's and James' rules given their relevance to the research.
Additional comments: I had to edit and re-read the manuscript several times to understand the material as written, and I do not believe the main manuscript is clear without referring to the Supplemental Materials frequently. The writing is often unsophisticated and casual. Moreover, preparation appears rushed (especially the Supplemental Materials), as formatting, names, and language are inconsistent and difficult to follow. I urge the authors to consult a native English speaker to increase the quality of the manuscript.


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

Input:
Title: BODY SIZE INFORMATION IN LARGE-SCALE ACOUSTIC BAT DATABASES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The standards of the journal have generally been met in the manuscript, with a clear and professional structure including stated goals and background, methods, results, and discussion that pertain to the stated goals. That said, I failed to find information regarding the ethical use of animals and some minor errors to note:
1.	Line 12 - I believe this should be Sorbonne (not Sorbone)
2.	Line 44 - 'calculation algorithms' is redundant - recommend deleting 'calculation'
3.	Lines 62 and 67- At ^the intraspecific level
4.     In all materials (manuscript + supplementary), please be sure to define your acronyms before you use them for the first time - e.g., QCF, MNHN
5.	Style point - e.g. and i.e. are typically followed by a comma
6.	Line 201 and other - 'precipitations' should be 'precipitation'
7.	Line 240 [This pattern of body size]……has
8.	Line 261 - may also ^have the potentiality to allow detecting fine scale….
9.	Line 285 - 'form' should be 'from'
10.	Line 286 - 'that aim ^to at assess…'

I do, however, recommend that a greater attention to detail, proper English spelling and grammar is given to the supplementary material and data files. For example, in Appendix I - page 4 - this sentence is repeated 'The results of the cross-validation on more that 58% of the data indicate that for 8% of the calls identified as P. kuhlii it was not possible to refute potential contact of P. nathusius and among the P. pipistrellus less than 0.1% could be P. nathusii'. Also, Figure A2.1 legend needs to reference panel A correctly.

Specific comments for the data files:
1.	description of field names (headings) should include units
2.	the wind speed field consist of French words describing conditions - an English translation of each of the words in the description of the field would be helpful
3.	Is time provided in UTC?
Experimental design: This manuscript takes advantage of the established relationship between body size and call frequencies in bats to apply the acoustic data collected through a citizen science bat acoustic monitoring project throughout France to a relevant ecological question about whether intraspecific variation in body size varies systematically with climate. Given that this is the first study (of which I and the authors are aware) that attempts this approach, the authors endeavor to confirm the validity of the findings against a comparable analysis using actual body size data collected from bat capture efforts from throughout France. Both sets of data appear to be robust and collected over the same time period and spatial range. However, the distribution of points does vary significantly between the datasets, with the capture dataset largely from northwestern France and the acoustic dataset largely from central-northern France. The geographic biases in the datasets are the greatest weakness in the study, in my opinion, but do not negate the findings. Bat data are extremely challenging to collect, capture data in particular, and thus sparsity and patchiness of data availability are the norm for ecological studies involving bats. Studies such as this that evaluate the potential diverse applications of the (relatively) more easily acquired acoustic data are therefore of great value to the study of bat ecology.

Manuscript appears to have been prepared to journal standards, with some minor comments:

1. Line 96 - Please clarify what is intended by the phrase 'capture points'. I presume that refers to unique combinations of data and site, but it is not clear. How many sites across how many degrees of latitude were included? I recommend that Figure A1.1 be included in  the main manuscript.

2. Please provide more details about the WorldClim analysis - especially, how many years of data were used? Restricted to time period of capture and acoustical data would be the most compelling, of course.

3. Please provide more details about the suitability of the data for the linear mixed effects model approach, including any evaluation of the variance, independence, and distribution of the errors.
Validity of the findings: In this study, the authors apply traditional principal components techniques to reduce a diversity of relevant geographic and climate parameters into two principal components variables. These are then incorporated into linear mixed-effects models to evaluate whether actual measures of body size (empirical forearm lengths) and an acoustic body size correlate (characteristic low frequency) from Pipistrellus pipistrellus respond in similar ways to the geographic and climate principal components, while evaluating or incorporating the potential impacts of sex, temperature, year, and sampling location on the response. Significant, consistent relationships between body size and both measures of body size are found, although the slopes of the relationships are not striking. The authors thoroughly explore the potential biases in these findings in their analyses, as captured in the supplementary material, and they sufficiently explore the implications of their findings in the discussion. In short, their results suggest that the acoustic body size correlate is a valid proxy when actual body size data are unavailable.

Related comments:
1. I am struggling with the evaluation of the geographic biases in the data section of the Appendix and how it is conveyed in the manuscript. I appreciate that the authors are transparent about the geographic biases in the data, and I appreciate also that these are often unavoidable. I am concerned that merely splitting the data into two subsets and comparing results does not control for the effects of widely varying sample sizes. Subsampling of the dense data areas and/or evaluating the data subsets against the assumptions of linear mixed effects models would likely help to address this concern. Alternatively, acknowledging the bias and avoiding the subsetting analyses altogether would be acceptable from my perspective as well. If the spatial subset analysis is retained, I would consider rephrasing the concluding sentence capture din Lines 226-228. I do not agree that the 'main results were maintained when the analyses were conducted on spatial data subsets'. For example, the forearm length-climate relationship was nonexistent in the eastern dataset. Again, I think this could merely be insufficient data and therefore not readily interpretable or able to negate the main findings of this study.

2. As the authors note, the likely increased abundance of juveniles in the second acoustic survey could alter the intraspecific variation of body sizes and call frequencies. I recommend only using the first survey in the analysis, since the capture data did not include forearm lengths from juveniles.
Additional comments: I enjoyed this manuscript and, with the above comments and a few below addressed, believe it could be an important contribution to bat ecology, as the abundance of acoustic data increases significantly year over year.

A few minor comments:
1.     Line 41 - I would argue that acoustic methods have already demonstrated their utility for biodiversity assessment, so recommend deleting 'promising'
2.	Line 45 - ability to automatically identify species varies across taxonomic groups - recommend rephrasing to reflect the significant amount of research still remaining to be done in this area for many species
3.	Line 53 - Brown's 1995 Macroecology book is a more appropriate reference for the sentence than Gaston & Blackburn 1996/Pyron 1999)
4.	To further bolster the strength of the relationship between call frequency and body size, I recommend adding in a reference to Body Size Predicts Echolocation Call Peak Frequency Better than Gape Height in Vespertilionid Bats. Jeneni Thiagavel, Sharlene E. Santana & John M. Ratcliffe Scientific Reports 7, 828 (2017) doi:10.1038/s41598-017-00959-2


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

Input:
Title: BODY SIZE INFORMATION IN LARGE-SCALE ACOUSTIC BAT DATABASES
Review round: 2
Reviewer: 1

Output:
Basic reporting: Generally, I think that this is a well-written manuscript with interesting implications. I believe that they find support for their hypothesis: they do show that acoustic data collected by a citizen science program can be used to detect broad scale patterns in species traits.

Please see the annotated copy of the manuscript to see where I made some minor changes and where I made comments.
Experimental design: The methods and results are much easier to follow. One thing I noticed this time is that the climate data overlaps with the sampling data for one year only, which might play a role in the strength of their findings. Also, there is only one year of overlap between the mist net and acoustic datasets, which might also introduce inconsistencies.
Validity of the findings: I believe that the authors must be more explicit about the limitations of the study given that the sampling locations are so different. Mist net results are most applicable to longitudinal sampling and acoustic results are most applicable to latitudinal sampling.

Also, I feel that they inflate the strength of the correlation between the two datasets. According to my interpretation of their results, although the first two PCA axes show similar loadings between the mist net dataset and acoustic dataset, the PCA axes that are significantly related to their two response variables are not the same. That is, PCA axis 1 is significant for forearm length, but PCA axis 2 is significant for frequency. Thus, the strongest climatic variables that concur between the two models are:
(1) large forearm/large body & low frequency/large body both relate to low temp range/low variation in temp, but stronger for large forearm, and
(2) large forearm/large body & low frequency/large body both relate to low temp in summer, but stronger for low frequency.
Additional comments: Thank you very much for addressing all of my recommendations. Again, please see the annotated copy of the manuscript.


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

Input:
Title: BODY SIZE INFORMATION IN LARGE-SCALE ACOUSTIC BAT DATABASES
Review round: 2
Reviewer: 2

Output:
Basic reporting: I appreciate the authors' efforts to improve the quality and clarity of the writing in this revision. I also appreciate that the authors incorporated my comments, regarding literature cited, figure selection, and raw data improvements.
Experimental design: I appreciate the authors additional efforts in response to the reviewers to evaluate the dataset for appropriateness for the model used and to further evaluate potential biases in the data.
Validity of the findings: Manuscript is much clearer and stronger in its intent and results, and I think will be a valuable contribution to acoustic ecology.
Additional comments: The authors efforts in the revision process are greatly appreciated and have resulted in a robust manuscript.


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

Input:
Title: FOXR1 IS A NOVEL MATERNAL-EFFECT GENE IN FISH THAT IS REQUIRED FOR EARLY EMBRYONIC SUCCESS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The underlying claim that foxr1 is a evolutionarily conserved gene necessary for early embryonic development seems sound given the multiple mutant offspring analysis. The results relate to this hypothesis, but there is need for some work on the description of experimentation (see section below) before a reader can be convinced that care in experimentation has been taken to warrant all the claims the authors make about mechanism. Additionally speculative parts of the paper need to be addressed.

The UF in Figure three should be described as unfertilized in the figure legend for clarity.
Experimental design: The recording of how the protein sequence for the different foxr genes were obtained should be clarified. The statement “amino acid data were extracted” in the methods section is insufficient to help the reader understand how the databases were queried (BLAST stringencies should be described or method cited).  This will be helpful for someone interpreting the lack of Foxr1 in many species (lines 211-214) and to reproduce the results.

The verification of Cas9 knockout is insufficiently documented, given the tendency of smaller PCR templates to be amplified when in the same reaction as larger templates (in this case, mutant and wild-type respectively.

Description of the qPCR experiments in Figures 4 and 6 line 267-268 and 299-304 should be presented with scatter plots to help the reader understand the distribution of the data and a statement of which mutant(s) produced the experimental embryos. This is especially important given the difference in developmental potential through the blastula stages of two developmental types of presumptive heterozygotes, clutches could have been divided into groups of non-cellularized, cellularized with developmental defects, and normally developing embryos.

Description of male transmission of mutated foxr1 genes is lacking or confusing (lines 271 and 364-365). How many F0 males were mated? What were the results? Why no qPCR data on testes, given 2 males were dissected for tissue analysis?

Line 295 should read Fig 5M rather than 4M.

The gels shown in Figure 5 and in peerj-27280-Uncropped_image_Figure_5 do not appear to be the same exposure of the same gel. The bands look dissimilar.
Validity of the findings: The Cas9 deletion approach and the ability of the authors to detect fertilization by assaying a transgene only from sperm is a compelling assay for fertilization in non-developing embryos and supports the conclusion of a maternal effect for foxr1.

I am not expertly qualified to comment on the phylogenetic analysis of this paper, but the tree seems like a reasonable fit to the data. Would a second BLAST search for homologs using another species than zebrafish be warranted to validate the results found searching only with zebrafish proteins?

I think the authors meant the F1 generation at line 272 since the F0 generation were viable and fertile (excepting foxr1-2) demonstrating no direct phenotype assayed by the authors.

A speculation at lines 217-218 and again at 330-333 is that the different sequences found in fish comes from a gene duplication event is not supported by the data. This is troublesome given that syntenic analysis is obviously done on one of the Foxr1 protein sequences from gar and medaka etc., but not the other. Given the apparent ability of the authors to compare syntenic regions, an explanation about the other sequences and their synteny is warranted. Only sequence obtained from different databases resulted in unique isoforms of the sequence. When speculating, the authors should provide more than one interpretation, and where possible how one would test between the possibilities (lines 340-342 makes the vague statement “Further analyses on the two copies” without explaining the analyses).

The word penetrance (line 276) is used incorrectly in a genetics paper as penetrance is the condition when an phenotype-linked allele is present, but the affected organism does not exhibit a phenotype. The authors had made the point in the paragraph preceding that all surviving embryos were WT. This indicates that the F0 generation were mosaic and many eggs did not have foxr1 disruption. I suggest the word frequency instead.

A comment on the differences seen between qPCR levels and RNA-seq levels in bone is warranted.

The sentence of lines 387-388 is confusing with verb subject disagreement.

The expression claim in lines 388-389 needs citation.

The conclusion statement on 393 and repeated at 399 “via the p21 and mTOR pathways” has been correlated, but not tested in this paper.

Opossum Foxr2 seems given the wrong color in Figure 1. Should it be blue like Armadillo?
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: FOXR1 IS A NOVEL MATERNAL-EFFECT GENE IN FISH THAT IS REQUIRED FOR EARLY EMBRYONIC SUCCESS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is a well-designed and described study from a prominent fish reproduction biologist.
The major findings included:
The presence of foxr1 in a number of species.
In zebrafish, foxr1 has predominately ovarian specific expression.
In zebrafish oocytes, foxr1 is expressed early and accumulates through oogenesis.
When foxr1 is knocked out, oocyte are still fertilized but have problems undergoing normal cell division. This is due to an increase in p21 and a decrease in rictor.

I thought this was a well written paper with a good depth to the introduction and very straightforward study design and results:
Minor issues
A few awkward or confusing sentences:
*note: my complete review download has different line numbers than the individual paper download.

Line 43: In vertebrates, maternal products including transcripts, proteins, and other biomolecules are necessary for kick-starting early embryonic development until the mid-blastula transition (MBT) when the zygotic genome is activated [1].

Line58: foxl2 and its relatives are known to be key players in ovarian differentiation and oogenesis in vertebrates; it is essential for mammalian ovarian maintenance and through knockout experiments, it was demonstrated that foxl2is a critical regulator of sex determination by regulating ovary development and maintenance also in Nile tilapia, medaka, and zebrafish[9]

Line 333: It is also possible that foxr1 was duplicated in the ancestral actinopterygii and subsequent gene losses in bowfin as well as in the teleosts especially following the multiple gene duplication events such as the teleost-specific whole genome duplication (TGD) and salmonid-specific whole genome duplication (SSGD).

Line 345: Previous reports have demonstrated the predominant expression of foxr1 mRNA in the ovary of medaka, eel, and tilapia[8,11,13], but in the male germ cells and spermatids in mouse and human[24].

Vasa::gfp line wasn’t described when it was first mentioned, but was later in the text. Helpful to know why this was used earlier for people outside of the field of maternal-effect genes

Problem with citation 4
Years missing in citations 9 and 11
Experimental design: Well designed. I have a few minor comments:
No section in the methods on animal husbandry.
npm2b mutants are mentioned on Line 288 but without any background as to what or why they were mentioned. This extends to Fig. 5M
Tissue samples came from 2 males and 3 females. It is not clear why only 2 males were used. Aren’t you treating them as separate groups since you are looking at a sex specific expected phenotype? Furthermore, the way they are reported in Fig. 3, it appears that they are just grouped together. Clarity in the description in the methods would probably clarify the design better and make more clear Fig. 3. Furthermore in the legend for Fig. 3, it says that tissues were harvested from 3 to 4 WT zebrafish?
Fig. 4C: there are no WT controls to compare this information to. You seems to have this information for 4B, therefore it should be added to Fig.4C for comparison.
Fig. 4C the cross indicates that all zebrafish in these groups showed the exact same phenotype of a blastodisc sitting atop an enlarged syncytium? Or is this just an example of a developmental defect. Clarity would be good for this.
Fig. 5M. Why was npm2b used as a control. This isn’t clear since it is also missing from the paper.
Validity of the findings: I am concerned about the uncropped blot. I can’t tell what I am looking at in the cropped version (compared to the uncropped blot). I think just a label on the uncropped blot would allow reassurance.
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: FOXR1 IS A NOVEL MATERNAL-EFFECT GENE IN FISH THAT IS REQUIRED FOR EARLY EMBRYONIC SUCCESS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript “foxr1 is a novel maternal-effect gene in fish that regulates
embryonic cell growth via p21 and rictor” by Cheung et al. identifies a function for the transcription factor foxr1 in oogenesis and early embryogenesis. They demonstrate that foxr1 mRNA is provided maternally, consistent with this role. Further, through loss-of-function studies using Crispr-Cas9, they find that the number of normal embryos produced from a foxr1 Crispr-Cas9 edited female and a male WT at the foxr1 locus (carrying the vasa::gfp transgene, which should be expressed in the fertilized F1 progeny) is significantly reduced. Phenotypes of the progeny include failure to undergo any cleavages and abnormal cleavages, both of which result in death of the embryos by 24 hours post spawning. As expected from a mosaic parent, a proportion of the embryos are normal. Overall, this is a well done study that will be of interest to the scientific community, and especially to those interested in maternal effect genes and early development. However, there are some changes needed before the manuscript is ready for publication.
Experimental design: The experimental designs are strong. However, in a few places, additional information is needed.
A. How many biological replicates were done for the RNA-seq experiments?
B. For the GFP PCR on F1 progeny with no cell divisions (Figure 5), were the PCRs done on single embryos or groups? This may affect the interpretation of the results.
Validity of the findings: Major concerns.
1. The claim that Foxr1 functions through regulation of p21 and rictor is not supported by the data in this manuscript. The authors demonstrate that expression of these two genes is altered in the F1 progeny of the foxr1 Crispr-Cas9 edited female X vasa::gfp male. There is no functional data to demonstrate that these changes in gene expression are causing the observed phenotypes in the progeny. The authors should change their title to focus only on their research on Foxr1, they should take the p21 and rictor reference out of the title. There also several places in the Discussion that need to be revised to make it clear that Foxr1 acting through p21 and rictor is a hypothesis or working model only. These places are marked in the manuscript pdf.

2. The Results and Discussion sections are somewhat redundant with each other. The authors should follow the conventions of these sections and make sure text is in the appropriate section.

Minor concerns
1. In several places, the figure legends are not complete or need clarification. These places are marked on the pdf of the manuscript.

2. Similarly, there are several places in the text that need to be revised to improved clarity. These places are also marked in the pdf of 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: FOXR1 IS A NOVEL MATERNAL-EFFECT GENE IN FISH THAT IS REQUIRED FOR EARLY EMBRYONIC SUCCESS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The underlying claim that foxr1 is a evolutionarily conserved gene necessary for early embryonic development seems sound given the multiple mutant offspring analysis. Care has been taken to separate speculation from supported claims. BLAST analysis appears sound. Description of Cas9-mediated gene knockout is described sufficiently. The data plots are easily readable and typographical errors have been corrected. F0 generation fish were viable giving rise to fertilizable eggs without ability to develop. Gene deletion was confirmed by PCR and fertilization by transgene detection in non-developing embryos and supports the conclusion of a maternal effect for foxr1. The authors have rightly backed off of any causation claims involving rictor and p21. This manuscript is acceptable for publication.


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

Input:
Title: FOXR1 IS A NOVEL MATERNAL-EFFECT GENE IN FISH THAT IS REQUIRED FOR EARLY EMBRYONIC SUCCESS
Review round: 2
Reviewer: 2

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: I am fine with the changes made to the paper and think it is ready to go.


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: FOXR1 IS A NOVEL MATERNAL-EFFECT GENE IN FISH THAT IS REQUIRED FOR EARLY EMBRYONIC SUCCESS
Review round: 2
Reviewer: 3

Output:
Basic reporting: The manuscript is now acceptable for publication, with the exception of a few sentences that still need to be revised:
Questions:
1. Figure legend 1:
"The Foxr1 sequences are in red, Foxr2 sequences are in blue, those of Foxn1 are in green, and Foxn3 sequences are in purple. a and b denote multiple copies of Foxr1 sequences in fish."
-a and be are also included for Xenopus tropicalis-does this mean the same as for the fish?.

2. Section 310
"In addition, two of the mutants produced developmentally incompetent eggs with two phenotypes; those with a non-cellularized morphology (Fig 5E-H), and another population that developed albeit with an abnormal morphology (Fig 5I-L). "
Are these two mutants included in the three mutants that are discussed earlier in this paragraph, or are these different fish?

Suggested revisions
Section 235
Remarkably, despite the wide-ranging presence of Foxr1, no related sequences were observed in invertebrates and chondrichthyans (dogfish and sharks) or in certain vertebrate species such as chicken (Gallus gallus).

Section 300
Since neither males nor the females could transmit the mutated genes to future generations (ie. all the surviving embryos were WT), all of our observations were obtained from the F1 generation, which were the fertilized eggs derived from the mosaic female foxr1 mutants.

Section 305
The frequency of the mutation in the mosaic females is demonstrated in Fig 4C, and it was observed that 3 of the mosaic fish produced abundant non-developing eggs that remained non-cellularized, reflecting their failure to undergo cell division, suggesting that they had germ-line transmission of the mutation (Fig 5E-H). The eggs derived from these 3 foxr1 mutant females did not undergo...

Section 315
These embryos underwent developmental arrest at around 4 hpf or the MBT and appeared to regress with further expansion of the syncytium (Fig 5J-K) until death by 24 hpf.

Section 340
These results were in line with the growth arrested phenotype that was observed in the uncellularized and developmentally challenged eggs from the foxr1 mutant females.
Experimental design: All of my concerns have been addressed.
Validity of the findings: All of my concerns have been addressed.
Additional comments: All of my concerns have been addressed in the revised manuscript.


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

Input:
Title: THE EFFECT OF WALKING SPEED ON THE FOOT INTER-SEGMENT KINEMATICS, GROUND REACTION FORCES AND LOWER LIMB JOINT MOMENTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1.1	The paper generally uses clear and unambiguous English through; however, the paper should be further reviewed for grammar / correct use of English. A few notable examples: Lines 104, 161-162, and elsewhere: The present tense should be used.

1.2 Literature references need to be expanded further to fully cover the topic of intra-foot kinetics: A number of researchers have investigated intra-foot kinetics using multi-segment foot models. I have provided a few references below:

-	PC Dixon, H Böhm, L Döderlein. Ankle and midfoot kinetics during normal gait: a multi-segment approach. Journal of biomechanics 45 (6), 1011-1016
-	Bruening, D. A., Cooney, K. M. and Buczek, F. L. (2012). Analysis of a kinetic multi-segment foot model. Part I: Model repeatability and kinematic validity. Gait and Posture 35, 529–534 (listed in the reference list, but not described in the text)
Experimental design: The methods are generally described with sufficient detail & information to replicate; however, a few additional details could improve the manuscript:

2.1	Line 99: The authors discuss the use of a modified plug-in gait model. In which way was the model modified? These details are important for assessing this paper.

2.2	Line 109: “non-wanted effects” is vague and should be revised to allow the reader to understand the rational for the actions taken.

2.3	Line 140: It is unclear what the term “extrinsic moment data” refers to. If the authors are describing external moments, this term in preferred in the literature.

2.4	Knee and ankle moment calculations: Why were these computed via Keenan et al.? Is this approach different to the one implemented in the Vicon modeller? The authors should explain further their analysis choice.

2.5	Data filtering: Why was a single filter used for kinematic and kinetic data? Often settings are tuned for each data type.

2.6	Line 162 and elsewhere: One-way ANOVA tests do not “prove” that differences exist. The word proved used in this context is a little strong. The ANOVA simply suggests that you can are confident in rejecting the null hypothesis. This section and potentially others should be revised.

2.7	FF/TB angles: These angles are generated by the OFM modeller, but are rarely reported in the literature as they are difficult to interpret. The authors should justify the importance of assessing this measure.
Validity of the findings: The results appear valid based on the methods implemented. Statistical approach is sound.
Additional comments: This paper misses a great opportunity to assess the effect of speed on intra-foot kinetics. It is clear that adding these variables would represent a significant amount of extra work for the authors, but it would greatly improve the manuscript. At the very least, the authors should include in their limitations that these intra-foot kinetic parameters are important and could be investigated in future studies.


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

Input:
Title: THE EFFECT OF WALKING SPEED ON THE FOOT INTER-SEGMENT KINEMATICS, GROUND REACTION FORCES AND LOWER LIMB JOINT MOMENTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is a well written article.
The research in this field is important, however the data reported in this article is not new.
The message (increasing rom with increasing speed) of the article had published before by various autors. Adding the ground reaction forces is something new. I would like to emphasie this more in this article.
Experimental design: I would like to see a more specific aim of this article with a more specific hypothesis. The goal of the article should include a statement about ground reaction forces with different speeds, while this is new.
I would like to know, why there were 10 subjects included.
Which foot was analysed
Why slow and fast speed was 25% higher and lower compared to normal speed
How angles were calculated. It is known that the angles are calculated by the anatomical postion of patients (sagittal flexion/extension, frontal inversion/eversion, transverse abduction/adduction). However some models, for example in van hoeve et al angles were calculated with the foot in line with the tibia resulting in  (sagittal flexion/extension, frontal abduction/addution, transverse inverion/eversion).
36 markers were used?? Was there a static trial. Were there markers removed after static trials.
How is it possible that the speed was highly comparable between subjects, while they were walking at self selected speed?
Why was the stance presented and not stance and stride?
Why was the stance not divided in a loading phase, midstance and push-off phase.
Validity of the findings: How do the autors reflect on the findings that several parameters have lower ROM with higher speed. This is not in line with dubbeldam et al. 2010 and van Hoeve et al. 2017. They found higher ROM or equal ROM with higher speed. I understand that there will be some compensation in the foot however to autors did not find this in comparable research.
Please explain in the discussion.
Additional comments: Introduction:
Significant relevance have been found between walking speed and spatial-temporal
53 parameters, lower limbs kinematics, kinetics and electromyography activities in both healthy and 54 pathology adults (Andriacchi, Ogle and Galante, 1977; Chiu and Wang, 2007; Chung and Wang, PeerJ reviewing PDF | (2018:05:28076:0:0:CHECK 6 May 2018)
Manuscript to be reviewed 55 2010; Dubbeldam et al., 2010; Holden, Chou and Stanhope, 1997; Tulchin et al., 2009).

I feel this article should be citated while it is higly comparable.
Medicine (Baltimore). 2017 Sep;96(35):e7907. doi: 10.1097/MD.0000000000007907.
The effect of age and speed on foot and ankle kinematics assessed using a 4-segment foot model.
van Hoeve S1, Leenstra B, Willems P, Poeze M, Meijer K.


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

Input:
Title: THE EFFECT OF WALKING SPEED ON THE FOOT INTER-SEGMENT KINEMATICS, GROUND REACTION FORCES AND LOWER LIMB JOINT MOMENTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have made sufficient changes to warrant publication


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

Input:
Title: INVESTIGATING DIVERSITY OF PATHOGENIC MICROBES IN COMMERCIAL BAIT TRADE WATER
Review round: 1
Reviewer: 1

Output:
Basic reporting: This article describes findings from a microbial DNA analysis of 96 water samples collected from holding tanks in bait shops located in eight states in the Great Lakes Region.  Findings were compared to data from a single location near Beaver Island which is located in the northernmost area of Lake Michigan.  This article is well-written and the abstract appropriately summarizes the research hypothesis, methods and findings.  The number and selection of articles cited seems appropriate.
Experimental design: The research described is original and the question being addressed is relevant to the quality and protection of the Great Lakes ecosystem.  The experimental design used to select water samples for analysis appears valid, however it is unclear why so many of the bait shops were located in Michigan and why so many of the Michigan locations appear to be more than 50 miles from the nearest Great Lake shoreline.  The sampling of the ‘control’ area in northern Lake Michigan is not described, however an earlier article by Hengy et al. is referenced.

This section includes a detailed description of the genetic analysis.  This is not an area I am qualified to evaluate and should be reviewed by others who work in this area.

The genetic analysis was limited to a relatively small number of pathogens.  A USGS report on pathogens found at beaches on the Great Lakes assessed common fecal bacteria including E. coli, Enterococci, Cryptosporidium, and norovirus, among others of public health concern (https://pubs.usgs.gov/fs/2013/3071/pdf/fs2013-3071.pdf).  Yet none of these were included in this article which summarizes findings for 12 pathogens, many of which seem less likely to be of concern to the Great Lakes.  The selection of pathogens merits additional explanation.
Validity of the findings: The author’s findings are interesting and appear valid.
Additional comments: This is an interesting article that describes original research.  However, there are limitations in the study design the most important of which seems to be the selection and number of pathogens included in the analysis.  The relevance of the authors’ findings to the condition of the Great Lakes ecosystem, the safety of public source waters, and to the health of wildlife, domestic animals, anglers and beachgoers is not clear.   For example, genetic sequencing does not indicate whether the identified organisms are viable or whether a pathogen can survive and replicate in the cold waters of these lakes.  Perhaps the discussion section could be expanded to include ideas for future research that would answer questions regarding the potential for the identified pathogens, as well as pathogens not included in this study but likely to be found in bait shop tanks, to survive in the Great Lakes and infect aquatic biota, public water supplies, animals, swimmers and consumers of fish from these lakes.

Minor editorial suggestions:
	1.  In line 103, the word ‘identify’ should be changed to ‘identity’.
	2.  The numbers in Table 1 do not exactly match the dots of the map in Fig 1.  Perhaps the dots in Fig 1 are too large to show all sampling sites.
	3.  Tables 2, 3 and 4 should be revised to be more easily compared.  I suggest listing pathogens in alphabetical order and use of the same font for each table.  Tables 3 and 4 should show the number of positive samples.  Percent can be shown in parentheses, but this likely isn’t necessary due to the small number of samples included in this study.


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

Input:
Title: INVESTIGATING DIVERSITY OF PATHOGENIC MICROBES IN COMMERCIAL BAIT TRADE WATER
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comments
Experimental design: Really, my only concern with the manuscript is that the authors use a single wild location in Lake Michigan as a basis for comparison of all the bait shops spread around the region. I don't think it's appropriate to make broad statements about the amount of pathogen signal present in bait water vs lake water when using only one location in the wild. I suggest using at least a few more lake sites, possibly from a few other of the Great Lakes.
Validity of the findings: As stated above, the findings need more grounding against natural/field conditions. Otherwise the science of the manuscript is sound.
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: FINE-SCALE DIFFERENTIATION BETWEEN BACILLUS ANTHRACIS AND BACILLUS CEREUS GROUP SIGNATURES IN METAGENOME SHOTGUN DATA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is clearly written and well structured, and meets the journal's criteria.
Experimental design: 1. There are now many classifiers available which vary in their specificity and sensitivity, recently reviewed by McIntyre et al (as cited in the manuscript). McIntyre also specifically addresses this question of identification of B. anthracis in these same samples, and reports that when combining tools to reduce false positives, 'two of six such pairs still reported anthrax', with the focus on pointing out that false positives are a problem, but does not elaborate on the 4 of the six pairs which do not report anthrax.  While the authors of this manuscript include comparisons with Kraken and MetaPhlAn results (from the Afshinnekoo paper), a direct comparison with the findings of McIntyre with relation to these samples would seem appropriate to gaining a fuller understanding of the limitations and utility of the different methods.

2. A comparison of the results of this analysis with KrakenHLL was performed using 'the standard Kraken database'. A number of different pre-built databases are currently available for Kraken (4Gb and 8Gb minikraken databases which available with and without DUST masking: https://ccb.jhu.edu/software/kraken/). The use of a DUST-masked database should significantly reduce the false positive rate, however it is not clear from the manuscript which of these databases was used. This should be clarified, and results from using a DUST masked database should be provided. Since the authors describe the identification of a number of misclassified B. cereus genomes in the databases, a better comparison with Kraken would be to use a custom database including these taxonomical corrections.

3. The authors have gone to admirable lengths in providing access to scripts and code used to carry out the analysis described in this paper, however identifying precisely how a given program was run requires the reader to locate the appropriate script in the github repository then find the appropriate command-line in the script. It would be preferable if basic parameters of programs (i.e. which bwa algorithm?) used in the analysis were included directly in the methods

4. A number of Entrez queries used to download reference data are provided, however the results of these queries are highly variable with time. Providing a list of the accessions (including sequence version) of the sequences included in these reference sets in the github repository would greatly assist anyone attempting to replicate or build on this analysis.
Validity of the findings: No comment
Additional comments: This is a paper which clearly demonstrates the dangers associated with making assumptions on the basis of general metagenomics classification methods. It is weakened by only comparing results with  Kraken and MetaPhlan, while the data of McIntyre et. al is suggestive of reduced false positive rates when using certain classifiers in combination.

The paper would also benefit from discussion of how readily this approach can be adapted for other organisms.